(12) Patent Application Publication (10) Pub. No.: US 2015/0368710 A1 Fuller Et Al

US 2015036871 OA1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0368710 A1 Fuller et al. (43) Pub. Date: Dec. 24, 2015

(54) CHEMICAL METHODS FOR PRODUCING (22) Filed: Mar. 23, 2015 TAGGED NUCLEOTDES Related U.S. Application Data (71) Applicants: Carl W. Fuller, Berkeley Heights, NJ (US); Shiv Kumar, Belle Mead, NJ (60) Provisional application No. 61/969,628, filed on Mar. (US); Jingyue Ju, Englewood Cliffs, NJ 24, 2014. (US); Randall Davis, Pleasanton, CA (US); Roger Chen, Saratoga, CA (US) Publication Classification (51) Int. C. (72) Inventors: Carl W. Fuller, Berkeley Heights, NJ (US); Shiv Kumar, Belle Mead, NJ CI2O I/68 (2006.01) (US); Jingyue Ju, Englewood Cliffs, NJ C07H 17/02 (2006.01) (52) U.S. C. (US); Randall Davis, Pleasanton, CA CPC ...... CI2O I/6874 (2013.01); C07H 17/02 (US); Roger Chen, Saratoga, CA (US) (2013.01); C12O 1/6806 (2013.01) (73) Assignees: The Trustees of Columbia University in the City of New York, New York, NY (57) ABSTRACT (US); Genia Technologies, Inc., This disclosure provides systems and methods for attaching Mountain View, CA (US) nanopore-detectable tags to nucleotides. The disclosure also provides methods for sequencing nucleic acids using the dis (21) Appl. No.: 14/666,124 closed tagged nucleotides. Patent Application Publication Dec. 24, 2015 Sheet 1 of 40 US 2015/036871.0 A1

FIG. I. Patent Application Publication Dec. 24, 2015 Sheet 2 of 40 US 2015/036871.0 A1

Patent Application Publication Dec. 24, 2015 Sheet 3 of 40 US 2015/036871.0 A1

f C YN-so o=-c-A-d-- Follo 8. {C} C. : 3. Ra R (dCp)-(PO)-dC

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w o to C N ---x . . {}r-i-le-for-creas C {- C- C- fi :- s '...'s & 32 (dGp)-(PO)-tiG

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(dTp)-(PO)-dT

FIG. 4 Patent Application Publication Dec. 24, 2015 Sheet 5 of 40 US 2015/036871.0 A1

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Ce: {{}rrent Reactigs 388to: Ca

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FIG. 7 Patent Application Publication Dec. 24, 2015 Sheet 8 of 40 US 2015/036871.0 A1

Nucleic Sample Nucleic Acid Preparation Acid Extraction Sequencing

FIG. 8 Patent Application Publication Dec. 24, 2015 Sheet 9 of 40 US 2015/036871.0 A1

FIG. 9A Patent Application Publication Dec. 24, 2015 Sheet 10 of 40 US 2015/036871.0 A1

Patent Application Publication Dec. 24, 2015 Sheet 11 of 40 US 2015/036871.0 A1

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Patent Application Publication Dec. 24, 2015 Sheet 19 of 40 US 2015/036871.0 A1

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acid-haide Arairie fortation of aide

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Patent Application Publication Dec. 24, 2015 Sheet 35 of 40 US 2015/036871.0 A1

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Patent Application Publication Dec. 24, 2015 Sheet 37 of 40 US 2015/036871.0 A1

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FIG 38 US 2015/036871.0 A1 Dec. 24, 2015

CHEMICAL METHODS FOR PRODUCING of these four PEG-coumarin tagged dG nucleotides by DNA TAGGED NUCLEOTDES polymerase. See also, U.S. Patent Application Publication 0001. This application claims benefit under 35 U.S.C. Nos. US 2013/0244340A1 and US 2013/0264207 A1. S119(e) of U.S. Provisional Patent Application Ser. No. 0010 Recognized herein is the need for improved compo 61/969,628, filed Mar. 24, 2014, the contents of which are sitions and methods for nucleotide identification and nucleic hereby incorporated herein by reference. acid sequencing. 0002 This application incorporates-by-reference nucle otide and/or amino acid sequences which are present in the SUMMARY file named “150805 0575 85625 SequenceListing JAK. txt, which is 52 kilobytes in size, and which was created Aug. 0011 Provided herein are nucleotides with attached tags 5, 2015 in the IBM-PC machine format, having an operating and methods for attaching tags to nucleotides. The tags can be system compatibility with MS-Windows, which is contained attached by chemical reactions, such as "click chemistry. in the text file filed Aug. 5, 2015 as part of this application. 0012. In an aspect, the present disclosure provides a 0003. This invention was made with government support tagged nucleotide, comprising: (a) a poly-phosphate moiety under Grant number 5R01HG007415 awarded by the having a terminal phosphate; and (b) a tag covalently coupled National Institutes of Health. The government has certain to the terminal phosphate of the nucleotide by a triazole, a rights in the invention. 1.2-diazine, a disulfide, a secondary amine, a hydrazone, a thio-acetamide, or a maleimide-thioadduct. TECHNICAL FIELD 0013. In some embodiments of the tagged nucleotide, the 0004. This application relates to tagged nucleotide com tag is covalently coupled to the terminal phosphate by a positions, methods of preparing and using the disclosed triazole. In some embodiments, the triazole has the structure: tagged nucleotide compositions for sequencing nucleic acids, and in particular, nanopore-based sequencing methods. R2 A INCORPORATION BY REFERENCE N - 0005 All publications, patents, and patent applications mentioned in this specification are herein incorporated by R t/ reference to the same extent as if each individual publication, patent, or patent application was specifically and individually wherein R comprises a tag, and R comprises a nucleotide; or indicated to be incorporated by reference. wherein R comprises a nucleotide, and R comprises a tag. In Some embodiments, the triazole has the structure: BACKGROUND 0006 Nucleic acid sequencing is the process for determin ing the nucleotide sequence of a nucleic acid. Such sequence information may be helpful in diagnosing and/or treating a Subject. For example, the sequence of a nucleic acid of a Subject may be used to identify, diagnose and potentially develop treatments for genetic diseases. As another example, research into pathogens may lead to treatment for contagious diseases. Since some diseases are characterized by as little as one nucleotide difference in a chain of millions of nucle wherein R and R combine to form a cyclic moiety; and otides, highly accurate sequencing is essential. wherein R and R combined comprise a tag, and R com 0007. There are methods available that may be used to prises a nucleotide; or wherein R and R combined comprise sequence a nucleic acid. Such methods, however, are expen a nucleotide, and R comprises a tag. In some embodiments, sive and may not provide sequence information within a time the triazole is formed by a reaction between an azide and an period and at an accuracy that may be necessary to diagnose alkyne. and/or treat a Subject. 0014. In some embodiments of the tagged nucleotide, the 0008. In some instances, methods of nucleic acid sequenc tag is covalently coupled to the terminal phosphate by a ing that pass a single stranded nucleic acid molecule through 1.2-diazine. In some embodiments, the 1,2-diazine comprises a nanopore have insufficient sensitivity. Nucleotide bases a dihydropyridazine moiety. In some embodiments, the 12 (e.g., adenine (A), cytosine (C), guanine (G), thymine (T) diazine or dihydropyridazine moiety is formed by reaction and/or uracil (U)) may not provide a sufficiently distinct between a tetrazine and a trans-cyclooctene. signal from each other. In particular, the purines (i.e., A and 0015. In some embodiments of the tagged nucleotide, the G) are of a similar size, shape and charge to each other and poly-phosphate moiety is at the 5'-position of the nucleotide. provide an insufficiently distinct signal in Some instances. In some embodiments, the poly-phosphate moiety comprises Also, the pyrimidines (i.e., C.T and U) are of a similar size, at least 3 phosphates, at least 4 phosphates, at least 5 phos shape and charge to each other and provide an insufficiently phates, at least 6 phosphates, or at least 7 phosphates. In some distinct signal in Some instances. embodiments, the poly-phosphate moiety comprises from 4 0009 Kumar et al. (2012) describes using a nanopore to to 6 phosphates. In some embodiments, the poly-phosphate distinguish four different length PEG-coumarin tags attached moiety comprises 6 phosphates. via a terminal 5'-phosphoramidate to a dG nucleotide, and 0016. In some embodiments, the covalent coupling separately demonstrates efficient and accurate incorporation between the tag and the terminal phosphate can comprise a US 2015/036871.0 A1 Dec. 24, 2015

linker or a spacer moiety. In some embodiments, the linker or nucleotide to the tag, wherein the first reactive functional spacer moiety comprises an alkyl group of at least 2 carbons group is selected from (i) the group consisting of a thiol, an to about 12 carbons. imidazole, an amine, an alkyne and a diene, and the second 0017. In some embodiments of the tagged nucleotide, the reactive functional group is selected from (ii) the group con tag comprises nucleotides, oligonucleotides, peptides, poly sisting of a maleimide, a haloacetamide, an aldehyde, an ethylene glycol (PEG), oligo-Saccharides, carbohydrates, isothiocyanate, an isocyanate, a vinyl Sulphone, an azide and peptide nucleic acids (PNA), vinyl polymers, other water a tetrazine, or vice versa (i.e. the first reactive functional soluble polymers, or any combination thereof. group is selected from (ii), and the second reactive functional 0018. In some embodiments of the tagged nucleotide, the group is selected from (i)). tag comprises an oligonucleotide. In some embodiments, the 0023. In some embodiments, the first reactive functional oligonucleotide tag comprises at least 7 monomer units, at group is different than the second reactive functional group. least 10 monomerunits, at least 15 monomer units, at least 20 0024. In some embodiments, the first reactive functional monomer units, at least 25 monomer units, at least 30 mono group is selected from the group consisting of a thiol, an mer units, at least 35 monomer units, at least 40 monomer imidazole, an amine, an alkyne and a diene. units, or at least 50 or more monomer units. 0025. In some embodiments, the second reactive func 0019. In some embodiments of the tagged nucleotide, the tional group is selected from the group consisting of a male tag comprises an oligonucleotide wherein the oligonucle imide, a haloacetamide, an aldehyde, an isothiocyanate, an otide comprises an unnatural nucleotide. In some embodi isocyanate, a vinyl Sulphone, an azide and a tetrazine. ments, the unnatural nucleotide comprises a group selected 0026. In some embodiments, the first reactive functional from the group consisting of an L-nucleotide, a 2, 5'-linkage, group is an alkyne and the second reactive functional group is an C-D-nucleotide, a non-naturally occurring internucleotide an azide. linkage, a non-naturally-occurring base, a non-naturally 0027. In some embodiments, the alkyne is a cyclooctyne. occurring Sugar moiety, and any combination thereof. In 0028. In some embodiments, the first reactive functional Some embodiments, the unnatural nucleotide comprises a group is selected from the group consisting of a maleimide, a non-naturally occurring base is selected from the group con haloacetamide, an aldehyde, an isothiocyanate, an isocyan sisting of nitropyrrole, nitroindole, nebularine, Zebularine, ate, a vinyl Sulphone, an azide and a tetrazine. benzene, and benzene derivatives. In some embodiments, the 0029. In some embodiments, the second reactive func unnatural nucleotide comprises a non-naturally occurring tional group is selected from the group consisting of a thiol, internucleotide linkage selected from the group consisting of an imidazole, an amine, an alkyne and a diene. a phosphotriester, phosphorothioate, methylphosphonate, 0030. In some embodiments, the first reactive functional boronophosphate, phosphoramidate and a morpholino moi group is an azide and the second reactive functional group is ety. an alkyne. 0020. In some embodiments of the tagged nucleotide, the 0031. In some embodiments, the alkyne is a cyclooctyne. tag comprises an oligonucleotide wherein the 5'-end of the 0032. In some embodiments, the reaction is facilitated by oligonucleotide is covalently coupled to the terminal phos a heterogeneous catalyst comprising copper, ruthenium, sil phate of a poly-phosphate moiety. In some embodiments, the ver, or any combination thereof. oligonucleotide with the 5'-end covalently coupled to the 0033. In some embodiments, the reaction is not facilitated terminal phosphate further comprises a chemical modifica by a heterogeneous catalyst. tion of its 3' terminus that protects it from exonuclease deg 0034. In another aspect, the disclosure provides a kit for radation. In some embodiments, the chemical modification of sequencing nucleic acid comprising at least one tagged nucle its 3' terminus is selected from phosphorylation, and covalent otide. coupling with a C-alkyl to C2-alkyl spacers. In other 0035. In some embodiments of the invention, the tag is embodiments of the tagged nucleotide, the tag comprises an selected from the group consisting of the tags listed in Table oligonucleotide wherein the 3'-end of the oligonucleotide is 4. covalently coupled to the terminal phosphate of a poly-phos 0036. In some embodiments of the invention, the tagged phate moiety. In some embodiments, the oligonucleotide with nucleotide is selected from the group consisting of the tagged the 3'-end covalently coupled to the terminal phosphate fur nucleotides listed in Table 4. ther comprises a chemical modification of its 5' terminus that 0037. In some embodiments of the invention, the tag is protects it from exonuclease degradation. In some embodi selected from the group consisting of the tags listed in Table ments, the chemical modification of its 5' terminus is selected 5. from phosphorylation, and covalent coupling with a C-alkyl 0038. In some embodiments of the invention, the tag com to C2-alkyl spacers. prises a chemical modification selected from the group con 0021. In some embodiments of the tagged nucleotide, the sisting of the chemical modifications listed in Table 6. tag comprises an oligonucleotide wherein the oligonucle 0039. In some embodiments of the invention, the tagged otide comprises a linker comprising a cyanine dye moiety. In nucleotide comprises a cyanine dye moiety in a linker con Some embodiments, the cyanine dye moiety is a Cy3 moiety. necting the tag to the nucleotide, and the tagged nucleotide 0022. In another aspect, the disclosure provides a process has an improved rate of capture by a polymerase compared to for making a tagged nucleotide, comprising: (a) providing a a tagged nucleotide without a cyanine dye moiety. nucleotide comprising a poly-phosphate moiety that com 0040. The disclosure provides methods for determining prises a terminal phosphate, wherein the terminal phosphate the nucleotide sequence of a single-stranded nucleic acid is coupled to a linker that comprises a first reactive functional (DNA or RNA) that use the tagged nucleotides disclosed group; (b) providing a tag comprising a second reactive func herein. Thus, in another aspect, the disclosure provides a tional group; and (c) reacting the first reactive functional method for determining the nucleotide sequence of a single group with the second reactive functional group to link the stranded nucleic acid (DNA or RNA) comprising: US 2015/036871.0 A1 Dec. 24, 2015

(a) contacting the single-stranded nucleic acid, wherein the otide under conditions permitting the nucleic acid poly single-stranded nucleic acid is in an electrolyte solution in merase to catalyze incorporation of the tagged nucleotide into contact with a nanopore in a membrane and wherein the the primer if it is complementary to the nucleotide residue of single-stranded nucleic acid has a primer hybridized to a the single-stranded nucleic acid which is immediately 5' to a portion thereof, with a nucleic acid polymerase and at least nucleotide residue of the single-stranded nucleic acid hybrid four tagged nucleotides under conditions permitting the ized to the 3' terminal nucleotide residue of the primer, so as nucleic acid polymerase to catalyze incorporation of one of to form a nucleic acid extension product, wherein the tagged the tagged nucleotides into the primer if it is complementary nucleotide comprises a poly-phosphate moiety having a ter to the nucleotide residue of the single-stranded nucleic acid minal phosphate, a base which is adenine, guanine, cytosine, which is immediately 5' to a nucleotide residue of the single stranded nucleic acid hybridized to the 3' terminal nucleotide thymine, or uracil, or a derivative of each thereof, and a tag residue of the primer, so as to form a nucleic acid extension covalently coupled to the terminal phosphate of the nucle product, otide by a triazole, a 1,2-diazine, a disulfide, a secondary 0041 wherein each of the at least four tagged nucleotides amine, a hydrazone, a thio-acetamide, or a maleimide-thio comprises a poly-phosphate moiety having a terminal phos adduct, phate, a base which is adenine, guanine, cytosine, thymine, or wherein incorporation of a tagged nucleotide results in uracil, or a derivative of each thereof, and a tag covalently release of a polyphosphate having the tag attached thereto and coupled to the terminal phosphate of the nucleotide by a wherein if the tagged nucleotide is not incorporated, itera triazole, a 1,2-diazine, a disulfide, a hydraZone, a secondary tively repeating the contacting with a different tagged nucle amine, a thio-acetamide, or a maleimide-thioadduct, otide until a tagged nucleotide is incorporated, with the pro 0042 wherein (i) the type of base in each tagged nucle Viso that (1) the type of base in each tagged nucleotide is otide is different from the type of base in each of the other different from the type of base in each of the other three three tagged nucleotides, and (ii) either the number of phos tagged nucleotides, and (2) either the number of phosphates phates in the poly-phosphate moiety of each tagged nucle in the poly-phosphate moiety of each tagged nucleotide is otide is different from the number of phosphates in the poly different from the number of phosphates in the poly-phos phosphate moiety of the other three tagged nucleotides, or the phate moiety of the other three tagged nucleotides, or the number of phosphates in the poly-phosphate moiety of each number of phosphates in the poly-phosphate moiety of each tagged nucleotide is the same and the type of tag on each tagged nucleotide is the same and the type of tag on each tagged nucleotide is different from the type of tag on each of tagged nucleotide is different from the type of tag on each of the other three tagged nucleotides, the other three tagged nucleotides; 0043 wherein incorporation of the tagged nucleotide (b) determining which tagged nucleotide has been incorpo results in release of a polyphosphate having the tag attached rated into the primer to form a nucleic acid extension product thereto; in step (a) by applying a Voltage across the membrane and (b) determining which tagged nucleotide has been incorpo measuring an electronic change across the nanopore resulting rated into the primer to form a nucleic acid extension product from the polyphosphate having the tag attached theretogen in step (a) by applying a Voltage across the membrane and erated in step (a) entering into, becoming positioned in, and/ measuring an electronic change across the nanopore resulting or translocating through the nanopore, wherein the electronic from the polyphosphate having the tag attached theretogen change is different for each value of n, or for each different erated in step (a) entering into, becoming positioned in, and/ type oftag, as appropriate, thereby identifying the nucleotide or translocating through the nanopore, wherein the electronic residue in the single-stranded nucleic acid complementary to change is different for each different number of phosphates in the poly-phosphate moiety, or for each different type oftag, as the incorporated tagged nucleotide; and appropriate, thereby identifying the nucleotide residue in the (c) iteratively performing steps (a) and (b) for each nucleotide single-stranded nucleic acid complementary to the incorpo residue of the single-stranded nucleic acid being sequenced, rated tagged nucleotide; and wherein in each iteration of step (a) the tagged nucleotide is (c) iteratively performing steps (a) and (b) for each nucleotide incorporated into the nucleic acid extension product resulting residue of the single-stranded nucleic acid being sequenced, from the previous iteration of step (a) if it is complementary wherein in each iteration of step (a) the tagged nucleotide is to the nucleotide residue of the single-stranded nucleic acid incorporated into the nucleic acid extension product resulting which is immediately 5' to a nucleotide residue of the single from the previous iteration of step (a) if it is complementary stranded nucleic acid hybridized to the 3' terminal nucleotide to the nucleotide residue of the single-stranded nucleic acid residue of the nucleic acid extension product, which is immediately 5' to a nucleotide residue of the single 0046 thereby determining the nucleotide sequence of the stranded nucleic acid hybridized to the 3' terminal nucleotide single-stranded nucleic acid. residue of the nucleic acid extension product, 0044 thereby determining the nucleotide sequence of the 0047. In some embodiments of the methods, each poly single-stranded nucleic acid. phosphate moiety comprises at least 3 phosphates, at least 4 0045. In another aspect of the methods, the disclosure phosphates, at least 5 phosphates, at least 6 phosphates, at provides a method for determining the nucleotide sequence of least 7 phosphates, or in Some embodiments at least 8 phos a single-stranded nucleic acid (DNA or RNA) comprising: phates. In some embodiments, the poly-phosphate moiety (a) contacting the single-stranded nucleic acid, wherein the comprises from 4 to 6 phosphates. In some embodiments, the single-stranded nucleic acid is in an electrolyte solution in poly-phosphate moiety comprises 6 phosphates. contact with a nanopore in a membrane and wherein the 0048. In some embodiments of the methods, each tag is single-stranded nucleic acid has a primer hybridized to a covalently coupled to the terminal phosphate by a triazole. In portion thereof, a nucleic acid polymerase and a tagged nucle Some embodiments, each triazole has the structure: US 2015/036871.0 A1 Dec. 24, 2015

BRIEF DESCRIPTION OF THE DRAWINGS ?: 0063. The novel features of the invention are set forth with N particularity in the appended claims. A better understanding R ? Y. of the features and advantages of the present invention will be U NM. obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the prin ciples of the invention are utilized, and the accompanying 0049 wherein R comprises the tag, and R comprises the drawings (also referred to as “Figures' or “FIGS.) of which: nucleotide; or 0064 FIG. 1 shows a tag attached to the terminal phos 0050 wherein R comprises the nucleotide, and R com phate of a nucleotide; prises the tag. 0065 FIG. 2 shows alternate tag locations: 0051. In some embodiments of the methods, each triazole 0.066 FIG. 3 shows an example of tagged nucleotides; has the structure: 0067 FIG. 4 shows an example of tagged nucleotides; 0068 FIG. 5 shows a structure of a tagged nucleotide. Tag 505 is attached to the terminal phosphate: 0069 FIG. 6 shows a nucleotide (left) and a tag (right) capable of being joined by click chemistry; 0070 FIG.7 shows an example of the cell current readings for four cleaved tags; 0071 FIG. 8 schematically shows the operations of the sequencing method described herein; (0072 FIG. 9A, FIG.9B and FIG. 9C show examples of 0052 wherein R and R combine to form a cyclic moiety; nanopore detectors, where FIG. 9A has the nanopore dis and posed upon the electrode, FIG.9B has the nanopore inserted 0053 wherein R and R combined comprise a tag, and R in a membrane over a well and FIG.9C has the nanopore over comprises a nucleotide; or a protruding electrode; 0054 wherein RandR combined comprise a nucleotide, 0073 FIG. 10 illustrates a method for nucleic acid and R2 comprises a tag. sequencing: 0055. In some embodiments of the methods, each triazole 0074 FIG. 11 shows an example of a signal generated by is formed by a reaction between an azide and an alkyne. the passage of tags through a nanopore; 0056. In some embodiments of the methods, each tag is 0075 FIG. 12 shows an exemplary chip set-up comprising covalently coupled to the terminal phosphate by a 1,2-diaz a nanopore, ine 0076 FIG. 13 shows an array of nanopore detectors; 0057. In some embodiments, each tag comprises nucle 0077 FIG. 14 shows a computer system configured to otides, oligonucleotides, peptides, polyethylene glycol control a sequencer, (PEG), oligo-saccharides, carbohydrates, peptide nucleic (0078 FIG. 15 shows detectable TAG-polyphosphate and acids (PNA), vinyl polymers, other water-soluble polymers, detectable TAG: or any combination thereof. 007.9 FIG.16 shows an example of synthesis of coumarin PEG-dG4P tagged nucleotides; 0058. In some embodiments, each tag comprises a chemi 0080 FIG. 17 shows an example of characterization of the cal modification selected from the group consisting of the released tags by MALDI-TOF MS; chemical modifications listed in Table 6. I0081 FIG. 18 shows a histogram of cell current readings: 0059. In some embodiments, each tagged nucleotide is I0082 FIG. 19 shows a plot of current measured in pico selected from the group consisting of the tagged nucleotides amps versus time measured in seconds for 4 different tags; listed in Table 4. I0083 FIG. 20 shows examples of conjugation reactions; 0060. In some embodiments, each tagged nucleotide com I0084 FIG. 21 shows exemplary click chemistry reactions prises a cyanine dye moiety in a linker connecting the tag to useful for making the tagged nucleotides of the present dis the nucleotide, and the tagged nucleotide has an improved closure, where (A) shows a click reaction between an azide rate of capture by a polymerase compared to a tagged nucle modified A compound and an alkyne-modified B compound otide without a cyanine dye moiety. to produce an A-B conjugate with a triazole covalent cou 0061. In some embodiments, the four tagged nucleotides pling, where (B) shows a click reaction between an azide are dA6P-Cy3-T-FldTT-FIdT-T-C3, dT6P-Cy3-T- modified A compound and an cyclooctyne-modified B com dSp8-To-C3, dG6P-Cy3-To-C6, and dC6P-Cy3-T-dSp3 pound (e.g., as in a Cu-free click reaction) to produce an A-B T-C3. conjugate with a triazole covalent coupling, and where (C) 0062. Additional aspects and advantages of the present shows a click reaction (e.g., an IEDDA click reaction) disclosure will become readily apparent to those skilled in between an tetrazine-modified A compound and a trans-cy this art from the following detailed description, wherein only clooctene-modified B compound to produce an A-B conju illustrative embodiments of the present disclosure are shown gate with a 1,2-diazine covalent coupling in the dihydropy and described. As will be realized, the present disclosure is ridazine tautomeric form; capable of other and different embodiments, and its several 0085 FIG.22 shows the result of a click reaction between details are capable of modifications in various obvious dA6P-N and DBCO-Cy3; respects, all without departing from the disclosure. Accord I0086 FIG. 23 shows a MALDI-TOF MS spectrum that ingly, the drawings and description are to be regarded as indicates the conversion of azido-nucleotide to the product, illustrative in nature, and not as restrictive. DBCO-Cy3-dT6P; US 2015/036871.0 A1 Dec. 24, 2015

I0087 FIG. 24 shows a click reaction between dT6P-N binds to the polymerase active site with its tag positioned to and Hexynyl-Cy3-Ts oligonucleotide to form a dT6P-Cy3 enter the nanopore for detection by current blockade. T2s tag: I0088 FIG. 25 shows examples of the synthesis of DETAILED DESCRIPTION 2'-Deoxyadenosine-5'-hexaphosphate and attachment of a 0102. While various embodiments of the invention have tag to the terminal phosphate using click chemistry; been shown and described herein, it will be obvious to those 0089 FIG. 26 shows an example of a click reaction skilled in the art that such embodiments are provided by way between dT6P-N3 and Oligo-Alkyne; of example only. Numerous variations, changes, and Substi 0090 FIG.27 shows an example of a thiol (disulfide bond) tutions may occur to those skilled in the art without departing coupling of a tag to a nucleotide; from the invention. It should be understood that various alter 0091 FIG. 28 shows mass spectra of Tag-Nucleotide natives to the embodiments of the invention described herein dT6P-Cy3-Ts and an extension reaction; and may be employed. 0092 FIG. 29 shows examples of monomers that can be 0103) The term “nanopore.” as used herein, generally incorporated into oligonucleotides using amidite chemistry. refers to a pore, channel or passage formed or otherwise 0093 FIG. 30 shows four different tagged nucleotides provided in a membrane. A membrane may be an organic prepared using azido-alkyne click chemistry and which com membrane, Such as a lipid bilayer, or a synthetic membrane, prise four different oligonucleotide-Cy3 tags. Such as a membrane formed of a polymeric material. The 0094 FIG. 31 depicts (A) denaturing gel images of nanopore may be disposed adjacent or in proximity to a samples from DNA polymerase extension reactions using sensing circuit, such as, for example, a complementary metal four different tagged nucleotides which comprise different oxide semiconductor(CMOS) or field effect transistor (1-ET) oligonucleotide-Cy3 tags, and which reactions carried out circuit. A nanopore may have a characteristic width or diam using Bst2.0 DNA polymerase; and (B) MALDI-TOF MS eter on the order of 0.1 nanometers (nm) to about 1000 nm. analysis of the four different oligonucleotide-Cy3 tagged Some nanopores are proteins. C-hemolysin is an example of nucleotides used in the reactions. a protein nanopore. 0095 FIG.32 depicts exemplary L-nucleotides that can be 0104. The term “nucleic acid, as used herein, generally used in the oligonucleotide tags of the present disclosure. refers to a molecule comprising one or more nucleotide Sub 0096 FIG. 33 depicts exemplary C-D-nucleosides, B-D- units. A nucleotide may include one or more subunits selected nucleosides and 2.5-linked nucleotides that can be used in the from adenine (A), cytosine (C), guanine (G), thymine (T) and oligonucleotide tags of the present disclosure. uracil (U). In some examples, a nucleic acid is deoxyribo 0097 FIG. 34 depicts exemplary unnatural internucle nucleic acid (DNA) or ribonucleic acid (RNA), or derivatives otide linkages and non-natural Sugars that can be used in the thereof. A nucleic acid may be single-stranded or double oligonucleotide tags of the present disclosure. Stranded. 0098 FIG. 35 depicts an SDS-PAGE gel image showing 0105. The term “tag” as used herein, generally refers to an results demonstrating that 3'-chemical modification of oligo atom or molecule that enables the detection or identification nucleotide tags can protect the tag from exonuclease degra of a molecular complex that is coupled to the tag. A tag can dation by Phi29 polymerase. provide a detectable signature. Such as an electrostatic, elec 0099 FIG.36 depicts current level traces corresponding to trochemical and/or optical signature (light). tag capture events measured under slightly different condi 0106. The term “nucleotide,” as used herein refers to a tions using a nanopore array chip, a primer (SEQID NO: 118) nucleoside-5'-polyphosphate compound, or structural analog and four different oligonucleotide tagged nucleotides (dT of a nucleoside-5'-polyphosphate, which is capable of acting Tagl is dT6P-Cy3-dT-dSps-dTo-C3; dC-Tag2 is dC6P as a Substrate or inhibitor of a nucleic acid polymerase to Cy3-dT-dSp-dT-C3; dG-Tag3 is dG6P-Cy3-dTo-C6; extend a growing nucleic acid chain. Exemplary nucleotides dA-Tag4 is dA6P-Cy3-dT-FldT-dT-FldT-dT-C3) to include, but are not limited to, nucleoside-5'-triphosphates sequence a portion of a DNA template (SEQ ID NO: 120). (e.g., dATP, dCTP, dGTP, dTTP, and dUTP): nucleosides Conditions used for both (A) and (B) were: 150 mM KC1, 20 (e.g., dA, dC, dG, dT, and dU) with 5'-polyphosphate chains mM HEPES, pH 7.5 buffer; 3.0 mM SrC1 on trans side of of 4 or more phosphates in length (e.g., 5'-tetraphosphos pore; 160 mV potential was applied and maintained. The phate, 5'-pentaphosphosphate, 5'-hexaphosphosphate, following cis side of the pore conditions differed: (A) 0.1 mM 5'-heptaphosphosphate, 5'-octaphosphosphate); and struc MnCloncis side; (B) 3.0 mM MgCl2-0.7 mM SrC1 on cis tural analogs of nucleoside-5'-triphosphates that can have a side. modified base moiety (e.g., a Substituted purine or pyrimidine 0100 FIG. 37 depicts a current level trace corresponding base), a modified Sugar (e.g., an O-alkylated Sugar), and/or a to tag capture events measured under slightly different con modified polyphosphate moiety (e.g., a polyphosphate com ditions using a nanopore array chip and oligonucleotide prising a thio-phosphate, a methylene, and/or other bridges tagged nucleotides for single molecule, real time, electronic between phosphates). sequencing by synthesis of a 12-base homopolymeric region 0107 The term "tagged nucleotide, as used herein refers of a double hairpin template shown above trace. Conditions to any nucleoside-5'-polyphosphate with a nanopore-detect used were 150 mMKC1, 3.0 mM MgCl2 on cis side of pore, able tag attached to the polyphosphate moiety, base moiety, or 3.0 mMSrC1 on trans side of pore, and 100 mV potential was Sugar moiety. A nanopore-detectable tag includes any applied and maintained. Tagged nucleotides were as molecular group or moiety (e.g., a linker, oligomer, polymer) described in FIG. 36. that can enter into, become positioned in, be captured by, 0101 FIG.38 depicts attachment of primer (SEQID NO: translocate through, and/or traverse a nanopore and thereby 121) to the nanopore and adding template (SEQID NO: 122), result in a detectable change in current through the pore. tagged nucleotides, and DNA polymerase for DNA sequenc Exemplary nanopore-detectable tags include, but are not lim ing. As illustrated in the figure, the tagged 'A' nucleotide ited to, natural or synthetic polymers, such as polyethylene US 2015/036871.0 A1 Dec. 24, 2015 glycol, oligonucleotides, polypeptides, carbohydrates, pep ecules (also "tags' herein) may be detected Subsequent to tide nucleic acid polymers, locked nucleic acid polymers, any release as the tag flows through oradjacent to the nanopore. In of which may be optionally modified with or linked to chemi Some cases, an enzyme attached to or in proximity to the cal groups, such as dye moieties, or fluorophores, that can nanopore may aid in detecting tags or other by-products result in detectable pore current changes. 0108. The term "oligonucleotide, as used herein refers to released upon the incorporation of one or more nucleotides. an oligomer of nucleotide monomer units wherein the oligo See, for example, U.S. Pat. No. 8,889,348; U.S. Patent Appli mer optionally includes non-nucleotide monomer units, and/ cation Publication No. US 2013/0264207A1; and PCT Inter or other chemical groups attached at internal and/or external national Application Publication Nos. PCT/US 13/35630 and. positions of the oligomer. The oligomer can be natural or PCT/US 13/35635, each of which is hereby incorporated synthetic and can include naturally-occurring oligonucle herein by reference in its entirety. otides, or oligomers that include nucleosides with non-natu rally-occurring (or modified) bases, Sugar moieties, phos 0112 Methods described herein may be single-molecule phodiester-analog linkages, and/or alternative monomer unit methods. That is, the signal that is detected is generated by a chiralities and isomeric structures (e.g., 5'-to-2 linkage, single molecule (i.e., single nucleotide incorporation) and is L-nucleosides, C-anomer nucleosides). Exemplary oligo not generated from a plurality of clonal molecules. The nucleotides useful as nanopore-detectable tags in the compo method may not require DNA amplification. sition and methods of the present disclosure include the oli 0113 Nucleotide incorporation events may occur from a gonucleotide tag structures shown in Table 4. mixture comprising a plurality of nucleotides (e.g., deoxyri 0109 The term “nucleotide analog, as used herein refers bonucleotide triphosphate (dNTP where N is adenosine (A), to a chemical compound that is structurally similar to a cytidine (C), thymidine (T), guanosine (G), or uridine (U) and nucleoside-5'-triphosphate and capable of serving as a Sub derivatives thereof). Nucleotide incorporation events do not strate or inhibitor of a nucleic acid polymerase to extend a necessarily occur from a solution comprising a single type of growing nucleic acid chain. A nucleotide analog may have a modified base moiety, for example a substituted purine or nucleotide (e.g., dATP). Nucleotide incorporation events do pyrimidine base, a modified Sugar Such as an O-alkylated not necessarily occur from alternating Solutions of a plurality Sugar, and/or a modified polyphosphate moiety, for example, of nucleotides (e.g., dATP, followed by dCTP, followed by a polyphosphate comprising a thiophosphate, a methylene, dGTP, followed by dTTP, followed by dATP). Additionally, and/or other bridges between phosphates. It can have more as described throughout the present disclosure, the nucleotide than three phosphates in the polyphosphate chain, and it can incorporation events also can occur from a mixture of tagged be detectably tagged on any of the base, Sugar or polyphos nucleotides, wherein the tagged nucleotide can comprise phate moieties. 5'-polyphosphate chains of 4 or more phosphates in length 0110. Described herein are methods, devices and systems (e.g., 5'-tetraphosphosphate, 5'-pentaphosphosphate, useful for sequencing nucleic acids using a nanopore. The 5'-hexaphosphosphate, 5'-heptaphosphosphate, 5'-Octaphos methods may accurately detect individual nucleotide incor phosphate), and comprise further chemical moieties in the poration events, such as upon the incorporation of a nucle tag. otide by a nucleic acid polymerase into a growing strand that 0114 Chemical Conjugation Methods Such as “Click is complementary to a template nucleic acid strand. An Chemistry” enzyme (e.g., DNA polymerase) may incorporate nucleotides to a growing polynucleotide chain, wherein the added nucle 0115 Described herein are methods for attaching tags to otide is complimentary to the corresponding template nucleic nucleotides using chemical conjugation. In some embodi ments, the tag is attached to the nucleotide using a "click acid strand which is hybridized to the growing strand. These chemistry” reaction or "click reaction.” Click reactions are nucleotide incorporation events include capturing the nucle fast, irreversible reactions between pairs of specific chemical otide, reading the associated tag in the pore, and releasing the groups, such as azides and alkynes (or cyclooctynes), or tag from the nucleotide and the released tag then passes tetrazines and trans-cyclooctenes. The specific pairs of through a nanopore. In this way, the incorporated base may be chemical groups used in click reactions provide covalent identified (i.e., A, C, G, T or U) because a unique tag is first linkages that comprise specific chemical groups. Such as tria read and then released from each type of nucleotide (i.e., A.C. Zole, or 1,2-diazine (or its tautomer, dihydropyridazine) as G, Tor U). part of the covalent linkage. FIG. 21 depicts general reaction schemes illustrating three exemplary click reactions useful in 0111 Nucleotide incorporation events may be detected preparing the tagged nucleotide conjugates of the present with the aid of a nanopore in real-time (i.e., as they occur) or disclosure. These three exemplary reactions are described following a sequencing reaction by analyzing the nanopore further below. data. In some instances, an enzyme (e.g., DNA polymerase) attached to or in proximity to the nanopore may facilitate the 0116. An exemplary click reaction between azide and alkyne is the azide-alkyne Huisgencycloaddition. The azide flow of a nucleic acid molecule through or adjacent to the alkyne Huisgen cycloaddition is a 1,3-dipolar cycloaddition nanopore, and position the tag of a complimentary nucleotide between a compound with an azide group and a compound in the nanopore for detection. Thus, a complimentary tagged with a terminal or internal alkyne group to yield a product nucleotide binding to an enzyme (prior to release of the tag) compound with a 1,2,3-triazole covalent linkage. The exem can result in the positioning of the tag in the pore of the plary azide-alkyne Huisgen click reaction follows the general nanopore, which can then be detected by a change in the scheme of FIG. 21, scheme (A), and is further detailed in the current level through the nanopore. Or one or more tag mol scheme below. US 2015/036871.0 A1 Dec. 24, 2015

modified compound and a cyclooctyne-modified compound (e.g., modified with dibenzyl-cyclooctyne "DBCO) to yield a product conjugate of the two compounds comprising a -- covalent triazole linkage. See e.g., Jewett and BertoZZi (2010). A general scheme for the use of the Cu-free azide cyclooctyne click reaction to conjugate two compounds A 1 and B with a triazole is depicted in FIG. 21, scheme (B). In Some embodiments, this Cu-free click-reaction can be used to He attach tags to nucleotides in accordance with the methods of GE) G. the present disclosure and provide tagged nucleotides com NEN-N prising a triazole in the covalent linkage between the tag and 2 the nucleotide. N I0121 Another click chemistry reaction useful for provid N%2 NN ing the tagged nucleotides of the present disclosure is the inverse-electron demand Diels-Alder (IEDDA) reaction. See y 5 e.g., Reiner et al. (2014) and U.S. Patent Application Publi O cation Nos. 2013/0266512 A1 and 2013/00852.71 A1. The IEDDA click-reaction uses the fast, irreversible reaction between a tetrazine-modified compound and trans-cy 3 clooctene modified compound to provide a conjugate product that comprises a covalent 1,2-diazine linkage, or more spe 0117. In the exemplary azide-alkyne cycloaddition reac cifically, the tautomeric equivalent of a 1,2-diazine, a dihy tion scheme above (e.g., carried out at 98°C. in 18 hours), the dropyridazine. A general scheme for the use of the IEDDA azide group of compound 2 reacts with alkyne group of com click reaction between tetrazine and trans-cyclooctene for pound 1 to afford a product composition 3 which is a mixture conjugating two compounds A and B with a 1,2-diazine (di of 1,4-triazole and 1,5-triazole adducts. hydropyridazine tautomer) group is depicted in FIG. 21. 0118 Copper-catalyzed azide-alkyne cycloaddition reac scheme (C). Accordingly, in some embodiments, this IEDDA tion also provides click reaction products coupled a covalent click-reaction also can be used to attach tags to nucleotides in triazole linkage but can proceed with an enormous rate accel accordance with the methods of the present disclosure and eration of between about 107-fold and 10-fold compared to provide tagged nucleotides comprising a 1,2-diazine (dihy un-catalyzed 1,3-dipolar cycloaddition. Further, this Cu-cata dropyridazine tautomer) in the covalent linkage between the lyzed click reaction can take place over a broad temperature tag and the nucleotide. range, can be insensitive to aqueous conditions and a pH 0.122 Connection of the nucleotide polyphosphate to the range from about 4 to about 12, can tolerate a broad range of tag can also be achieved by the formation of a disulfide functional groups, and can yield single isomers under appro (forming a readily cleavable connection), formation of an priate conditions. See e.g., Himo et al. (2005), which is amide, formation of an ester, by alkylation (e.g., using a hereby incorporated herein by reference in its entirety. The Substituted iodoacetamide reagent) or forming adducts using Cu-catalyzed chemical reaction follows the general scheme aldehydes and amines or hydrazines. Numerous conjugation for conjugating two compounds (A and B) shown in FIG. 21. chemistries can be found in Hermanson (May 2, 2008), which scheme (A), and is further detailed in the scheme below. is incorporated herein by reference in its entirety. I0123 Tagged Nucleotides 0.124. In some cases, a tagged nucleotide comprises a tag 0.25-2 mol-% CuSO4·5H2O (or label) that is separated from the nucleoside during a poly 5-10 mol-96 sodium ascorbate merase-catalyzed nucleotide incorporation event. The tag R N3 + E-R -- H2O/tBuOH (1:1), r.t., 6-12 h. may be attached to the 5'-phosphate or 5'-polyphosphate R N chain of the nucleotide. In some instances, the tag does not nN.1 SN comprise a fluorophore. The tag can be detectable by a nan opore and identified (e.g., distinguished from other tags) by its charge, shape, size, or any combination thereof. Examples R of tags include various polymers. Each type of nucleotide R: alkyl, CHOBn (i.e., A, C, G, TU) generally comprises a uniquely recogniz R": Ph, CO2H able tag. 0.125 Tags of the present disclosure may be molecules that 0119 Because of its tolerance for aqueous conditions the may be detectable using electrostatic, electrochemical, and/or Cu-catalyzed azide-alkyne click reaction has been used for optical approaches. In some examples, a tag may provide an covalent conjugation of biological molecules. See e.g., Wang electronic signature that is unique to a given nucleic acid et al. (2003) and Presolski et al. (2011). This Cu-catalyzed molecule (e.g., A, C, G, T, U). azide-alkyne click-reaction also can be used to attach tags to 0.126 Tags may be located on any suitable position on the nucleotides in accordance with the methods of the present nucleotide. FIG. 1 shows a potential tagged nucleotide, where disclosure and provide tagged nucleotides comprising a tria R can be OH and R can be H (i.e., for deoxy-ribonucle Zole in the covalent linkage between the tag and the nucle otides) or OH (i.e., for ribonucleotides), although other otide. choices for R and R are acceptable. In FIG. 1, X is any 0120 Copper-free click-reactions also have been devel suitable linker. In some cases, the linker is cleavable. oped that utilize cycloaddition reaction between an azide Examples of linkers include without limitation, O, NH, S or US 2015/036871.0 A1 Dec. 24, 2015

CH. The linker may also contain, for example, O, N, S, or P if present, is generally —H or —OH. Also, Z is generally O, atoms. The linker can also be a detectable moiety, directly or S or BH, and n is any integer including 1, 2, 3, 4, 5, 6, or 7. indirectly, Such as amino acids, peptides, proteins, carbohy In some cases, the A is O, S, CH, CHF, CFF, or NH. drates, PEGs of different length and molecular weights, (0132. With continued reference to FIG. 2, a set of 4 dis organic or inorganic dyes, fluorescent and fluorogenic dyes, tinct tagged nucleotides can be used wherein each type of drugs, oligonucleotides, mass tags, chemiluminiscent tags base on the tagged nucleotide is generally different from the and may contain positive or negative charges, as discussed in type of base on each of the other three tagged nucleotides, and U.S. patent application Ser. No. 13/994,431, which has here the type oftag on each tagged nucleotide is generally different inabove been incorporated herein by reference in its entirety. from the type of tag on each of the other three tagged nucle 0127. In some embodiments, the suitable linker comprises otide. Suitable bases include, but are not limited to adenine, a fluorescent cyanine dye (or “CyDye), such as Cy3 and guanine, cytosine, uracil or thymine, or a derivative of each Cy3.5. In such embodiments, the CyDye moiety in the linker thereof. In some cases, the base is one of 7-deazaguanine, may be used to provide an additional moiety which can be 7-deazaadenine or 5-methylcytosine, or non-naturally occur used to detect the tagged nucleotide, or the CyDye moiety ring bases Such as nitropyrrole, nitroindole, nebularine, Zebu may not be detected and simply provide further structure that enhances the ability to detect the tag moiety attached to the larine, benzene, or derivatives thereof (see e.g., FIG. 29). linker. Indeed, the presence of a CyDye moiety in the linker I0133. In cases where R is —O—CHN, the nucleotide portion of an oligonucleotide tag can enhance the capture and can be used in methods that further comprise treating the detection of the tagged nucleotide by a nanopore. Example 15 incorporated tagged nucleotide so as to remove the —CHN demonstrates how an oligonucleotide tag with a Cy3 moiety and result in an OH group attached to the 3' position thereby in the linker portion enhances the nanopore capture and detec permitting incorporation of a further tagged nucleotide. tion of the tagged nucleotide when bound to a DNA poly I0134) In cases where R is —O-2-nitrobenzyl, the tagged merase linked to a nanopore. Accordingly, in Some embodi nucleotide can be used in methods that further comprise treat ments, the disclosure provides a tagged nucleotide wherein ing the incorporated tagged nucleotide so as to remove the the tag comprises a CyDye moiety, and in some embodiments 2-nitrobenzyl and result in an OH group attached to the 3' the CyDye moiety is Cy3. In some embodiments of the tagged position thereby permitting incorporation of a further tagged nucleotide, the tag comprises an oligonucleotide and a linker, nucleotide. and the linker further comprises a CyDye moiety. 0128. Examples of suitable chemical groups for the posi 0.135 A tag may be any chemical group that is capable of tion Zinclude O, S, or BH. The base can be any base suitable being detected in or with the aid of a nanopore. In some cases, for incorporation into a nucleic acid including adenine, gua a tag comprises one or more of ethylene glycol, an amino nine, cytosine, thymine, uracil, or a derivative thereof. Uni acid, a carbohydrate, a peptide, a dye, a chemiluminescent Versal bases (i.e., bases that are capable of pairing with more compound, a mononucleotide, a dinucleotide, a trinucleotide, than one of A, C, T. G., and U) are also acceptable in some a tetranucleotide, a pentanucleotide, a hexanucleotide, an cases (e.g. 2'deoxyinosine derivatives, nitroindole deriva oligonucleotide (of greater length than 6-mer), a polynucle tives). otide an aliphatic acid, an aromatic acid, an alcohol, a thiol 0129. The number of phosphates (n) is any suitable integer group, a cyano group, a nitro group, an alkyl group, an alkenyl value (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) (e.g., a number group, an alkynyl group, an azido group, or a combination of phosphates such that the nucleotide may be incorporated thereof. into a nucleic acid molecule by a polymerase). In some 0.136. It is also contemplated that the tag further comprises instances, all types of tagged nucleotides have the same num appropriate number of lysines or arginines to balance the ber of phosphates, but this is not required. In some applica number of phosphates in the compound. tions, there is a different tag for each type of nucleotide and 0.137 In some cases, the tag is a polymer. Polyethylene the number of phosphates is not necessarily used to distin glycol (PEG) is an example of a polymer and has the structure guish the various tags. However, in some cases more than one as follows: type of nucleotide (e.g., A. C. T. G or U) may have the same tag and the ability to distinguish one nucleotide from another is determined at least in part by the number of phosphates (with various types of nucleotides having a different value for n). In some embodiments, the value for n is 1, 2, 3, 4, 5, 6, 7, -N1-a-ha-'-no-nu 8, 9, 10, or greater. 0130 Suitable tags are described below. In some 0.138 Any number of ethylene glycol units (W) may be instances, the tag has a charge which is opposite in sign used. In some instances, W is an integer between 0 and 100. relative to the charge on the rest of the nucleotide. When the In some cases, the number of ethylene glycol units is different tag is attached, the charge on the overall compound may be for each type of nucleotide. In an embodiment, the four types neutral. Release of the tag may result in two molecules, a of nucleotides comprise tags having 16, 20, 24 or 36 ethylene charged tag and a charged nucleotide. The charged tag enters glycol units. In some cases, the tag further comprises an a nanopore and is thereby detected in some cases. additional identifiable moiety. Such as a coumarin based dye. 0131 More examples of suitable tagged nucleotides are In some cases, the polymer is charged. In some instances, the shown in FIG. 2. The tag may be attached to the Sugar moiety, polymer is not charged and the tag is detected in a high the base moiety, the polyphosphate moiety or any combina concentration of salt (e.g., 3-4M). In some cases, the polymer tion thereof. With reference to FIG. 2, Y is a tag and X is a is an oligonucleotide comprising ribonucleotides and/or linker (in Some cases cleavable). Furthermore, R, if present, deoxyribonucleotides. In addition, the polymer can be a is generally —OH, —OCHN or—O-2-nitrobenzyl, and R. polypeptide comprising amino-acid subunits. US 2015/036871.0 A1 Dec. 24, 2015

0.139. In some cases, a tag comprises multiple PEG chains. prising an L-nucleotide covalently coupled, directly or with a In an example, a tag has the structure as follows: further linker moiety, to the terminal phosphate through a ~~-oh-i-o-O ~N~~N~~ O o~...~-oh-i-o-O ~~N~oh-i-o-O wherein R is NH, OH, COOH, CHO, SH, or N, and W is an triazole, a 1,2-diazine, a disulfide, a amide, a hydraZone, a integer from 0 to 100. See, for example, U.S. patent applica thio-acetamide, or a maleimide-thio adduct. tion Ser. No. 13/994,431, which has hereinabove been incor 0.143 Naturally occurring nucleosides have a f-D con porated herein by reference in its entirety. figuration with respect to the 1'-position of ribose and the nucleic acid base. In another embodiment, the oligonucle 0140. As noted above, in some embodiments the tag of the otide tag can comprise an O.-D-nucleoside (FIG. 33A). The tagged nucleotide can itself comprise an oligonucleotide. In oligonucleotide-tag may comprise all or a mixture of C-D- Some embodiments, the oligonucleotide tag can comprise nucleotides and B-D-nucleotides in a ratio such that they do naturally occurring bases (e.g., A, C, G, T), non-naturally not hybridize with the nucleic acid template being sequenced occurring (or modified) nucleoside bases, or mixtures (FIG. 33B). Accordingly, the present disclosure provides a thereof. Some exemplary non-naturally-occurring (or modi tagged nucleotide, comprising: (a) a nucleotide polyphos fied) bases are illustrated in FIG. 29 and include, but are not phate moiety having a terminal phosphate; and (b) an oligo limited to nitropyrrole, nitroindole, nebularine, Zebularine, nucleotide tag comprising an O-D-nucleotide covalently and benzene, and derivatives thereof. In some embodiments, coupled, directly or with a further linker moiety, to the termi the oligonucleotide tag can comprise a naturally-occurring nal phosphate of the nucleotide through a triazole, a 12 phosphodiester inter-nucleotide linkage, or can have non diazine, a disulfide, an amide, a hydraZone, a thio-acetamide, naturally occurring internucleotide linkages such as phospho or a maleimide-thio adduct. triester, phosphorothioate, methylphosphonate or borono 0144. In an another embodiment, the present disclosure phosphate. In some instances, the inter-nucleotide linkage is provides an oligonucleotide tag comprising an unnatural Syn a morpholino moiety. thetic nucleoside as described by Kim et al. (2005), Sefah et al. (2014) and Romesberg et al. (J. Am. Chem. Soc. 2014 and 0141. As described further below, oligonucleotide tags Nucleic Acids Research 2014). The unnatural synthetic can be detected by a nanopore due to their presence in the pore nucleosides described in these publications do not form causing a detectable electric current change in the sensor H-bonds with the naturally occurring nucleosides (adenine, associated with the nanopore. It is not necessary, however, for guanine, cytosine, thymine, uracil, deazapurines or deriva the oligonucleotide to hybridize. Indeed, hybridization of the tives thereof) and thus, do not hybridize with natural nucleic oligonucleotide tag to the template sequence could create acid templates. An oligonucleotide tag comprising Such problems in providing the appropriate current blockade sig unnatural synthetic nucleosides may be a deoxyribonucle nal necessary for nanopore sequencing. Accordingly, in some otide or a ribonucleotide and may comprise all unnatural embodiments, the oligonucleotide tags can comprise nucle nucleosides or a mixture with some naturally occurring otides with one or more unnatural bases (e.g., Such as noted nucleosides. above) or an unnatural Sugar moiety (described further 0145. In another aspect, the present disclosure provides a below). Such non-naturally occurring bases and Sugar moi tagged nucleotide wherein the tag comprises an oligonucle eties do not form hydrogen bonds with natural nucleotides otide with at least one 2',5'-linkage (rather than the naturally and thus, do not hybridize to the nucleic acid template being occurring 3',5'-linkage) between a pair of nucleotides in the Sequences. tag. FIG.33B shows a comparative illustration of 2',5'-linked 0142. Additionally, in some embodiments the oligonucle and a 3',5'-linked oligonucleotide. Such 2',5'-linked oligo otide tag can comprise an L-nucleotide (rather than a nucleotides bind selectively to complementary RNA but not D-nucleotide). Exemplary L-nucleosides that can be used in to DNA templates (Bhan et al. (1997)). Thus, an oligonucle the oligonucleotide tags of the present disclosure are shown in otide tag comprising of 2.5'-linked oligonucleotide would FIG. 32A. L-nucleic acids do not, in general, recognize not bind to the nucleic acid template being sequenced. It is single-stranded, natural DNA and RNA (see e.g., Asseline et contemplated that an oligonucleotide tag can comprise only al. (1991) and Garbesi et al. (1993)). It is contemplated that 2',5'-linked nucleotides or can comprise a mixture of 2',5'- oligonucleotide tags can comprise all L-nucleotides or mix linked and 3',5'-linked nucleotides. Accordingly, the present tures of L- and D-nucleotides in ratios such that they do not disclosure provides an oligonucleotide tag comprising: (a) a hybridize with the nucleic acid template being sequenced. nucleotide polyphosphate moiety having a terminal phos Accordingly, the present disclosure provides a tagged nucle phate; and (b) a tag comprising a chain of 1-1002',5'-linked otide comprising: (a) a nucleotidepolyphosphate moiety hav nucleotide units that is covalently coupled, directly or with a ing a terminal phosphate; and (b) an oligonucleotide tag com further linker moiety, to the terminal phosphate of the nucle US 2015/036871.0 A1 Dec. 24, 2015

otide by a triazole, a 1,2-diazine, a disulfide, an amide, a bond to a hydrogen atom contained therein is replaced by a hydrazone, a thio-acetamide, or a maleimide-thioadduct. bond to non-hydrogen or non-carbon atom, provided that 0146 In another aspect, the present disclosure provides a normal valencies are maintained and that the Substitution(s) tagged nucleotide wherein the tag comprises an oligonucle result(s) in a stable compound. Substituted groups also otide with at least one modified Sugar and/or phosphate moi include groups in which one or more bonds to a carbon(s) or ety. Exemplary modified Sugar and/or phosphate moieties hydrogen(s) atom are replaced by one or more bonds, includ that can be used in the oligonucleotide tags of the present ing double or triple bonds, to a heteroatom. disclosure are depicted in FIG. 34. It is contemplated that an 0151 FIG.5 shows a nucleoside with a tag 505 attached to oligonucleotide tag can comprise only modified Sugar and/or the terminal phosphate. As shown here, the base can be any phosphate moieties or can comprise a mixture of modified base (e.g., A.T. G. C. U, or derivatives thereof), R can be any Sugar and/or phosphate moieties and naturally occurring chemical group (e.g., H, OH), in can be any integer (e.g., 1, 2, (e.g., ribose) nucleotides in the oligonucleotide tag. Accord 3, 4, 5, 6, 7, 8, 9, 10, or more), X can be any chemical group ingly, the present disclosure provides an oligonucleotide tag (e.g., O, NH, S) and Y can be any functional group which comprising: (a) a nucleotide polyphosphate moiety having a makes a covalent bond with X and is attached to the tag. terminal phosphate; and (b) a tag comprising a chain of 1-100 Examples of tags include, but are not limited to oligonucle nucleoside units comprising a modified Sugar and/or phos otides of any size (e.g., with 2-100 bases, 5-50 bases, or 2-40 phate moiety that is covalently coupled, directly or with a bases). In some cases, the oligonucleotide tag has nitropyr further linker moiety, to the terminal phosphate of the nucle role, nitroindole, nebularine, Zebularine, benzene, or deriva otide by a triazole, a 1,2-diazine, a disulfide, an amide, a tives thereof as a homopolymer or heteropolymer. In some hydrazone, a thio-acetamide, or a maleimide-thioadduct. cases, the tag has a phospotriester, phosphodiester, phospho 0147 In the tagged nucleotide embodiments provided by ramidate, phosphorothioate, methylphosphonate or borono the present disclosure it is contemplated that a natural or phosphate internucleotide linkages. In some instances, the synthetic oligonucleotide tag can be covalently coupled internucleotide linkage is a morpholino moiety. through either its 5' or 3' end, directly or through a linker 0152. In some embodiments, the tag is attached to the moiety, to a terminal phosphate of the nucleotide. In some nucleotide using azide-alkyne Huisgen cycloaddition, also embodiments, the oligonucleotide tag is covalently coupled known as "click chemistry'. For example, FIG. 6 shows a through its 5' end, directly or through a linker moiety, to a nucleotide having 6 phosphates with a 6 carbon spacer and a terminal phosphate of the nucleotide. In Such embodiments, it reactive azide group attached to the terminal phosphate (left) is contemplated that the 3'-hydroxyl at the other end of the being reacted with a tag having a 6 carbon spacer and a oligonucleotide tag is modified so as to protect the oligo reactive alkyne group (right) using click chemistry. As nucleotide from potential exonuclease degradation. In some described elsewhere herein, FIG. 21, schemes (A), (B), and embodiments, the 3'-hydroxyl terminus of the oligonucle (C) illustrates three exemplary click chemistry reactions for otide tag is protected from exonuclease activity by chemical conjugating two compounds (A and B). Any one of these modification. Exemplary chemical modifications of the three exemplary click reactions can be adapted for use in 3'-hydroxyl terminus can include phosphorylation, or cova conjugating a tag to a nucleotide and thereby make the tagged lent coupling with C-alkyl to C2-alkyl spacers having ter nucleotides of the present disclosure. Specific illustrations of minal hydroxyl groups. the use of such click reactions are provided in the Examples. 0148. In some examples, a tag is chosen from the mol 0153. In an aspect, a tagged nucleotide is formed by pro ecules (dCp)m, (dGp)m, (dAp)m, and (dTp)m or a combina viding a nucleotide comprising a poly-phosphate tail com tion of one or more units of (dCp), dGp), (dAp) and (dTp). prising a terminal phosphate. The terminal phosphate of the FIG.3 and FIG. 4 show these molecules attached to a nucle nucleotide can be covalently connected to an alkane or similar otide. Here, m is, independently, an integer from 0 to 100, linker to an azide. The tag can be covalently bound to the and wherein when m is 0 the terminal phosphate of the dNPP nucleotide terminal phosphate-azide using the "click” reac is bonded directly to the 3'O atom of the nucleoside shown on tion to form a triazole. The triazole can be formed by a the left hand side of the structure. In some cases, the value of reaction between an azide and an alkyne. In some embodi n is different for each type of base. ments, the poly-phosphate tail comprises at least 3 phos phates, at least 4 phosphates, at least 5 phosphates, at least 6 0149. In some instances, a tag is a hydrocarbyl, substituted phosphates, or at least 7 phosphates. In some embodiments, or unsubstituted, such as an alkyl, alkenyl, alkynyl, and hav the poly-phosphate moiety comprises from 4 to 6 phosphates. ing a mass of 3000 Daltons or less. In some embodiments, the poly-phosphate moiety comprises 0150. As used herein, the term “alkyl includes both at least 6 phosphates. The tag can comprise nucleotides, oli branched and straight-chain Saturated aliphatic hydrocarbon gonucleotides, polyethylene glycol (PEG), oligo-saccha groups having the specified number of carbon atoms and may be unsubstituted or substituted. As used herein, “alkenyl rides, carbohydrates, peptide nucleic acids (PNA), vinyl refers to a non-aromatic hydrocarbon radical, straight or polymers, other water-soluble polymers, peptides, or any branched, containing at least 1 carbon to carbon double bond, combination thereof. and up to the maximum possible number of non-aromatic 0154. In some cases, the triazole has the structure: carbon-carbon double bonds may be present, and may be unsubstituted or substituted. The term “alkynyl refers to a hydrocarbon radical straight or branched, containing at least ?: 1 carbon to carbon triple bond, and up to the maximum 1\N possible number of non-aromatic carbon-carbon triple bonds R-- N may be present, and may be unsubstituted or substituted. The term “substituted” refers to a functional group as described above such as an alkyl, or a hydrocarbyl, in which at least one US 2015/036871.0 A1 Dec. 24, 2015 wherein R comprises the tag, and R comprises the nucle and Subsequently or concurrently contacting the product with otide; or wherein R comprises the nucleotide, and R com NH-OH so as to form a compound having the structure: prises the tag.

O155 In some cases, the triazole has the structure: O O O HN N-B-O-P-O-P-O-I II CBSEBASE s H O R2 O- O- O R \ Y R R2 M. R3 N 0.161 The product of operation b) may then be reacted with a tag having a -COR group attached thereto under wherein R and R combine to form a cyclic moiety; and conditions permitting the tag to bond indirectly to a terminal wherein R and R combined comprise the tag, and R com phosphate thereby forming the tagged nucleotide having the prises the nucleotide; or wherein R and R combined com Structure: prise the nucleotide, and R2 comprises the tag. 0156 Also provided herein is a method for making a TAG tagged nucleotide, comprising providing a nucleotide com prising a poly-phosphate tail, where the poly-phosphate tail comprises a terminal phosphate. The terminal phosphate can comprise either an azide group or an alkyne group. The method includes providing a tag molecule comprising either R R2 an azide group or an alkyne group, where the nucleotide and the tag molecule do not each comprise an azide group, and wherein R is OH, wherein R is Hor OH, wherein the base is where the nucleotide and the tag molecule do not each com adenine, guanine, cytosine, thymine, uracil, a 7-deaZapurine prise an alkyne group. The method can also include reacting or a 5-methylpyrimidine. the azide group with the alkyne group to link the nucleotide to 0162 Connection of the nucleotide polyphosphate to the the tag molecule. In some cases, the reaction is facilitated by tag can also be achieved by the formation of a disulfide a catalyst comprising salts of copper, ruthenium, silver, or any (forming a readily cleavable connection), formation of an combination thereof. amide, formation of an ester, by alkylation (e.g., using a 0157. In some cases, the reaction does not require a cata Substituted iodoacetamide reagent) or forming adducts using lyst. A catalyst may not be needed when the alkyne is a aldehydes and amines or hydrazines. Numerous conjugation cyclooctyne, (e.g., a dibenzylcyclooctyne). chemistries can be found in Hermanson (2008), which is 0158. In some cases, tags can be attached to the terminal incorporated herein by reference in its entirety. phosphate by (a) contacting a nucleoside triphosphate with 0163 Specific examples of reactive groups on the terminal dicyclohexylcarbodiimide/dimethylformamide under condi phosphates or the Oligonucleotide Tags and groups with tions permitting production of a cyclic trimetaphosphate; (b) which groups can reactare provided in Table 1. These reactive contacting the product resulting from operation (a) with a groups with which they can react can be present either on the nucleophile so as to form an —OH or —NR, functionalized linker or on the tag. compound; and (c) reacting the product of operation (b) with a tag having a -COR group attached thereto under condi TABLE 1 tions permitting the tag to bond indirectly to a terminal phos Possible Reactive Substituents and Functional phate thereby forming the tagged nucleotide. Groups Reactive Therewith 0159. In some cases, the nucleophile is HN R-OH, HN R NH, RS-R-OH, R'S R NH, or Reactive Groups Functional Groups Succinimidyl esters Primary amino, secondary amino Anhydrides, acid halides Amino and Hydroxyl groups Carboxyl Amino, Hydroxy, Thiols NHTFA. Aldehyde, Isothiocyanate & Isocyanates Amino groups HN Vinyl sulphone & Dichlorotriazine Amino groups Haloacetamides Thiols, Imidazoles Maleimides Thiols, Hydroxy, Amino Thiols Thiols, Maleimide, Haloacetamide 0160 In some instances, the method comprises, in opera Phosphoramidites, Activated Phosphates Hydroxy, Amino, Thiol groups tion b), contacting the product resulting from operation a) AZide Alkyne with a compound having the structure: Tetrazine Dienes 0164. Another aspect of the present disclosure provides a NHTFA method for sequencing a nucleic acid sample with the aid of HN a nanopore in a membrane adjacent to a sensing electrode. The method comprises providing tagged nucleotides having a tag linked to a terminal phosphate by a triazole into a reaction chamber comprising the nanopore, where an individual US 2015/036871.0 A1 Dec. 24, 2015

tagged nucleotide of the tagged nucleotides contains a tag least 600, at least 700, at least 800, at least 800, at least 1000, coupled to a nucleotide that is detectable with the aid of the at least 1500, at least 2000, at least 2500, at least 3000, at least nanopore. A polymerization reaction is carried out with the 3500, at least 4000, at least 4500, at least 5000, at least 6000, aid of a polymerase, thereby incorporating an individual at least 7000, at least 8000, at least 9000, at least 10000, at tagged nucleotide of the tagged nucleotides into a growing least 20000, at least 40000, at least 60000, at least 80000, at Strand complementary to a single stranded nucleic acid mol least 100000, and the like bases are sequenced. In some ecule from the nucleic acid sample. Using the nanopore, a tag instances the sequenced bases are contiguous. In some cases, associated with the individual tagged nucleotide is detected the nucleic acid sample may be partitioned prior to sequenc upon forming a ternary complex at the polymerase active 1ng. which allows the tag to enter and become positioned in the 0172 A tag may be released in any manner. A tag can be adjacent pore, and/or Subsequent to the polymerase incorpo released during or Subsequent to the incorporation of a nucle rating the individual tagged nucleotide into the growing otide into a polynucleotide chain. In some cases, the tag is strand, whereby the tag is detected with the aid of the nanop attached to the polyphosphate moiety of a nucleotide (e.g., ore when the tag has been cleaved from the nucleotide. FIG. FIG. 15) and incorporation of the nucleotide into a nucleic 7 shows cell current readings for four different tags that are acid molecule results in release of a polyphosphate having the attached using click chemistry. The four different tags are tag attached thereto (e.g., separating it from the rest of the individually resolvable and can correspond to A residues, T nucleotide and growing nucleic acid strand). The incorpora residues, G residues and C residues. tion may be catalyzed by at least one polymerase, which can 0.165 Methods for Molecular Sensing and/or Identifica be attached to the nanopore. In some instances, at least one tion phosphatase enzyme is also attached to the pore. The phos 0166 The present disclosure provides methods for phatase enzyme may cleave the phosphates from the released molecular sensing and/or identification. Such methods may polyphosphate tag. In some cases, the phosphatase enzymes be used to detect various types of biological species. Such as are positioned such that polyphosphate product of the poly nucleic acids, proteins and antibodies. In some embodiments, merase interacts with the phosphatase enzymes prior to the methods for molecular identification are used to sequence tag entering the pore. nucleic acid molecules. 0173. In some cases, the tag is not attached to polyphos 0167. In an example, a method for sequencing nucleic phate (see, e.g., FIG. 2). In these cases, the tag is attached by acids includes retrieving a biological sample having the a linker (X), which can be cleavable. Methods for production nucleic acid to be sequenced, extracting or otherwise isolat of cleavably capped and/or cleavably linked nucleotides are ing the nucleic acid sample from the biological sample, and in disclosed in U.S. Pat. No. 6,664,079, which is entirely incor Some cases preparing the nucleic acid sample for sequencing. porated herein by reference. The linker need not be cleavable. 0168 FIG. 8 schematically illustrates a method for 0.174. The linker may be any suitable linker and can be sequencing a nucleic acid sample. The method comprises cleaved in any suitable manner. The linkers may be photo isolating the nucleic acid molecule from a biological sample cleavable. In an embodiment UV light is used to photochemi (e.g., tissue sample, fluid sample), and preparing the nucleic cally cleave the photochemically cleavable linkers and moi acid sample for sequencing. In some instances, the nucleic eties. In an embodiment, the photocleavable linker is a acid sample is extracted from a cell. Some exemplary tech 2-nitrobenzyl moiety. niques for extracting nucleic acids are using lysozyme, Soni (0175. The -CHN group may be treated with TCEP cation, extraction, high pressures or any combination thereof. (tris(2-carboxyethyl)phosphine) so as to remove it from the The nucleic acid is cell-free nucleic acid in Some cases and 3'-O atom of a nucleotide, thereby creating a 3'OH group. does not require extraction from a cell. 0176). In some instances, a polymerase draws from a pool 0169. In some cases, a nucleic acid sample may be pre of tagged nucleotides comprising a plurality of different pared for sequencing by a process that involves removing bases (e.g., A, C, G, T, and/or U). It is also possible to contact proteins, cell wall debris and other components from the the polymerase with the various types of tagged nucleotides nucleic acid sample. There are many commercial products comprising different bases individually and serially. In this available for accomplishing this, such as, for example, spin case, it may not be necessary that each type of nucleotide have columns. Ethanol precipitation and centrifugation may also a unique tag since only one nucleotide is present during any be used. given reaction. 0170 The nucleic acid sample may be partitioned (or frac 0177 FIG. 15 shows that incorporation of the tagged tured) into a plurality of fragments, which may facilitate nucleotide into a nucleic acid molecule (e.g., using a poly nucleic acid sequencing, such as with the aid of a device that merase to extend a primer base paired to a template) can includes a plurality of nanopores in an array. However, frac release a detectable TAG-polyphosphate in some embodi turing the nucleic acid molecule(s) to be sequenced may not ments. In some cases, the TAG-polyphosphate is detected as be necessary. it passes through the nanopore. 0171 In some instances, long sequences are determined 0178. In some cases, the method distinguishes the nucle (i.e., "shotgun sequencing methods may not be required). otide based on the number of phosphates comprising the Any suitable length of nucleic acid sequence may be deter polyphosphate (e.g., even when the TAGs are identical). Nev mined. For instance, at least about 400, about 500, about 600, ertheless, each type of nucleotide can have a unique tag. about 700, about 800, about 800, about 1000, about 1500, (0179. With reference to FIG. 15, the TAG-polyphosphate about 2000, about 2500, about 3000, about 3500, about 4000, compound may be treated with phosphatase (e.g., alkaline about 4500, about 5000, about 6000, about 7000, about 8000, phosphatase) before passing the tag into and/or through a about 9000, about 10000, about 20000, about 40000, about nanopore and measuring the ionic current. 60000, about 80000, or about 100000, and the like bases may 0180 Tags may flow through a nanopore after they are be sequenced. In some instances, at least 400, at least 500, at released from the nucleotide. In some instances, a Voltage is US 2015/036871.0 A1 Dec. 24, 2015 applied to position the tags in and pull the tags through the perature. The method may be performed in any suitable solu nanopore. At least about 85%, at least 90%, at least 95%, at tion and/or buffer. In some instances, the buffer is 300 mM least 99%, at least 99.9 or at least 99.99% of the released tags KCl buffered to pH 7.0 to 8.0 with 20 mM HEPES. In some may enter into, become positioned in, and/or translocate embodiments, the buffer does not comprise divalent cations. through the nanopore. In some cases, the method is unaffected by the presence of 0181. In some instances, the tags reside in the nanopore for divalent cations. a period of time where they are detected. In some instances, a 0187. In another embodiment, a “SpyCatcher approach Voltage is applied to pull the tags into the nanopore, detect the may be used to attach a polymerase to a nanopore protein. In tags, or any combination thereof. The tags can be released Such an approach, two fragments of the collagen adhesion upon nucleotide incorporation events. domain (CnaB2) of the Streptococcus pyogenes fibronectin 0182. In some embodiments, the nanopore current change binding protein FbaB recognize each other and Subsequently event is monitored and detected while the tag is still attached generate a peptide bond between the e-amino group of a to the tagged nucleotide rather than when the tag Subse lysine in one fragment (i.e., the “SpyCatcher') and the car quently is released from the nucleotide and passes through the boxyl side group of an aspartic acid in the other fragment (i.e., nanopore channel. In Such embodiments, the tag is detected the “SpyTag”). See e.g., Zakeri and Howarth (2010). JACS while the tagged nucleotide is in a ternary complex at the 132:4526-7. Accordingly, in some embodiments, a DNA polymerase active site with its complementary template polymerase can be attached to a nanopore by attaching a nucleotide, i.e., prior to nucleotide incorporation and phos SpyTag to an aspartic acid residue of a pore protein monomer phoryl transfer. In such embodiments, the long “tail” of the (e.g., C.-hemolysin), attaching a SpyCatcher on the N-termi tag becomes positioned in (or “captured by) the pore of the nus of a DNA polymerase (e.g., Phi29 or Bst2.0 DNA poly adjacent nanopore during formation of the ternary complex merase), and allowing the covalent peptide linkage to form and results in a change in the current level through the nan via the SpyTag and the SpyCatcher. opore (i.e., a current blockade event). Detection of the tag 0188 In another embodiment, a covalent conjugate of a while attached in the ternary complex can be facilitated by the polymerase and a nanopore protein can be prepared using an use of polymerases and reaction conditions (e.g., pH, metal inverse electron demand Diels-Alder (IEDDA) reaction as salts, etc.) that slow the rate of nucleotide incorporation Such described in U.S. Provisional Application No. 62/130,326, that it is slower than the rate of tag capture and current which is hereby incorporated by reference. In such an blockade measurement at the nanopore. Additionally, appro embodiment, the conjugate is prepared by attaching a linker priate covalent tethering of the polymerase to the nanopore comprising trans-cyclooctene (TCO) group to a monomer of can result in rapid tag capture on the order of microseconds. nanopore forming protein (e.g., C.-hemolysin) and attaching a 0183 The tag may be detected in the nanopore (at least in linker comprising a 6-methyl-tetrazine (6-Me-TZ) group to a part) because of its charge. In some instances, the tag com polymerase (e.g., Bst2.0 DNA polymerase). Upon mixing pound is an alternatively charged compound which has a first under mild aqueous conditions, the 6-Me-TZ modified poly net charge and, after a chemical, physical or biological reac merase and the TCO-modified nanopore rapidly (1 h) and tion, a different Second net charge. In some instance, the nearly quantitatively form a covalent linkage that provides a magnitude of the charge on the tag is the same as the magni conjugate of a polymerase and nanopore protein that can be tude of the charge on the rest of the compound. In an embodi used in nanopore sensing applications. ment, the tag has a positive charge and removal of the tag 0189 In some cases, current may be measured at different changes the charge of the compound. applied Voltages. In order to accomplish this, a desired poten 0184 In some cases, as the tag enters, becomes positioned tial may be applied to the electrode, and the applied potential in, passes into and/or through the nanopore, it may generate may be subsequently maintained throughout the measure an electronic change. In some cases the electronic change is a ment. In an implementation, an op-amp integrator topology change in current amplitude, a change in conductance of the may be used for this purpose as described herein. The inte nanopore, or any combination thereof. grator maintains the Voltage potential at the electrode by 0185. The nanopore may be biological or synthetic or a means of capacitive feedback. hybrid nanopore. It is also contemplated that the pore is (0190. A voltage potential “V” may be applied to the proteinaceous, for example wherein the pore is an O.-hemol chamber which provides a common electrical potential (e.g., ysin protein. An example of a synthetic nanopore is a solid 350 mV) for all of the cells on the chip. The integrator circuit state pore or graphene. may initialize the electrode (which is electrically the top plate 0186. In some cases, polymerase enzymes and/or phos of the integrating capacitor) to a potential greater than the phatase enzymes are attached to the nanopore. A variety of common liquid potential. For example, biasing at 450 mV. techniques for preparing fusion proteins or protein conjugates may give a positive 100 mV potential between electrode and may be employed. Fusion proteins or disulfide crosslinks are liquid. This positive Voltage potential may cause a current to examples of methods for attaching to a proteinaceous nanop flow from the electrode to the liquid chamber contact. In this ore. In the case of a solid state nanopore, the attachment to the instance, the carriers are: (a) K" ions which flow through the Surface near the nanopore may be viabiotin-streptavidin link pore from the electrode (trans) side of the bilayer to the liquid ages. In an example the DNA polymerase is attached to a solid reservoir (cis) side of the bilayer and (b) chlorine (Cl-) ions surface via gold surface modified with an alkanethiol self on the trans side which reacts with the silver electrode accord assembled monolayer functionalized with amino groups, ing to the following electro-chemical reaction: Ag+Cl wherein the amino groups are modified to NHS esters for ->AgCl--e. attachment to amino groups on the DNA polymerase. The (0191 In some cases, K' flows out of the enclosed cell method may be performed at any suitable temperature. In (from trans to cis side of bilayer) while Cl is converted to some embodiments, the temperature is between 4°C. and 10° silver chloride. The electrode side of the bilayer may become C. In some embodiments, the temperature is ambient tem desalinated as a result of the current flow. In some cases, a US 2015/036871.0 A1 Dec. 24, 2015 silver/silver-chloride liquid spongy material or matrix may and this, in turn, is in the vicinity of a temperature control serve as a reservoir to supply Cl ions in the reverse reaction element 909. The temperature control element 909 may be a which occur at the electrical chamber contact to complete the thermoelectric heating and/or cooling device (e.g., Peltier circuit. device). Multiple nanopore detectors may form a nanopore 0.192 In some cases, electrons ultimately flow onto the top array. side of the integrating capacitor which creates the electrical 0198 With reference to FIG. 9B, where like numerals current that is measured. The electrochemical reaction con represent like elements, the membrane 905 can be disposed verts silver to silver chloride and current will continue to flow overa well 910, where the sensor 902 forms part of the surface only as long as there is available silver to be converted. The of the well. FIG.9C shows an example in which the electrode limited supply of silver leads to a current dependent electrode 902 protrudes from the treated semiconductor surface 903. life in some cases. In some embodiments, electrode materials (0199. In some examples, the membrane 905 forms on the that are not depleted (e.g., platinum) are used. bottom conductive electrode 902 and not on the semiconduc 0193 Devices and Systems for Molecular Sensing and/or tor 903. The membrane 905 in such a case may form coupling Identification interactions with the bottom conductive electrode 902. In 0194 The present disclosure provides systems for some cases, however, the membrane 905 forms on the bottom molecular sensing and/or identification. Such systems may be conductive electrode 902 and the semiconductor 903. As an used to detect various types of biological species, such as alternative, the membrane 905 can form on the semiconduc nucleic acids, proteins and antibodies. In some embodiments, tor 903 and not on the bottom conductive electrode 902, but systems for molecular sensing and/or identification are used may extend over the bottom conductive electrode 902. to sequence nucleic acid molecules. 0200 Nanopores may be used to sequence nucleic acid 0.195 A system for nucleic acid sequencing can include a molecules indirectly, in some cases with electrical detection. nanopore formed or otherwise embedded in a membrane Indirect sequencing may be any method where an incorpo disposed adjacent to a sensing electrode of a sensing circuit, rated nucleotide in a growing strand does not pass through the Such as an integrated circuit. The integrated circuit may be an nanopore. The nucleic acid molecule may pass within any application specific integrated circuit (ASIC). In some Suitable distance from and/or proximity to the nanopore, in examples, the integrated circuit is a field effect transistor or a Some cases within a distance Such that tags released from complementary metal-oxide semiconductor (CMOS). The nucleotide incorporation events are detected in the nanopore. sensing circuit may be situated in a chip or other device having the nanopore, or off of the chip or device, such as in an 0201 Byproducts of nucleotide incorporation events may off-chip configuration. The semiconductor can be any semi be detected by the nanopore. “Nucleotide incorporation conductor, including, without limitation, Group IV (e.g., sili events' are the incorporation of a nucleotide into a growing con) and Group III-V semiconductors (e.g., gallium ars polynucleotide chain. A byproduct may be correlated with the enide). incorporation of a given type nucleotide. The nucleotide 0196. In some cases, as a nucleic acid or tag flows through incorporation events are generally catalyzed by an enzyme, or adjacent to the nanopore, the sensing circuit detects an Such as DNA polymerase, and use base pair interactions with electrical signal associated with the nucleic acid or tag. The a template molecule to choose amongst the available nucle nucleic acid may be a subunit of a larger strand. The tag may otides for incorporation at each location. be a byproduct of a nucleotide incorporation event or other 0202. A nucleic acid sample may be sequenced using interaction between a tagged nucleotide and the nanopore or tagged nucleotides. In some examples, a method for sequenc a species adjacent to the nanopore, Such as an enzyme that ing a nucleic acid molecule comprises (a) incorporating (e.g., may hold a tagged nucleotide such that the tag enters or polymerizing) tagged nucleotides, wherein a tag associated becomes positioned in the pore, and then cleave the tag from with an individual nucleotide is released upon incorporation, the nucleotide upon incorporation of the nucleotide into the and (b) detecting the tag during the incorporation process, nucleic acid extension product. A detected signal may be either while it attached and bound in the nucleotide-enzyme collected and stored in a memory location, and later used to complex or upon its release, with the aid of a nanopore. In construct a sequence of the nucleic acid. The collected signal Some instances, the method further comprises directing the may be processed to account for any abnormalities in the tag attached to or released from an individual nucleotide detected signal, such as errors. through the nanopore. The released or attached tag may be 0.197 FIG.9 shows an examples of a nanopore detector (or directed by any Suitable technique, in some cases with the aid sensor) having temperature control, as may be prepared of an enzyme (or molecular motor) and/or a Voltage differ according to methods described in U.S. Patent Application ence across the pore. Alternatively, the released or attached Publication Nos. 2011/0193570 A1, 2013/0244340 A1, and tag may be directed through the nanopore without the use of US 2013/0264207 A1, each of which is incorporated by ref an enzyme. For example, the tag may be directed by a Voltage erence herein in its entirety. With reference to FIG. 9A, the difference across the nanopore as described herein. nanopore detector comprises a top electrode 901 in contact 0203. In some cases, the byproduct passes through the with a conductive solution (e.g., salt solution) 907. Abottom nanopore and/or generates a signal detectable in the nanop conductive electrode 902 is near, adjacent, or in proximity to ore. Released tags are an example of byproducts. In some a nanopore 906, which is inserted in a membrane 905. In some cases, the byproducts are protons (i.e., a pH change). In other instances, the bottom conductive electrode 902 is embedded cases, the byproducts are phosphates (e.g., phosphates in a semiconductor 903 in which is embedded electrical cir released during nucleotide incorporation events). For cuitry in a semiconductor substrate 904. A surface of the example, each of the different types of nucleotides may com semiconductor 903 may be treated to be hydrophobic. A prise a different number of phosphates, and detection of the sample being detected goes through the pore in the nanopore released phosphates allows one to determine the identity of 906. The semiconductor chip sensor is placed in package 908 the incorporated nucleotide. US 2015/036871.0 A1 Dec. 24, 2015

0204 An example of the method is depicted in FIG. 10. 0210. With continued reference to FIG. 10, the enzyme Here, the nucleic acid strand 1000 passes across or in prox draws from a pool of nucleotides (filled circles at indication imity to (but not through as indicated by the arrow at 1001) the 1005) attached to tags (open circles at indication 1005). Each nanopore 1002. An enzyme 1003 (e.g., DNA polymerase) type of nucleotide is attached to a different tag so that when extends a growing nucleic acid strand 1004 by incorporating the tags are released and pass through the nanopore 1006, one nucleotide at a time using a first nucleic acid molecule as they may be differentiated from each other based on the signal a template 1000 (i.e., the enzyme catalyzes nucleotide incor that is generated in the nanopore. poration events). 0205 The enzyme 1003 may be attached to the nanopore 0211 FIG. 11 shows an example of different signals being 1002. Suitable methods for attaching the enzyme to the nan generated by different tags as they pass through the nanopore. opore include cross-linking such as the formation of intra Four different signal intensities (1101,1102.1103 and 1104) molecular disulfide bonds, or via another covalent conjuga are detected. These correspond to four different tags. For tion reaction, Such as an inverse electron demand Diels-Alder example, the tag released by incorporation of adenosine (A) (IEDDA) reaction as described in U.S. Provisional Applica may generate a signal with an amplitude 1101. A tag released tion No. 62/130,326, which is hereby incorporated by refer by incorporation of cytosine (C) may generate a signal with a ence. The nanopore and the enzyme may also be a fusion higher amplitude 1103. A tag released by incorporation of protein that is encoded by a single polypeptide chain. Meth guanine (G) may generate a signal with a yet higher amplitude ods for producing fusion proteins are known in the art and 1104. And a tag released by incorporation of thymine (T) may include fusing the coding sequence for the enzyme in frame generate a signal with a yet higher amplitude 1102. The lack and adjacent to the coding sequence for the nanopore (with of signal during periods when there is no tag passing through out a stop codon in between) and expressing this fusion the nanopore are indicated by 1105. sequence from a single promoter. In some cases, phosphatase 0212. The rate of nucleotide incorporation events is gen enzymes are also attached to the nanopore. erally slower than (or equal to) the rate at which tags mol 0206 Generally, the polymerase used in the methods of ecules released during the nucleotide incorporation events the present disclosure can include any naturally-occurring or pass through and/or are detected by the nanopore. Generally, non-naturally occurring (e.g., engineered) enzyme that has the rate of nucleotide incorporation events is not greater than 5'-->3' DNA polymerase activity and strong strand displace the rate at which tags molecules released during the nucle ment activity but lacks 5'->6' exonuclease activity. In some otide incorporation events pass through and/or are detected cases, the DNA polymerase is 9° N polymerase or a variant by the nanopore (i.e., otherwise the nucleotide incorporation thereof, E. coli DNA polymerase I, Bacteriophage T4 DNA events are not detected accurately and/or in the correct polymerase, Sequenase, Taq DNA polymerase, DNA poly sequence). merase from Bacillus Stearothermophilus, Bst 2.0DNA poly 0213. The present disclosure provides various devices for merase, 9° N polymerase (exo-)A485L/Y409V, Phi29 DNA molecular identification and/or sensing. FIG. 12 is a sche Polymerase (cp29 DNA Polymerase), T7 DNA polymerase, matic diagram of a nanopore device 100 (or sensor) that may DNA polymerase II, DNA polymerase III holoenzyme, DNA be used to sequence a nucleic acid and/or detect a tag as polymerase IV. DNA polymerase V, or Vent DNA poly described herein. The nanopore containing lipid bilayer may CaS. be characterized by a resistance and capacitance. The nanop 0207 Generally, the polymerase requires the presence of a ore device 100 includes a lipid bilayer 102 formed on a lipid primer strand that hydridizes to the template DNA strand that bilayer compatible surface 104 of a conductive solid substrate is extended by the enzyme and thereby sequenced. Accord 106, where the lipid bilayer compatible surface 104 may be ingly, in another possible configuration of the nanopore isolated by lipid bilayer incompatible surfaces 105 and the device of the present disclosure, the primer strand is attached conductive solid substrate 106 may be electrically isolated by to the pore protein, the template DNA strand is hybridized to insulating materials 107, and where the lipid bilayer 102 may this attached primer Strand, and the polymerase binds to the be surrounded by amorphous lipid 103 formed on the lipid template primer hybrid and thereby is non-covalently bound bilayer incompatible surface 105. The lipid bilayer 102 may to the nanopore device. Such an embodiment is depicted in be embedded with a single nanopore structure 108 having a FIG.38. The tag attached to the complementary tagged nucle nanopore 110 large enough for passing of the tags being otide is attracted to the lumen of the nanopore by the electro characterized and/or small ions (e.g., Na', K", Ca", Cl) static field gradient, ensuring that it can be detected and between the two sides of the lipid bilayer 102. A layer of water identified by monitoring current in the pore. molecules 114 may be adsorbed on the lipid bilayer compat 0208. A nucleic acid sample may be sequenced using ible surface 104 and sandwiched between the lipid bilayer tagged nucleotides. In some examples, a method for sequenc 102 and the lipid bilayer compatible surface 104. The aque ing a nucleic acid molecule comprises (a) polymerizing ous film 114 adsorbed on the hydrophilic lipid bilayer com tagged nucleotides, wherein a tag associated with an indi patible surface 104 may promote the ordering of lipid mol vidual nucleotide is released upon polymerization, and (b) ecules and facilitate the formation of lipid bilayer on the lipid detecting the released tag with the aid of a nanopore. bilayer compatible surface 104. A sample chamber 116 con 0209. In some instances, the method further comprises taining a solution of the nucleic acid molecule 112 and tagged directing the tag released from an individual nucleotide nucleotides may be provided over the lipid bilayer 102. The through the nanopore. The released tag may be directed by Solution may be an aqueous solution containing electrolytes any Suitable technique, in Some cases with the aid of an and buffered to an optimum ion concentration and maintained enzyme (or molecular motor). Alternative, the released tag at an optimum pH to keep the nanopore 110 open. The device may be directed through the nanopore without the use of an includes a pair of electrodes 118 (including a negative node enzyme. For example, the tag may be directed by a Voltage 118a and a positive node 118b) coupled to a variable voltage difference across the nanopore as described herein. Source 120 for providing electrical stimulus (e.g., voltage US 2015/036871.0 A1 Dec. 24, 2015 bias) across the lipid bilayer and for sensing electrical char various phospholipids such as palmitoyl-oleoyl-phosphati acteristics of the lipid bilayer (e.g., resistance, capacitance, dyl-choline (POPC) and dioleoyl-phosphatidyl-methylester and ionic current flow). (DOPME), diphytanoylphosphatidylcholine (DPhPC) 0214. The surface of the positive electrode 118b is or dipalmitoylphosphatidylcholine (DPPC), phosphatidylcho forms a part of the lipid bilayer compatible surface 104. The line, phosphatidylethanolamine, phosphatidylserine, phos conductive solid substrate 106 may be coupled to or forms a phatidic acid, phosphatidylinositol, phosphatidylglycerol, part of one of the electrodes 118. The device 100 may also and sphingomyelin. include an electrical circuit 122 for controlling electrical 0220. In addition to the CHL nanopore shown above, the stimulation and for processing the signal detected. In some nanopore may be of various other types of nanopores. embodiments, the variable voltage source 120 is included as Examples include Y-hemolysin, leukocidin, melittin, and a part of the electrical circuit 122. The electrical circuitry 122 various other naturally occurring, modified natural, and Syn may include amplifier, integrator, noise filter, feedback con thetic nanopores. A Suitable nanopore may be selected based trol logic, and/or various other components. The electrical on various characteristics of the analyte molecule Such as the circuitry 122 may be integrated electrical circuitry integrated size of the analyte molecule in relation to the pore size of the within a silicon substrate 128 and may be further coupled to a nanopore. For example, the CHL nanopore that has a restric computer processor 124 coupled to a memory 126. tive pore size of approximately 15 Angstroms. 0215. The lipid bilayer compatible surface 104 may be 0221 FIG. 13 shows that a plurality of nucleic acid mol formed from various materials that are suitable for ion trans ecules may be sequenced on an array of nanopore detectors. duction and gas formation to facilitate lipid bilayerformation. Here, each nanopore location (e.g., 1301) comprises a nan In Some embodiments, conductive or semi-conductive hydro opore, in some cases attached to a polymerase enzyme and/or philic materials may be used because they may allow better phosphatase enzymes. There is also generally a sensor at each detection of a change in the lipid bilayer electrical character array location as described elsewhere herein. istics. Example materials include Ag AgCl, Au, Pt, or 0222. In some examples, an array of nanopores attached to doped silicon or other semiconductor materials. In some a nucleic acid polymerase is provided, and tagged nucleotides cases, the electrode is not a sacrificial electrode. are polymerized with the polymerase. During polymeriza 0216. The lipid bilayer incompatible surface 105 may be tion, a tag is released and detected by the nanopore. The array formed from various materials that are not suitable for lipid of nanopores may have any Suitable number of nanopores. In bilayer formation and they are typically hydrophobic. In some instances, the array comprises about 200, about 400, some embodiments, non-conductive hydrophobic materials about 600, about 800, about 1000, about 1500, about 2000, are preferred, since it electrically insulates the lipid bilayer about 3000, about 4000, about 5000, about 10000, about regions in addition to separate the lipid bilayer regions from 15000, about 20000, about 40000, about 60000, about 80000, each other. Example lipid bilayer incompatible materials about 100000, about 200000, about 400000, about 600000, include for example silicon nitride (e.g., SiN.) and Teflon. about 800000, about 1000000, and the like nanopores. In 0217. In an example, the nanopore device 100 can be an Some instances, the array comprises at least 200, at least 400, alphaheolysin (CHL) nanopore device having a single alpha at least 600, at least 800, at least 1000, at least 1500, at least hemolysin (CHL) protein 108 embedded in a 2000, at least 3000, at least 4000, at least 5000, at least 10000, diphytanoylphosphatidylcholine (DPhPC) lipid bilayer 102 at least 15000, at least 20000, at least 40000, at least 60000, at formed over a lipid bilayer compatible Pt surface 104 coated least 80000, at least 100000, at least 200000, at least 400000, on an aluminum material 106. The lipid bilayer compatible Pt at least 600000, at least 800000, at least 1000000, and the like surface 104 is isolated by lipid bilayer incompatible silicon nanopores. nitride surfaces 105, and the aluminum material 106 is elec trically insulated by silicon nitride materials 107. The alumi 0223. In some cases, a single tag is released upon incor num 106 is coupled to electrical circuitry 122 that is inte poration of a single nucleotide and detected by a nanopore. In grated in a silicon substrate 128. A silver-silver chloride other cases, a plurality of tags is released upon incorporation electrode placed on-chip or extending down from a cover of a plurality of nucleotides. A nanopore sensor adjacent to a plate 128 contacts an aqueous solution containing nucleic nanopore may detect an individual released tag, or a plurality of released tag. One or more signals associated with plurality acid molecules. of released tags may be detected and processed to yield an 0218. The CHL nanopore is an assembly of seven indi averaged signal. vidual peptides. The entrance or vestible of the CHL nanop ore is approximately 26 Angstroms in diameter, which is wide 0224 Tags may be detected by the sensor as a function of enough to accommodate a portion of a dsDNA molecule. time. Tags detected with time may be used to determine the From the vestible, the CHL nanopore first widens and then nucleic acid sequence of the nucleic acid sample, Such as with narrows to a barrel having a diameter of approximately 15 the aid of a computer system (see, e.g., FIG. 14) that is Angstroms, which is wide enough to allow a single ssDNA programmed to record sensor data and generate sequence molecule (or the released tags) to pass through but not wide information from the data. enough to allow a dsDNA molecule to pass through. 0225. A nanopore based sequencing chip may incorporate 0219. In addition to DPhPC, the lipid bilayer of the nan a large number of autonomously operating or individually opore device may be assembled from various other suitable addressable cells configured as an array. For example an array amphiphilic materials, selected based on various consider of one million cells can be constructed of 1000 rows of cells ations, such as the type of nanopore used, the type of molecule by 1000 columns of cells. This array enables the parallel being characterized, and various physical, chemical and/or sequencing of nucleic acid molecules by measuring the con electrical characteristics of the lipid bilayer formed, such as ductance difference when tags released upon nucleotide stability and permeability, resistance, and capacitance of the incorporation events pass through the nanopore for example. lipid bilayer formed. Example amphiphilic materials include Moreover this circuitry implementation allows the conduc US 2015/036871.0 A1 Dec. 24, 2015

tance characteristics of the pore-molecular complex to be cation with the CPU 1405 through a communications bus determined which may be extremely valuable in distinguish (solid lines), such as a motherboard. The storage unit 1415 ing specific tags. can be a data storage unit (or data repository) for storing data. 0226. The integrated nanopore/bilayer electronic cell The computer system 1401 may be operatively coupled to a structures may apply appropriate Voltages in order to perform computer network (“network’) with the aid of the communi current measurements. For example, it may be necessary to cations interface 1420. The network can be the Internet, an both (a) control electrode voltage potential and (b) monitor internet and/or extranet, or an intranet and/or extranet that is electrode current simultaneously in order to perform cor in communication with the Internet. The network can include rectly. one or more computer servers, which can enable distributed 0227. Moreover it may be necessary to control cells inde computing. pendently from one another. The independent control of a cell 0234 Methods of the invention can be implemented by may be required in order to manage a large number of cells way of machine (or computer processor) executable code (or that may be in different physical states. Precise control of the Software) stored on an electronic storage location of the com piecewise linear Voltage waveform stimulus applied to the puter system 1401, such as, for example, on the memory 1410 electrode may be used to transition between the physical or electronic storage unit 1415. During use, the code can be states of the cell. executed by the processor 1405. In some cases, the code can 0228. In order to reduce the circuit size and complexity it be retrieved from the storage unit 1415 and stored on the may be sufficient to provide logic to apply two separate Volt memory 1410 for ready access by the processor 1405. In ages. This allows two independent grouping of cells and Some situations, the electronic storage unit 1415 can be pre corresponding state transition stimulus to be applied. The cluded, and machine-executable instructions are stored on state transitions are stochastic in nature with a relatively low memory 1410. probability of occurrence. Thus it may be highly useful to be able to assert the appropriate control Voltage and Subse 0235. The code can be pre-compiled and configured for quently perform a measurement to determine if the desired use with a machine have a processer adapted to execute the state transition has occurred. For example the appropriate code, or can be compiled during runtime. The code can be Voltage may be applied to a cell and then the current measured Supplied in a programming language that can be selected to to determine whetherapore has formed. The cells are divided enable the code to execute in a pre-compiled or as-compiled into two groups: (a) those which have had a pore form and no fashion. longer need to have the Voltage applied. These cells may have 0236. The computer system 1401 can be adapted to store a OV bias applied in order to effect the null operation user profile information, such as, for example, a name, physi (NOP) that is stay in the same state and (b) those which do cal address, email address, telephone number, instant mes not have a pore formed. These cells will again have the pore saging (IM) handle, educational information, work informa formation electric Voltage applied. tion, Social likes and/or dislikes, and other information of 0229. A substantial simplification and circuit size reduc potential relevance to the user or other users. Such profile tion may be achieved by constraining the allowable applied information can be stored on the storage unit 1415 of the Voltages to two and iteratively transitioning cells in batches computer system 1401. between the physical states. 0237 Aspects of the systems and methods provided 0230. For example, a reduction by at least a factor of 1.1, herein, such as the computer system 1401, can be embodied in 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 may be achieved programming. Various aspects of the technology may be by constraining the allowable applied Voltages. thought of as “products” or “articles of manufacture' typi 0231 Computer Control Systems cally in the form of machine (or processor) executable code 0232 Nucleic acid sequencing systems and methods of and/or associated data that is carried on or embodied in a type the disclosure may be regulated with the aid of computer of machine readable medium. Machine-executable code can systems. FIG. 14 shows a system 1400 comprising a com be stored on an electronic storage unit, such memory (e.g., puter system 1401 coupled to a nucleic acid sequencing sys ROM, RAM) or a hard disk. “Storage' type media can tem 1402. The computer system 1401 may be a server or a include any or all of the tangible memory of the computers, plurality of servers. The computer system 1401 may be pro processors or the like, or associated modules thereof. Such as grammed to regulate sample preparation and processing, and various semiconductor memories, tape drives, disk drives and nucleic acid sequencing by the sequencing system 1402. The the like, which may provide non-transitory storage at any time sequencing system 1402 may be a nanopore-based sequencer for the Software programming. All orportions of the Software (or detector), as described elsewhere herein. may at times be communicated through the Internet or various 0233. The computer system may be programmed to imple other telecommunication networks. Such communications, ment the methods of the invention. The computer system for example, may enable loading of the Software from one 1401 includes a central processing unit (CPU, also “proces computer or processor into another, for example, from a man sor herein) 1405, which can be a single core or multi core agement server or host computer into the computer platform processor, or a plurality of processors for parallel processing. of an application server. Thus, another type of media that may The computer system 1401 also includes memory 1410 (e.g., bear the software elements includes optical, electrical and random-access memory, read-only memory, flash memory), electromagnetic waves, such as used across physical inter electronic storage unit 1415 (e.g., hard disk), communica faces between local devices, through wired and optical land tions interface 1420 (e.g., network adapter) for communicat line networks and over various air-links. The physical ele ing with one or more other systems, and peripheral devices ments that carry Such waves. Such as wired or wireless links, 1425. Such as cache, other memory, data storage and/or elec optical links or the like, also may be considered as media tronic display adapters. The memory 1410, storage unit 1415, bearing the Software. As used herein, unless restricted to interface 1420 and peripheral devices 1425 are in communi non-transitory, tangible 'storage' media, terms such as com US 2015/036871.0 A1 Dec. 24, 2015

puter or machine “readable medium” refer to any medium salt) is converted to the tributylammonium salt by using 1.5 that participates in providing instructions to a processor for mmol (5 eq) of tributylamine in anhydrous pyridine (5 ml). execution. The resulting Solution is concentrated to dryness and co 0238 Hence, a machine readable medium, such as com evaporated with 5 ml of anhydrous DMF (x2). The dGTP puter-executable code, may take many forms, including but (tributylammonium salt) is dissolved in 5 ml anhydrous DMF, not limited to, a tangible storage medium, a carrier wave and 1.5 mmol 1, 1-carbonyldiimidazole added. The reaction medium or physical transmission medium. Non-volatile Stor is stirred for 6 hr, after which 12 ul methanol added and age media include, for example, optical or magnetic disks, stirring continued for 30 min. To this solution, 1.5 mmol Such as any of the storage devices in any computer(s) or the phosphoric acid (tributylammonium salt, in DMF) added and like. Such as may be used to implement the databases, etc. the reaction mixture stirred overnight at room temperature. shown in the drawings. Volatile storage media include The reaction mixture is diluted with water and purified on a dynamic memory, Such as main memory of Such a computer Sephadex-A25 column using 0.1 M to 1M TEAB gradient platform. Tangible transmission media include coaxial (pH 7.5). The dG4P elutes at the end of the gradient. The cables; copper wire and fiber optics, including the wires that appropriate fractions are combined and further purified by comprise abus within a computer system. Carrier-wave trans reverse-phase HPLC to provide 175 umol of the pure tetra mission media may take the form of electric or electromag phosphate (dG4P). 'P-NMR: 8, -10.7 (d. 1P, C-P), -11.32 netic signals, or acoustic or light waves Such as those gener (d. 1 P 8-P), -23.23 (dd. 2P, B, Y-P); ESI-MS (-ve mode): Calc. ated during radio frequency (RF) and infrared (IR) data 5872. Found 585.9 (M-2). communications. Common forms of computer-readable 0243 To 80 umol dG4P in 2 ml water and 3.5 ml 0.2M media therefore include for example: a floppy disk, a flexible 1-methylimidazole-HCl (pH 6) added 154 mg. EDAC and 260 disk, hard disk, magnetic tape, any other magnetic medium, a mg diaminoheptane. The pH of the resulting solution is CD-ROM, DVD or DVD-ROM, any other optical medium, adjusted to 6 with concentrated HCl and stirred at room punch cards paper tape, any other physical storage medium temperature overnight. This solution is diluted with water and with patterns of holes, a RAM, a ROM, a PROM and purified by Sephadex-A25 ion-exchange chromatography EPROM, a FLASH-EPROM, any other memory chip or car followed by reverse-phase HPLC to give -20 umol dG4P tridge, a carrier wave transporting data or instructions, cables NH. This is confirmed by ESI-MS data (-ve mode): talc. or links transporting Sucha carrier wave, or any other medium 699.1. Found (698.1, M-1). from which a computer may read programming code and/or data. Many of these forms of computer readable media may B. Synthesis of Coumarin-PEG-Acids and NHS be involved in carrying one or more sequences of one or more Esters instructions to a processor for execution. 0239 While preferred embodiments of the present inven 0244. The commercially available amino-dPEG-acids tion have been shown and described herein, it will be obvious Amino-d(PEG) 16, 20, 24, 36-acids; Quanta Biodesign are to those skilled in the art that such embodiments are provided reacted with 6-methoxycoumarin-NHS ester to provide the by way of example only. Numerous variations, changes, and corresponding coumarin-(PEG)-acid. Amino-PEG-acid (1 substitutions will now occur to those skilled in the art without eq) is dissolved in carbonate-bicarbonate buffer (pH 8.6), departing from the invention. It should be understood that followed by addition of coumarin-NHS (1 eq) in DMF, and various alternatives to the embodiments of the invention the reaction mixture stirred overnight. The coumarin-PEG described herein may be employed in practicing the inven acid is purified by Silica-gel chromatography using a CH2Cl2 tion. It is intended that the following claims define the scope MeOH (5-15%) mixture and the appropriate fractions com of the invention and that methods and structures within the bined. These compounds are analyzed by H NMR and Scope of these claims and their equivalents be covered MALDI-TOF MS analysis. Results are shown in Table 2. thereby. TABLE 2 EXAMPLES MALDI-TOF MS Data: Example 1 Coumarin- Coumarin- Coumarin- Coumarin Synthesis of a Coumarin-PEG-dG4P Tagged PEG16- PEG20- PEG24- PEG36 Nucleotide acid acid acid acid 0240. In this example, nucleotides are purified by reverse Expected MW 996 1,172 1,348 1877 phase HPLC on a 150x4.6 mm column (Supelco), mobile Observed MW 1,016 1,192 1,368 1,899 phase: A, 8.6 mM EtN/100 mM 1,1,1,3,3,3-hexafluoro-2- *Difference in observed values due to presence of sodium salt, propanol in water (pH 8.1); B, methanol. Elution is performed from 100% A isocratic over 10 min followed by a linear 0245. The coumarin-PEG-acids are converted to the cor gradient of 0-50% B for 20 mM and then 50% Bisocratic over responding NHS esters by reacting with 1.5 eq. of disuccin another 30 min. imidyl carbonate (DSC) and 2 eq of triethylamine in anhy 0241 The synthesis of coumarin-PEG-dG4P involves drous DMF for 2 h. The resulting NHS ester, which moves three synthesis operations, A, B, and C as shown in the slightly higher than the acid on silica-gel plates, is purified by scheme in FIG. 16. silica-gel chromatography using a CHCl-MeOH (5-15%) mixture and used in the next operation. A. Syntheses of 2'-deoxyguanosine-5'-tetraphosphate 0246 Coupling of Operation A and B Products to Form (dG4P) and dG4P-NH Coumarin-PEG-dG4P: 0242 First, the synthesis of 2'-dG4P is carried out starting 0247 dG4P-heptyl-NH2 from operation A) above is taken from 2'-dGTP. 300 umoles of 2'-dGTP (triethylammonium up in 0.1 M carbonate-bicarbonate buffer (pH 8.6) and to this US 2015/036871.0 A1 Dec. 24, 2015

stirred solution added one of the coumarin-PEG-NHS com (0252 (b) “Fake tag-TTT XXX” has sequence: Streptavi pounds (in DMF). The resulting mixture stirred overnight at din-Biotin-10T-TTT-3T-XXX-11T (SEQID NO. 2) room temperature and then purified on a silica-gel cartridge 0253 (c) “30T tag has sequence: Streptavidin-Biotin (15-25% MeOH in CHCl to remove unreacted coumarin acid or —NHS and then 6:4:1 isopropanol/NHOH/HO). 30T (SEQID NO. 3) This is further purified twice by reverse-phase HPLC to pro 0254 (d) “Fake tag-iFluorT has the sequence: Streptavi vide pure coumarin-PEG-dG4P. The structure is confirmed din-Biotin-10T-TTT-3TT-iFluorT-T-11T, where the T at by analysis on MALDI-TOF MS. Coumarin-PEG16-dG4P: position 18 is labeled with fluorescein. (SEQID NO. 4) retention time, 31.7 min; coumarin-PEG20-dG4P: retention 0255. The results are for one pore in an array capturing time, 32.2 min; coumarin-PEG24-dO4P: retention time, 33.0 multiple tag molecules from solution overtime. The detection min; coumarin-PEG36-dG4P: retention time, 34.3 min. conditions are 1MKC1, buffered with 20 mM HEPES, pH7.5 Results are shown in Table 3. at room temperature. Each molecule is captured and held in the pore while a Voltage is applied. The applied Voltage is TABLE 3 increased to +160 mV, a new molecule is captured, and the voltage is reduced below OV and the tag molecule falls out of MALDI-TOF MS Data: the pore. The cycle is then repeated. Four different tags are in Coumarin- Coumarin- Coumarin- Coumarin the sample mix at once. PEG16- PEG20- PEG24- PEG36 G4P G4P G4P G4P 0256. As shown in FIG. 18, the clear bands seen during the application of 160 mV become connected or slightly smeared Expected MW 1,673 1,850 2,025 2,554 in the histogram because the current during the ramp down is Observed MW 1,682 1858 2,036 2,569 also plotted. Despite this, distinct, repeatable capture bands can be seen for each tag. Example 2 (0257. As shown in FIG. 19, the horizontal axis of the plot is time (measured in seconds) vs. current (measured in pico amps (pA)) on the vertical axis. The applied Voltage wave Characterization of the Released Tags by form is not shown. The applied Voltage waveform starts MALDI-TOF MIS below OV and quickly increases to +160 mV and is held there 0248. The expected coumarin-PEG-NH, molecules are for approximately 2.3 seconds. The Voltage is then ramped confirmed by MALDI-TOF-MS analysis, following HPLC down to below OV. The current readings follow the applied purification (FIG. 17). MALDI-TOF-MS results indicate that voltage with a captured molecule's current being flat while the coumarin-PEG-NH2 tags generated by acid hydrolysis are the applied voltage is at +160 mV and then ramps down as the identical to the released tags produced during polymerase Voltage ramps down. reaction after alkaline phosphatase treatment. 0249. With reference to FIG. 17, coumarin-PEG-NH tags Example 4 generated by acid hydrolysis of coumarin-PEG16-dG4P yielding coumarin-PEG16-N1-19, coumarin-PEG20-dG4P Examples of Conjugation Reactions yielding coumarin-PEG20-NW, coumarin-PEG24-dG4P yielding coumarin-PEG24-NH9 and coumarin-PEG36 0258 Examples of conjugation reactions are shown in dG4P yielding coumarin-PEG36-NH2, are identical to the FIG. 20. As shown, (i.) amine reacts with NHS ester to form corresponding released tags generated in polymerase exten an amide, (ii.)amine reacts with acid halide to forman amide, sion reactions after treatment with alkaline phosphatase, as (iii.)amine or oxy-amine reacts with ketone to forman oxime, shown by MALDI-TOF-MS analysis. A composite image of (iv.) amine reacts with aldehyde to form Schiffs base and four separately obtained MS spectra is shown. The structures methyl amino by reduction, and (v.) hydrazine reacts with of the coumarin-PEG-NH2 tags are shown to the right. aldehyde to form a hydrazide. As shown, thiols react with Example 3 thiols, maleimide or halo-acetamides. Detection of Oligonucleotide Tags Example 5 0250) A nanopore array device (see e.g., FIG. 12) is used Examples of Click Chemistry to detect 4 distinct current levels for 4 different tags. As seen in FIG. 18, each of the tags can be distinguished from any of 0259 Examples of click chemistry using compounds with the other three (i.e., the histogram shows four distinct peaks azide, alkyne, alkene and tetrazine containing moieties are labeled in the graphic with the corresponding tag). Each tag is shown in FIG. 21. As shown, conjugation can be accom an oligonucleotide homopolymer of “T” approximately 30 plished to provide a triazole or 1,2-diazine (dihydropy bases in length, biotinylated on the 3' end with two regions in ridazine tautomer) linkage. AZide-containing molecule A the strand potentially modified. In each 30 base long mol reacts with alkyne-containing molecule B to form a conjugate ecule, the regions modified are: from the 3'end, base positions of A and B via a triazole. Also, azide-containing molecule A 11, 12, and 13 and positions 17, 18, and 19. As used here “x' can react with cyclooctyne-containing molecule B to form a is an abasic site (no base) and “T” is thymine. The four tags conjugate of A and B via a triazole fused with a cyclooctyl a. moiety. Alternatively, a tetrazine-containing molecule A 0251 (a) “Fake tag-XXX XXX has sequence: Strepta reacts with trans-cyclooctene-containing molecule B to form vidin-Biotin-10T-XXX-3T-XXX-11T (SEQID NO. 1) a conjugate of A and B via a dihydropyridazine. US 2015/036871.0 A1 Dec. 24, 2015 20

Example 6 Calif., USA) or Glen Research (Sterling, Va., USA). There are hundreds of non-standard phosphoramidite monomer unit Examples of Tagged Nucleotides “building blocks' published and commercially available 0260 Table 4 shows examples of tagged nucleotides that from custom oligonucleotide vendors that can be easily incor may act as polymerase Substrates. Exemplary tagged nucle porated into custom synthesized oligonucleotides useful as otides shown in Table 4 may be synthesized from a 5’-azido tags. Many of these non-standard monomer units are classi hexaphosphate-nucleotide (“dN6P-N3') and an alkyne-tag fied as spacers (e.g., "iSp’), dyes (e.g., “iCy3’), and linkers using either the azide-alkyne or azide-cyclooctyne "click” (e.g., "hexynyl). All oligonucleotide tag structures in Table 4 reaction (see e.g., FIG. 21). Further description of reagents are described using well-known oligonucleotide synthesis and conditions for the azide-alkyne and azide-cyclooctyne nomenclature to indicate the non-standard monomer units. click reaction syntheses are provided below in Examples (See e.g., the web-site of Integrated DNA Technologies at 7-11. www.idtdna.com for further details of commonly used oligo 0261 Table 4 includes numerous tag structures that com nucleotide nomenclature.) For example, non-standard mono prise a natural or unnatural oligonucleotide. These oligo mer units are enclosed in forward slashes “f” and an asterisk nucleotide tags are shown in 5' to 3' orientation and were “*” between units indicates a thiophosphate diester linkage. prepared by phosphoramidite synthesis, and are commer Thus, “/5Hexynyl/iSpC3//iCy3/T indicates 5'-hexyne cially available based on our design from custom oligonucle phosphate-dihydroxypropane-phosphate-cyanine 3 (dye)- otide vendors such as Integrated DNA Technologies (Cor phosphate-thymidine-3" (OH). A key of further selected alville, Iowa, USA) or TriLink Biotechnologies (San Diego, abbreviations is included in Table 4. TABLE 4 Tagged Nucleotide SEO ID Name Tag Structure (including alkyne or cyclooctyne moiety) No. dT6P - Cy3 DBCO-Cy3 dA6P - Cy3 DBCO-Cy3 dT6P - Cy3-Ts /5Hexynyl//icy3/TTTTT TTTTT TTTTT TTTTT TTTTT 5 dA6P-T* to ODD /5Hexynyl/Tk TT TTTTTTTTTTTTTTTTTTTTTTTTTT+ T 6 dG6P-Tso /5Hexynyl/TT TTTTT TTTTT TTTTT TTTTT TTTTT 7 dT6P-T-dSps-T16 /5Hexynyl/TTTTTT/idSp//idSp//idSp//idsp //idsp //idSp//idSp//idsp/TTTTT 8 TTTTT TTTTT T. dC6P-T-T*o-T /5Hexynyl/TTTTTTTTT T. T. T. T. T+ T TTTTTT TTTTT TTTT 9 dC6P-T-dSp-T2. /5Hexynyl/TTTT/idsp//idsp//idsp/TTTTT TTTTTTTTTT TTTTT TTT O dC6P-T7-dSp-Tao /5Hexynyl/ TT/idsp//idsp//idsp/TTTTT TTTTT TTTTT TTTTT TTTTT 1. dC6P-To-dSp-T7 /5Hexynyl/ TTTTT/idsp//idsp//idsp/TTTTT TTTTT TTTTT TT 2 dC6P-T13 - dSp-T /5Hexynyl/TTTTT TTTTT TTT/idsp//idsp//idsp/TTTTT TTTTT TTTT 3 dG6P-To-C6 /5Hexynyl/ TTTTT TTTTT TTTTT TTTTT TTTTT/3cs/ 4. dG6P - Cy3-To-C6 /5Hexynyl//icy3/TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT/3cs/ 5 dT6P-T-dSpo-T /5Hexynyl/TTTT/idSp//idSp//idSp//idsp //idsp //idSp//idSp//idSp//idsp //idsp/ 6 C6 TTTTT TTTTT TTTTT T/3cs/ dG6P- (T-Npy) - /5Hexynyl/TTTT/x//x/TTTT/x//x/TTTT/x//x/TTTT/x//x/TTTT/x//x/TTTT/ 7 C3 X//X//3SpC3/ X = NitroPyrrol dC6P- (T-Neb) - C3 /5Hexynyl/TTTT/x//X/TTTT/x//X/TTTT/x//X/TTTT/x//X/TTTT/x//X/TTTT/ 8 X//X//3SpC3/ dA6P-T-Sp18-T2- /5Hexynyl/TTTT/isp18/TTTTTTTTTT TTTTT TTTTTTT/3spc3/ 9 C3 dA6P-T-Sp182-To /5Hexynyl/TTTT/isp18//isp18/TTTTTTTTTT TTTTT TTTT/3spc3/ 2O C3 dA6P-T-Sp92-T22 /5Hexynyl/TTTT/isp9//isp9/TTTTT TTTTT TTTTTTTTTT TT/3spc3/ 21 C3

dT6P-dT-C7 NH- /5Hexyny tTAiUniAmMAAiUniAmM/AiUniAmM/AiUniAmM/AiUniAmM/A 22 dT8-C3 iUniAmM/TTTT TTTTTTTTTT TTT/3spc3/

US 2015/036871.0 A1 Dec. 24, 2015

TABLE 4 - continued Tagged Nucleotide SEQ ID Name Tag Structure (including alkyne or cyclooctyne moiety) No. dT6P-Cy3-dT. /5Hexynyl//iCy3/TTTT/alpha-dT//alpha-dT//alpha-dT/TTTTT TTTTT TTTTT 106 (alpha-dT) - dT-C3 TTTTT TTT/3SpC3/

Selected abbreviations wDBCO' = dibenzyl cyclooctyne “*" = thiophosphate diester ODD" = thiophosphates Only at odd-numbered linkages in sequence widSp' = furan amidite (abasic amidite) w3.gif 3-hexanol wNpy" 3-nitropyrrole “3SpC3" = 3'-propanol Neb' = nebularine iSp18 = polyethyleneglycol 18 atom length iSp3" = polyethyleneglycol 9 atom length wUniAmM = heptylamine amidite “Pyrd' = pyrrollidine amidite' wiAmMC6T' = aminohexyl dT amidite iFluorT' = fluorescein dT amidite wiAmMC2T = aminoethyl dT amidite wiSpC12' = dodecyl amidite wiSpcs' hexyl amidite wiSpc3 propyl amidite “dgéPaS' = Sp isomer of alpha-thic dig&P Rev" = oligonucleotide tag has 5'-phosphate and has alkyne group at its 3'- end wHP6' = hairpin structure videoxyl' = 2'-deoxyinosine i5NitInd' = 5-nitroindole wi5I-dU = 5-iodo deoxyuridine wi5Pyrene - dU = 5-pyrene-deoxyuridine wdT = L isomer of thymidine L111' = G-quadraplex structure L121' = G-quadraplex structure Pra' = propargylglycine wDab' = diaminobutyric acid 'U' = beta-alanine (in Context of peptide tags) “dT (mp) = thymidine methyl phosphonate pyrrollidine' = pyrrollidine amidite “alpha-dT = alpha anomer of thymidine

Example 7 7.5) and acetonitrilegradient. The tagged-nucleotide product, dT6P-Cy3-Ts is characterized by MALDI-TOF mass spec Synthesis of dT6P-DBCO-Cy3 troscopy and single base extension reaction. MALDI-TOF indicates a mass of 9179 Daltons. 0262 FIG.22 shows the result of a click reaction between dA6P-N and DBCO-Cy3. In this example, dT6P-N (500 Example 9 nmol, 100 ul HO) and DBCO-Cy3 (700 nmol, 100 ul DMF) are mixed together and stirred at room temperature for 2 Synthesis of 2'-Deoxythymidine-5'-hexaphosphate hours. FIG. 23 shows a MALDI-TOF mass spectrum that azide (dT6P-N.) indicates the conversion of azido-nucleotide to the product, DBCO-Cy3-dT6P. The product is characterized by MALDI 0264. Synthesis of Fmoc-6-Aminohexyltriphosphate: TOF mass spectroscopy and single base extension reaction. 0265 Fmoc-6-aminohexanol (1 g, 2.94 mmol) is The molecular weight is 1933 Daltons according to MALDI co-evaporated with anhydrous acetonitrile (2x20 ml) and TOF. then dissolved intriethyphosphate (10 ml). Phosphorous oxy chloride (550 ul, 5.88 mmol) is added to this solution once Example 8 cooled and stirred for 2 hours. To the reaction mixture, tribu tylammonium pyrophosphate (5 equivalents, 15 mmol, 0.5M Synthesis of dT6P-Cy3-dTs solution in anhydrous DMF) is added and stirred for 20 min utes. The solution is quenched with 0.1 M triethylammonium 0263 FIG.24 shows a click reaction between the 5'-azido bicarbonate buffer (200 ml, pH 7.5) and adjusted to pH~7. hexaphosphate-nucleotide, dT6P-N and the 5'-alkyne-oligo 0266 This solution is loaded on a Sephadex A-25 column nucleotide tag, 5'-Hexynyl-Cy3-Ts to form the tagged nucle and purified using 0.1 M to 1.0 M TEAB buffer (pH 7.0) otide, dT6P-Cy3-Ts. A solution of dT6P-N (750 nmol) is gradient. The appropriate fractions are pooled and further added to 5'-Hexynyl-Cy3-Ts oligonucleotide (obtained from purified on HPLC to provide pure triphosphate, P-NMR TriLink,500 nmol in 200 ul HO), followed by the addition of (DO) 8 -10.5 (d. 2P), –22.84 (t, 1P). copper bromide (50 ul, 0.1 M solution in 3:1 DMSO/t-BuOH) 0267 Synthesis of dT6P-NH: and TBTA (100 ul, 0.1 M solution in 3:1 DMSO/t-BuOH). 0268 Fmoc-aminohexyltriphosphate (200 mg 0.35 The reaction mixture is stirred at 40°C. for 16 hours. Purifi mmol) is co-evaporated with anhydrous acetonitrile (2x10 cation is performed by HPLC using 0.1 M TEAC buffer (pH ml) and then dissolved in anhydrous DMF (3 ml). Carbonyl US 2015/036871.0 A1 Dec. 24, 2015 26 diimidazole (CDI) (4 equivalents, 1.4 mmol) is added and Example 12 stirred at room temp for 4 hours. Methanol (6 equivalents, 85 ml) is added and further stirred for 30 minutes. To this, a Example of Thiol-Thiol (S-S) Coupling solution of 2'-deoxythymidine-5'-triphosphate (dTTP, tri ethyl or tributylammonium salt, 0.4 mmol) in DMF and 0274 FIG.27 shows an example of a thiol (disulfide bond) MgCl, (10 equivalent, 3.5 mmol) is added. The reaction mix coupling of a tag to a nucleotide. ture is stirred for 18 hours followed by the addition of 10% triethylamine in water (25 ml) to hydrolyze the Fmoc group. Example 13 The reaction mixture is stirred further for 16 hours and the DNA Polymerase Primer-Extension Reaction Using precipitated solid is filtered and the solution extracted with ether. The aqueous layer is concentrated and purified on Tagged Nucleotides HPLC using 0.1 M TEAC buffer (pH 7.5) and acetonitrile (0275 FIG. 28 shows an example of DNA polymerase gradient. This is characterized by P NMR and mass spec extension reaction using tagged-nucleotide hexaphosphates. troscopic data. 'P-NMR: d-10.63 (bs, 1P), -11.65 (bs, 1P), Extension reactions are carried out using a template-loop –23.35 (bm. 4P). primer in which the next complementary base on the template 0269. Synthesis of dT6P-N: is either A, G, C, or T, allowing extension by a single comple mentary nucleotide base. Each extension reaction is carried (0270. The prepared dT6P-NH. (10 umol) is dissolved in out in a thermal cycler at 65° C. for 25 minutes in 20 ul 0.1 M bicarbonate-carbonate buffer (500 pH 8.7) and azi reactions consisting of 3 LM template-loop primer, 2 units of dobutyric acid-NHS (25umol) in 200 ul DMF is added. The Therminatory DNA polymerase or Bst2.0 DNA polymerase reaction mixture is stirred overnight. The reaction mixture is (New England Biolabs) and 15uM of one of the oligonucle purified by HPLC using 0.1 M TEAC buffer (pH 7.5) and otide-tagged-dN6P nucleotides. The DNA extension prod acetonitrile gradient. ucts are precipitated with ethanol, purified through C18 Zip Tip columns (Millipore), and characterized by MALDI-TOF Example 10 MS analysis. As shown in FIG. 28, there is 100% extension of the primer (mol. Wt. 7983) with the addition of next nucle Synthesis of 2'-Deoxyadenosine-5'-Hexaphosphate otide TMP from the dT6P-Cy3-Ts tagged nucleotide (mol. and Attachment of Tag to the Terminal Phosphate wt. 8270). The other two peaks on the MALDI-TOF MS are Using Click Chemistry the intact tagged-nucleotide (mol. wt.8837) and the released product from the extension reaction (mol. wt. 9142). FIG. 29 0271 This example illustrates the general synthetic shows examples of monomers that can be incorporated into scheme for making a tagged nucleotide using a alkyne-azido oligonucleotides using amidite chemistry. cycloaddition click reaction. FIG. 25 shows the synthesis of 2'-deoxyadenosine-5'-hexaphosphate (“dA6P) and attach Example 14 ment of a tag to the terminal phosphate using the azide-alkyne click chemistry. Following the reaction arrows from begin Synthesis and Characterization of ning to end, reagents include (i) POCl and pyrophosphate, 5'-Oligonucleotide-Cy3-Tagged Nucleotides (ii) CDI and DMF, (iii) dATP, (iv) triethylamine and azido 0276. This example illustrates the synthesis of four differ butyrate NHS, and (v) TAG-alkyne. ent tags comprising oligonucleotides 5'-linked to a Cy3 moi 0272. As shown in FIG. 25, the synthesis of a tagged ety and covalently coupled to the terminal phosphate of four nucleotide, exemplified here for a tagged dATP (25), starts different nucleotide hexaphosphates, and the characterization with 6-Fmoc-aminohexanol (29), which reacts with phospho of these tagged nucleotides in polymerase extension reac rus oxychloride (POCl) and pyrophosphate with triethyl tions. phosphate as solvent at 0°C. to form 6-aminohexyltriphos 0277. The four tagged 2'-deoxy-5'-hexaphosphate nucle phate (30). The 6-aminohexyltriphosphate is activated by N. otides prepared and characterized in this example were: N carbonyl diimidazole (CDI) forming compound (31), dA6P-Cy3-T4-FldT-T-FldT-T-C, dT6P-Cy3-T2-dSps which reacts with the dATP to obtain the respective amino To-C, dG6P-Cy3-To-C, and dC6P-Cy3-T-dSp3-T-C. hexyl-dA6P (32). Then, the modified dA6P reacts with azido As shown in FIG. 30, each oligonucleotide tag is about 30 butyric acid-NHS to afford derivatives containing an azido bases long and includes dT nucleotide units and a mix of group (33). Finally, the azido derivatives and hexyne-deriva spacers and modified bases. These differences in the oligo tized tag (TAG-alkyne) react to obtain the targettagged nucle nucleotide tags are designed to create size and charge differ otide TAG-dA6P (25) through an alkyne-azido cycloaddition ences at the constriction site in the nanopore and thereby click reaction. provide unique current blockage characteristics under applied Voltage to the nanopore. For example, the abasic dSps Example 11 and dSps spacer residues have a smaller diameter than nucle otides in ssDNA, while the attached fluorescein on thy Click Reaction Between dT6P-N3 and Oligo-Alkyne midines in the FldT-T-FIdT tags have a larger diameter. 0278 Synthesis of Oligonucleotide-Cy3-Tagged Nucle 0273 FIG. 26 shows an example of a click reaction otides between the 5’-azido-hexaphosphate-nucleotide, dT6P-N 0279. Following the general reaction scheme shown in and the 5'-alkyne-oligonucleotide tag, 5'-Hexyn-Cy3-Ts. FIG. 25, 6-Fmoc-aminohexanol (29, 1 g, 2.94 mmol) was The reaction starts with dT6P-N to which 5'-Hexyn-Cy3-Ts coevaporated with anhydrous acetonitrile (2x20 ml) and then is added in the presence of Cubr/TBTA and DMSO to form dissolved in triethyl phosphate (10 ml). To this cooled and dT6P-Cy3-Ts. stirred solution was added fresh distilled phosphorous oxy US 2015/036871.0 A1 Dec. 24, 2015 27 chloride (550 ul, 5.88 mmol) and the mixture stirred for 2 hr 70% A/30% B for 30 minutes, and finally 0% A and 100% B at 0°C. Tributylammonium pyrophosphate (5 eq., 15 mmol. for another 45 min at room temperature at a flow rate of 1 0.5 M solution in anhydrous DMF) and tributylamine (15 ml/min. (Mobile phase: A, 0.1 M TEAA; B, 100% ACN). mmol) were added and the mixture was stirred for 20 min. 0284 DNA Polymerase Extension Reactions The solution was quenched with 0.1 M triethylammonium 0285 Screening for polymerase extension reaction activ bicarbonate buffer (TEAB, 200 ml, pH7.5) and adjusted to ity with these four oligo dN6Ps as substrates identified Bst2.0 DNA polymerase (Bst2.0 DNAP) as capable to carry out pH-7. This solution was loaded on a Sephadex A-25 column primer extension quickly and precisely at room temperature. and eluted using 0.1 M to 1.0 M TEAB buffer (pH 7.0) Additionally, Bst2.0 DNAP had the added advantage of lack gradient. The appropriate fractions were pooled and further ing 3' to 5’ exonuclease activity. purified on reverse phase HPLC on SUPELCOSILTM 0286 DNA polymerase extension reactions were per LC-18-T (Supelco) 3 LM, 15 cmx4.6 mm. Mobile phase: A, formed using these four oligonucleotide-Cy3-tagged nucle 8.6 mM EtN, 100 mM HFIP in water at pH 8.1; B, 100% otides, Bst2.0 DNAP, and “SimpleBell' primer-loop-tem methanol. Started from 100% A/0% B to 0% A? 100% B in 40 plate DNA (5'-GCGCTC GAG ATCTCC TCG TAAGAG minutes. The pure triphosphate, "P-NMR (DO) 8: -7.68 (d. GAGATCTCGAGC GCA CTG ACT GACTGACCT CAG 1P), -10.5 (d. 1P), –22.65 (t, 1P). The Fmoc-aminohexylt CTG CAC GTA AGT GCAGCT GAG GTCAG-3) (SEQID riphosphate produced (30, 200 mg, 0.35 mmol) was coevapo NO: 107). Each reaction was carried out at 65° C. for 30 rated with anhydrous acetonitrile (2x10 ml) and then dis minutes in 20 LL reactions consisting of 1.5 LM template loop-primer, 1x isothermal amplification buffer 20 mM Tris solved in anhydrous DMF (3 ml). CDI (4 eq., 1.4 mmol) was HCl, 10 mM (NH)SO4, 50 mM KC1, 2 mM MgSO, 0.1% added and the solution stirred at room temp for 4 hr. Methanol Tween R. 20, pH 8.8 (a 25°C., 4 units of Bst2.0 DNAP 2.25 (6 eq., 85ul) was added and further stirring was carried out for uM natural dNTPs or 3.75 uMoligonucleotide-tagged nucle 30 min. To the above product (31), a solution of the desired otides, with or without 1 mM MnSO. The DNA extension 2'-deoxynucleoside-5'-triphosphate (dNTP tributylammo products were denatured at 95°C. for 5 minutes and then fast nium salt, 0.5 mmol) in DMF and MgCl, (10 equivalents, 3.5 cooled to 4°C. The denatured extension products were sepa mmol) was added. The reaction mixture was stirred for 18 hr rated in 15% TBE-Urea Precast Gels (Bio-Rad) under 250 followed by the addition of 10% triethylamine in water (25 mV for 25 minutes. ml) to hydrolyze the Fmoc group and yield the dN6P-NH 0287. Results (32). The reaction mixture was stirred further for 16 hr and the 0288 The DNA polymerase extension products were precipitated solid was filtered and the solution extracted with separated on a denaturing gel and the gel image is shown in FIG. 31A. Lane 1 shows a negative control using only the ether. The aqueous layer was concentrated and purified on primer-loop-template DNA, lane 2 is a positive control fol reverse phase HPLC. lowing addition of the four natural dNTPs, and lane 3 is the 0280. The product dN6P-NH product was characterized extension reaction using the four oligonucleotide-Cy3 by 'P-NMR: 8-10.63 (bs, 1P), -11.65 (bs, 1P), -23.35 (bm. tagged nucleotides. The similar extension results in lanes 2 4P). MALDI-TOF MS data (not shown): dA6P-NH. (31); and 3 demonstrate that the primer-loop-template can be suc 832.02 (calculated 829), dT6P-NH. (not shown); 825.97 (cal cessfully extended by 48 bases using only the tagged nucle culated 820), dG6P-NH. (not shown); 848.33 (calculated otides and Bst2.0 DNAP. The release of the oligonucleotide 845), dC6P-NH. (not shown); 826.08 (calculated 828.0). tags during the reaction, was demonstrated by the observation (0281. The azide (33) of the dN6P-NH2 (32, 10umol) was of the lower bands in lane 3. prepared by dissolving 32 in 0.1 M bicarbonate-carbonate 0289. The oligonucleotide tagged dN6Ps also were puri buffer (500 ul, pH 8.7) and azidobutyric acid-NHS (25 mop in fied and measured by MALDI-TOF MS and their observed 200 ul DMF was added. The reaction mixture was stirred molecular weights correlated with the calculated numbers for overnight and purified by HPLC using 0.1 M TEAA buffer each (see FIG.31B). 0290 The results demonstrate that the Bst2.0 polymerase (pH 7.5) and an acetonitrile gradient. MALDI-TOF MS data is capable of carrying out full extension reactions with the (not shown): dA6P-N (33):963.75 (calculated 963.3 as Na" four oligonucleotide-tagged nucleotide hexaphosphate Sub salt), dT6P-N: 934.58 (calculated 932.3), dG6P-N: 960.27 strates that were synthesized via an azido-alkyne click reac (calculated 957.4), dC6P-N: 919.09 (calculated 917.4). tion that produces a triazole covalently coupling between the 0282. To 5'-hexynyl-modified oligonucleotide tag (ob tag and the terminal phosphate. tained from TriLink, 500 nmol in 200 ul H2O) a solution of dN6P-N (33) (750 nmol) was added followed by the addition Example 15 of copper bromide (50 ul, 0.1 M solution in 3:1 DMSO/t- Exonuclease Protection of Oligonucleotide Tags with BuOH) and TBTA (100 ul, 0.1 M solution in 3:1 DMSO/t- 3'-Modification BuOH). The reaction mixture was stirred at 40° C. for 16 hr 0291. This example illustrates how oligonucleotide tags followed by HPLC purification using 0.1 MTEAAbuffer (pH useful for tagging nucleotides in the embodiments of the 7.5) and an acetonitrile gradient, and the oligonucleotide present disclosure can be protected from exonuclease activity tagged-nucleotide (see FIG. 30, (25)-(28)) was characterized by chemical modification of the 3'-hydroxyl. Briefly, oligo by MALDI-TOF MS and extension reaction. MALDI-TOF nucleotides with varying 3'-modifications were prepared, MS data (FIG. 31B): dA6P-Cy3-T-FldT-T-FldT-T-C3 then incubated with Phi29 DNA polymerase (which has sig (25): 11834 (calculated 11835); dT6P-Cy3-T-dSps-T-C nificant exonuclease activity), and the incubated samples ana (26): 9806 (calculated 9808); dG6P-Cy3-T-C (27): 10825 lyzed by SDS-PAGE and HPLC to detect exonuclease deg (calculated 10826); and dC6P-Cy3-T-dSp-T-C (28): radation of the oligonucleotides. 10418 (calculated 10413). 0292 Materials and Methods: (0283 For samples (25, 26, 27, 28, 32 and 33), the follow 0293 Oligonucleotide chains of dT nucleotides with 5'-bi ing HPLC method was carried out on SUPELCOSILTM otin and various 3'-chemical modifications, as shown in Table LC-C18-T (Supelco) 3.0 um particle size, 15 cmx4.6 mm 5 below, were prepared using standard oligonucleotide Syn with 100% A/0% B in 4 min, then linear gradient change to thesis techniques. US 2015/036871.0 A1 Dec. 24, 2015 28

TABLE 5

Abbreviated SEQ Tag Name Tag Structure ID NO :

T30 /5Biosg/TT TTT TTT TTT TTT TTT TTT TTT TTT TTT T. O8 dSp /5Biosg/TT TTT TTT TTT TTT TTT TTT TTT TTT TTT T/3dsp/ O9

Phos /5Biosg/TT TTT TTT TTT TTT TTT TTT TTT TTT TTT T/3Phos/ 1O

C3 /5Biosg/TT TTT TTT TTT TTT TTT TTT TTT TTT TTT T/3spc3/ 11

C6 /5Biosg/TT TTT TTT TTT TTT TTT TTT TTT TTT TTT T/3spcs/ 12 dSpC3 /5Biosg/TTTTTTTTTTTTTT TTT TTT TTT TTT TTT T/dsp//3spc3/ 13

Tmp /5Biosg/TTT TTT TTT TmpTmpTmp TTT TmpTmpTmp TTT TTT TTT 14 TTT

0294 The various chemical modifications listed in the oligonucleotide structures of Table 5 are described in Table 6 below. TABLE 6

Abbreviation Chemical Modification fBiosg l DMTN NH

S NF-N-N- O - N(P): O-CNEt O folSp/

'.O O

O – O O n 3'

/SpC3/ '5 o 1N1 n OH

/SpC6/ is r 'Nu-1N1-1N OH US 2015/036871.0 A1 Dec. 24, 2015 29

TABLE 6-continued

Abbreviation Chemical Modification FTmpf O

NH R-O 1s O

f O -CH O a. R

PhOS R - O B O

O - O

0295 The oligonucleotide/exonuclease reaction samples polymerase attached to an O-hemolysin nanopore. Moreover, were prepared as follows: 1 uL oligonucleotide (200 uM the example illustrates that the inclusion of a cyanine dye concentration), 5 units of Phi29 DNA polymerase (New (“CyDye') moiety in the linker between the oligonucleotide England Biolabs, Ipswich, Mass., USA), and 1 uL 10xPhi29 tag and the nucleotide results in significantly improved rate of Reaction Buffer (New England Biolabs, Ipswich, Mass. capture by the polymerase-nanopore complex. USA) (50 mM Tris-HCl, pH 7.5, 10 mM MgCl, 10 mM 0299 The protein C-hemolysin self-assembles in the pres (NH)SO and 4 mMDTT) were combined in 10 uL volume ence of lipid bilayers to form heptameric nanopores. As dis of buffer. This reaction sample was incubated for 15 minat37 cussed and referenced elsewhere herein, these nanopores can C. The reaction was stopped by adding 5 uL of PAGE loading be modified with a DNA polymerase (with hybridized DNA dye (50% glycerol, 50 mM EDTA, 0.01% bromophenol primer and template) covalently attached adjacent to the pore. blue). A 3 ul. aliquot of the stopped reaction sample was The nanopore can be inserted in a lipid bilayer that is immo loaded on a. 15% polyacrylamide gel containing 50% urea bilized above an electrode containing well fabricated on a and buffered with TBE (MiniPROTEAN, Bio-Rad; Hercules, CMOS microchip, and the current level changes across the Calif., USA). Oligonucleotide products were stained using nanopore can be detected upon binding of tagged nucleotides Sybr Gold (Thermo-Fisher: USA) and photographed under at the polymerase active site. 300 nm UV illumination. The PAGE results were confirmed 0300. To perform the experiment described in this by HPLC analysis of the reaction samples. example, nanopores were prepared with a single biotin moi ety displayed near the C-terminus of each of the seven mono 0296 Results mers in the heptameric C-hemolysin pore. Then Streptavidin 0297. As shown in FIG.35, the T30 oligonucleotide reac (which has four biotin binding sites) and a biotinylated hair tion sample having no 3'-modification was completely pin BioSingleBell C primer/template DNA (5'-AGA GGA degraded by the exonuclease activity under these conditions. GAT CTC GAG CGC ACT GACTGC GTG ACC TCA GCT The T30 oligonucleotide having an un-modified 3'-terminus GCA CGT AAG TGC AGC TGA GGT CAC-3') (SEQ ID and internal methyl-phosphonate ("Tmp”) linkages also was NO: 115) were added. The presence of streptavidin allowed degraded, but only from the un-modified 3'-terminus to the formation of strong binding complex having one or more first Tmp linkage. On the other hand, the oligonucleotides hairpin primer/template molecules attached adjacent to the having 3'-modifications with phosphate, or alkyl carbon pore. This nanopore complex was purified to remove excess spacer groups (e.g., SpC3, SpC6, or dSp) remained intact, BioSingleBell C DNA primer/template, then DNA poly demonstrating their resistance to the exonuclease activity of merase was added and allowed to bind to the primer/template the Phi29 DNA polymerase. for at least 30 min. at room temperature. The resulting DNA Example 15 polymerase/nanopore/DNA complex was exposed to lipid bilayers on a Genia chip to form pores. The attached hairpin Oligonucleotide Tags Comprising a CyIDye Moieties DNA molecules do not interfere with ionic currents flowing have Improved Rate of Capture by Polymerase through the pores because their exposed 3' ends are double Attached to Nanopores Stranded and cannot enter the pore. 0298. This example illustrates the use of oligonucleotide 0301 The two tagged nucleotides used in this example are tagged nucleotides to detect capture of the nucleotide by a shown in Table 7 below. US 2015/036871.0 A1 Dec. 24, 2015 30

TABLE F Tagged Nucle otide SEQ ID Name Tag Structure (including alkyne) NO: /5Hexynyl/TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT/3 c6 / 14 dG6P-Cy3-dTso-c6 / 5Hexynyl//icy3/TTTTT TTTTT TTTTT TTTTT TTTTT 15 TTTTT/3C5/

0302 Both tagged nucleotides included were prepared TABLE 8 from a 2'-deoxyguanosine hexaphosphate nucleotide (“dG6P) using the alkyne/azide cyclo-addition click chem- Measurement dG6P-dTo-C6 dG6P-Cy3-dTo-C6 istry reaction as disclosed elsewhere herein. Briefly, the dG6P Mean Captures per Min. 12.8 46 was covalently coupled through its terminal phosphate to %time captured 7.3 41 either 5'-hexynyl-oligo-dT. or 5-hexynyl-Cy3-oligo-d R..". 31: 1 5 3.4 Both tags were modified at the 3'-terminus of the dTo with a Total Pores Measured 118 82 hexanol spacer (indicated by the abbreviation "3C6'). 0303 A mixture of tagged nucleotide (1 uM), polymerase, 0308 As noted above, the mean current blockade event primer template, and Sr.'" (3 mM) was added to the purified rate (as mean captures per minute) increased nearly 4-fold nanopore complex described above and any excess pore was with the Cy3 moiety present as part of the oligonucleotide tag. washed away. Under these conditions with Sr" present, the Similarly, the percentage of time captured also increased— tagged nucleotide binds to the polymerase active site along nearly 6-fold. On the other hand, the dwell time, which cor with the primer template, and presents its oligonucleotide tag responds to the time the tag spends in the nanopore, increased to the pore but does not undergo catalytic polymerization to only modestly ~1.5-fold, which also is favorable because it the primer template chain as it would in the presence of indicates that the presence of the Cy3 moiety does not cause catalytic metal ions. These non-catalytic binding events are a significant change in the rate of release of the tag by the readily observed as Sudden decreases in ionic current through nanopore. the pore lasting an average of about 300-600 m.sec. Example 16 0304 Results 0305 When the tagged nucleotide having the oligonucle Identification of Four Different Tagged Nucleotides by Differential Current Blockade Signals at otide tag with Cy3 in the linker is added to the nanopore array Nanopore-Polymerase Conjugate chip, significant current level changes or "current blockade' events that reduced the ionic current from ~12 p.A to about 5 0309 This example illustrates the use of a nanopore array pA were detected at a rate of about 46 per minute. Each of the chip to identify four different tagged nucleotides based on the distinct current blockade signals each provides when bound current blockade events indicated that a Cy3-dTo-C6 tag was to a complementary primer-template DNA strand at the active being captured in a nanopore. When the dG6P-dTo-C6 site of Bst2.0 DNA polymerase conjugated to the nanopore. tagged nucleotide that lacks the Cy3 moiety in the linker was The four different tagged nucleotides used in this examples Subsequently added to the same nanopore array chip, the rate were: dT6P-Cy3-T-dSps-To-C. dC6P-Cy3-T-dSp3-T- of blockade events indicating tag capture was substantially C; dG6P-Cy3-To-C; and dA6P-Cy3-T-FldT-T-FldT-T- reduced to about 13 per minute. As a control, the dG6P-Cy3 C. The Bst 2.0-C-HL nanopore conjugate was prepared dTo-C6 tagged nucleotide Subsequently was returned to the using the trans-cyclooctene (TCO) to 6-methyl-tetrazine nanopore array and the rate of blockage events indicating tag (6-Me-TZ) reagents and IEDDA click reaction and inserted in capture increased back to nearly the original level. a membrane as described in U.S. Provisional Application No. 62/130,326. 0306 A converse experiment also was performed, starting 0310 Briefly, the Bst2.0 DNA polymerase-nanopore con with dG6P-dTo-C6 tagged nucleotide that lacked the Cy3 in jugate binds the tagged nucleotides to form a complex in the the linker, then changing to the dG6P-Cy3-dTo-C6 tagged polymerase active site with the self-priming template. At the nucleotide with Cy3, and finally back to the dG6P-dTo-C6. same time, under an applied Voltage, the “tail” of the tag As would be expected if the Cy3 moiety were increasing the moiety becomes positioned in the pore of the adjacent rate of tag capture, the gain, the number, and the rate of C.-hemolysin nanopore. The positioning of the tag in the nucleotide captures in the nanopore was increased only when nanopore causes a current decrease (or "current blockade') as dG6P-Cy3-dTo-C6 tagged nucleotide was used and compared to the open nanopore current. For example, the decreased significantly when the tagged nucleotide without dG6P-Cy3-T-C tagged nucleotide when captured by the Cy3 was used. nanopore-conjugated Bst2.0 polymerase was found to pro 0307 Table 8 summarizes the results and shows a com duce a consistent current blockade of from about 15 p.A open parison of the capture rates, dwell times and waiting times pore current to about 7 p.A, with a duration of the current from the nanopore capture experiments carried out using blockade in the millisecond range. these tagged nucleotides with and without the Cy3 present in 0311. The general method of preparing of the nanopore the linker. polymerase conjugate included the steps of preparing a hep US 2015/036871.0 A1 Dec. 24, 2015

tameric complex of C-hemolysin (“C.-HL') wherein one of 100K cut-off desalting spin column to ~300 lug of 6:1 C.-HL the seven monomer units was the C-HL-C46 mutant. C.-HL pore complex in 100 uL volume. A 50 mM TCO-PEG C46 has the naturally occurring lysine at position 46 Substi maleimide (Jena Bioscience GmbH, Jena, Germany) stock tuted with a cysteine and an N-terminal 6-His tag for purifi solution was prepared in DMSO. The TCO-PEG-maleimide cation. The presence of the cysteine in this C.-HL-C46 mutant stock was added to the 6:1 C.-HL pore solution (described monomer unit allows for the attachment of a single TCO above) resulting in a reaction mixture having 100-fold molar maleimide linker reagent to the complex. This TCO-group excess of the maleimide reagent. This mixture was allowed to can then conjugate via an IEDDA click reaction with a TZ react overnight with rotation at 4°C. The resulting TCO group on a modified DNA polymerase. In this example, the PEG-C-HL reagent was purified on Sephadex G-50 and used single naturally-occurring cysteine residue of DNA poly in the IEDDA click reaction with the 6-Me-TZ-PEG-Bst 2.0 merase Bst 2.0 was modified with a 6-Me-TZ-maleimide polymerase reagent prepared as described below. reagent. This 6-Me-TZ-Bst 2.0 adduct was then combined 0316 Preparation of 6-Me-TZ-PEG-Bst 2.0 Reagent: with the TCO-C-HL adduct in a 10:1 ratio to provide a C-HL 0317 DNA polymerase Bst 2.0 (New England Biolabs, heptamer conjugate with polymerase Bst 2.0 enzyme. Mate Massachusetts, USA) in phosphate reaction buffer (100 mM rials and methods for the modification C.-HL-C46 with male sodium phosphate, 150 mM NaCl, pH 7.2) was concentrated imide linker reagents, and the formation of heptameric using a 10K cut-off desalting spin column to -580 ug in 100 C.-hemolysin pores incorporating C-HL-C46 also are uL volume. A 50 mM stock solution of 6-Me-TZ-PEG described in e.g., Valeva et al. (2001), and references cited maleimide (Jena Bioscience GmbH, Jena, Germany) in therein. DMSO was prepared. The 6-Me-TZ-PEG-maleimide stock 0312 Preparation of 6:1 C-HL:C-HL-C46 pore: The solution was added to the Bst 2.0 solution to yield a reaction K46C (lysine at position 46 substituted with cysteine) mutant mixture having 100-fold excess of the maleimide reagent. of a Staphyloccocus aureus C.-HL monomer with a 6-His tag Following incubation at 4°C. on a rotator overnight, 1 MDTT (“C.-HL-C46”) was prepared using standard protein engineer was added to a final concentration of 5 mM, and incubation ing techniques. (see e.g., Valeva et al. (2001) and Palmer et al. was carried out at room temperature to quench the reaction. (1993)) The C.-HL-C46 was purified as described in the pro The resulting 6-Me-TZ-PEG-Bst 2.0 reagent was purified on tocol for "PrepEase” His-tagged protein purification kits Sephadex G-50 and used in the IEDDA click reaction with the (USB-Affymetrix: USA) and exchanged into 1xPBS with 1 TCO-PEG-C-HL reagent as described below. mM tris-carboxyethyl-phosphine (TCEP) at pH 7.2 at 1.0 0318 IEDDAClickReaction of 6-Me-TZ and TCO Con mg/mL protein concentration. This purified C.-HL-C46 was jugates: mixed with wild-type C-HL in the presence of lipid to form 0319. The IEDDA click reaction between TCO-PEG-C- heptamers as follows. HL and 6-Me-TZ-PEG-Bst 2.0 was carried out using a 5:1 0313 To obtain the optimal 6:1 ratio of native C.-HL molar excess of 6-Me-TZ-PEG-Bst 2.0 reagent to the TCO monomers to the C.-HL-C46 mutant monomer, an 11:1 ratio PEG-C-HL reagent. Generally, the 6-Me-TZ-PEG-Bst 2.0 was used for oligomerization. Lipid (1,2-diphytanoyl-sn solution was added with mixing to a volume of the TCO glycero-3-phosphocholine, powder, Avanti Polar Lipids) was PEG-C-HL solution to provide the desired 5:1 mole excess added to a final concentration of 5 mg/mL in 50 mM tris, 200 in 1xRBS, 5 mM EDTA, at pH 7.0. The mixture was allowed mM NaCl, pH 8.0 for 30 minutes at 40° C. 5% octyl-beta to react at room temperature with rotation for 1 h. Then glucoside (B-OG) was added to pop vesicles, as assessed by samples from the reaction mixture was prepared for SDS clearing, to Solubilize the proteins. Then samples were con PAGE and Bioanalyzer (Agilent) analysis by spin filtering centrated using 100K MWCO filters and spun at 24000 RPM (100K) followed by purification on a Superdex 200 gel-fil for 30 minutes to pellet the precipitated protein. After equili tration column. Heat denatured samples were prepare by brating size-exclusion columns with 30 mM foG, 75 mM heating at 95°C. for 5 min under. Further purification of the KC1, 20 mM HEPES at pH 7.5, 500 uL of the concentrated conjugates was carried out using the His-tag on the C-HL samples were loaded at low pressure to separate heptameric C46 by using a Ni" column (PrepEase Histidine-tagged Pro 6:1 C.-HL pore complexes from monomers. After concentra tein Purification Mini Kit High Yield column; Affymetrix, tion to 5 mL in two consecutive size-exclusion columns, the CA, USA). The Ni" column was run according the manu samples were loaded on Mono S 5/50 GL columns (GE facturer's protocol. The C.-HL nanopore-BST 2.0 conjugate Healthcare; New Jersey, USA). Further FPLC was used to product was stored in 1xRBS buffer at 4°C. prior to further separate the 6:1 C.-HL:C-HL-C46 pores from those having use in preparing nanopore array. different subunit stoichiometries (e.g., 7:0, 5:2). The mobile 0320 264-Well Nanopore Array Microchip: phase consisted of: A, running buffer: 20 mM 2-(N-mor 0321. The nanopore current blockade measurements were pholino)ethanesulfonic acid (MES), 0.1% Tween(R) 20, at pH performed using a ~1x1 mm CMOS microchip that has an 5; B, elution buffer: 2MNaCl, 20 mMMES, 0.1%Tween(R) 20 array of 264 silver electrodes (5 um diameter) within shallow at pH 5. Purification was performed from 100% A isocratic wells (chip fabricated by Genia Technologies, Mountain over 21 minutes followed by a linear gradient of 0-100% B for View, Calif., USA). Methods for fabricating and using such 20 minutes and then 100% Bisocratic over another 2 minutes. nanopore array microchips can also be found in U.S. Patent The flow rate was 1 ml/min. Pure native 7:0 C.-HL pores Application Publication Nos. 2013/0244340 A1 and US eluted first and the 6:1 C.-HL:C.-HL-C46 pore complexes 2013/0264207 A1, each of which is hereby incorporated by eluted with a retention time of from about 24.5 minto about reference herein. Each well in the array is manufactured using 25.5 min. a standard CMOS process with surface modifications that 0314 Preparation of TCO-PEG-C-HL Reagent: allow for constant contact with biological reagents and con 0315. A solution of 6:1 C-HL pore complex was ductive salts. Each well can Support a phospholipid bilayer exchanged into a phosphate reaction buffer (100 mM sodium membrane with the nanopore conjugate embedded therein, phosphate, 150 mM NaCl, pH 7.2) and concentrated using a and is individually addressable by computer interface. All US 2015/036871.0 A1 Dec. 24, 2015 32 reagents used are introduced into a simple flow cell above the histogram of current blockade event dwell times observed for chip using a computer-controlled Syringe pump. The chip each different tagged nucleotide was fit to the exponential Supports analog to digital conversion and reports electrical function y=Ae” and the reciprocal of constant Bused as the measurements from all electrodes independently at a rate of calculated average dwell time. Current blockade events with over 1000 points per second. Current blockade measurements average dwell times longer than 10 ms and a blockade ampli can be made asynchronously at each of 264 addressable nan tude from 0.6 to 0.2 were deemed to be indicative of produc opore-containing membranes in the array at least once every tive capture of the tagged nucleotide by the Bst2.0 poly millisecond (msec) and recorded on the interfaced computer. merase conjugated to the nanopore (i.e., binding of the tagged 0322 Formation of Lipid Bilayer on Chip: nucleotide with the complementary template base at the poly 0323. The phospholipid bilayer membrane on the chip was merase active site and the “tail of the tagged nucleotide prepared using 1,2-diphytanoyl-sn-glycero-3-phosphocho positioned in the adjacent pore). line (Avanti Polar Lipids). The lipid powder was dissolved in decane at 15 mM and then painted in a layer across the 264 0330 Experiments were carried out wherein the current wells on the chip. A thinning process then was initiated by blockade levels of each of the four different tagged nucle pumping air through the cis side of the wells, thus reducing otides were measured when exposed to an array of its comple multi-lamellar lipid membranes to a single bilayer. Bilayer mentary SimpleBell DNA template bound to a membrane formation was tested using a ramping Voltage from 0 to 1000 embedded nanopore-polymerase conjugate on the array. The mV. A typical single bilayer would temporarily open at an results were analyzed for distinct preferred current blockade applied voltage of between 300 to 500 mV. signatures associated with each different tagged nucleotide. 0324 Nanopore-Conjugate Insertion in Membrane: 0325. After the lipid bilayer formed on the 256 wells of the 0331 Additionally, “mismatch” control experiments was chip, a solution (150 mM KC1, 3 mM SrC1, 20 mM Hepes, carried out wherein only tagged nucleotides that were not pH 7.5 at 25°C.) containing 0.05 ug of the Bst2.0-C-HL complementary to the SimpleBell DNA template were nanopore conjugate (as described above), 3 LM of the desired included in the solution exposed to the nanopore array. Spe “SimpleBell' DNA templates, and 30 M of one or more of cifically, the SimpleBell template used on the array had the four tagged nucleotides was added to the cis side of the adenine in the next position on the template and the three chip. The Bst2.0-C-HL nanopore conjugate in the mixture mismatch tagged nucleotides applied were: dA6P-Cy3-T- spontaneously inserts into the lipid bilayer. Since Sri" was the FldT-T-FldT-T-C, dG6P-Cy3-To-C, and dC6P-Cy3-T- only metalion present in this experiment, the ternary complex dSp-T-C. The conditions used in the mismatch experi at the DNA polymerase was able to form at the active site but ment were as described above for detecting the current the nucleotide was not incorporated and the 5'-phosphate blockade signatures for the complementary tagged nucle linked tag was not released. otides. 0326. The SimpleBell DNA template is an 83-mer self priming single-strand that have the sequence 5'-GCG CTC 0332 Results GAGATCTCC TCG TAAGAG GAG ATC TCGAGC GCA 0333. As shown in Table 9 below, the four different oligo CTG ACT GXCTGA CCT CAG CTG CAC GTA AGT GCA nucleotide tagged nucleotides each exhibited distinct block GCT GAG GTCAG-3' (SEQID NO: 116), where X, the first ade amplitudes and average dwell times. open position on the template, could be any one of the four bases A, C, G or T. The four SimpleBell DNA templates used TABLE 9 in these nanopore experiments differed only in the first avail able position on the template for binding to the complemen blockade amplitude avg. dwell tary nucleotide and incorporation by the polymerase. Tagged nucleotide (II) time (ms) 0327. The four different tagged nucleotides used in the dT6P-Cy3-T2-dSps-To-C OS to 0.6 16.9 nanopore experiments were: dT6P-Cy3-T-dSps-T20-C, dC6P-Cy3-T-dSp-T-C O4 to OS 29.7 dG6P-Cy3-To-C O.3 to O4 28.6 dC6P-Cy3-T-dSp-T-C; dG6P-Cy3-To-C6; and dA6P dA6P-Cy3-T-FldT-T-FldT-T-C O2 to O.3 16.9 Cy3-T-FldT-T-FldT-T-C. (See also, Table 4 above.) Each of the four tagged nucleotides had a Cy3 moiety linked to an oligonucleotide tag made up of varying 30-mer sequences 0334. The current level changes in the nanopore for the comprising dT nucleotides, fluoro-modified base dT nucle mismatch tagged nucleotides, however, were significantly otides (FldT), abasic spacers (dSp), and a 3’ exonuclease different from the blockade events measured for the comple protective group. mentary tagged nucleotides. The plot of the mismatch current 0328 Nanopore Current Level Measurements: level changes showed very few large changes indicative of a 0329. The same solution used for inserting nanopore con current blockade, and the majority of the mismatch “events' jugate and DNA template (150 mMKC1,3 m MSrC1, 20 mM were very close to the open pore current level. Further, the Hepes, pH 7.5 at 25°C.) was also used as the electrolyte mismatch dwell time histogram for the measured current Solution for the nanopore current blockade measurements. A level changes showed that the majority of events were shorter 100 mV (cis VS. trans) Voltage was applied across the chip than 20 msec, which corresponds to the background signal board between two Ag/AgCl electrodes placed on either side range for complementary tagged nucleotides. Of the 1041 of the membrane and pore. Numerous current blockade total mismatch “events' detected, only 34.9% events for the events were plotted for each of the different tagged nucle mismatch nucleotides were in the usual range for a current otides with the application of voltage across the pore. Plots blockade and only 19.8% exhibited the typical dwell times for were recorded based on the two types of current blockade a current blockade. Based on these results, the overall error events observed: (1) blockade amplitude, I, as a ratio of the rates due to tagged nucleotides mismatches was estimated at pore current Io, and (2) average dwell time in milliseconds. A 6.9%. US 2015/036871.0 A1 Dec. 24, 2015 33

Example 17 0341 Sequencing Using the Nanopore Array Chip: 0342. Following the insertion of the 6:1 C.-HL-Phi29 con Sequencing on a Nanopore Array Chip Using Four jugate with self-priming DNA template in the lipid bilayer Different Tagged Nucleotides membrane on the array, the buffer solution on the cis side of 0335. This example illustrates the use of four different the membrane, which contained only SrCl metal ion salt, tagged nucleotides on a nanopore array chip to detect the was replaced with a buffer solution that included a buffer sequence of DNA template. The four different tagged nucle solution of 150 mMKC1, 3 mM MgCl, 3 mMSrC1, 20 mM otides (dT6P-Cy3-T-dSps-To-C; dC6P-Cy3-T-dSp HEPES, pH 7.5 at 25° C., and either 0.1 mM MnC1 (see T-C; dG6P-Cy3-To-C; and dA6P-Cy3-T-FldT-T-FldT current trace of FIG. 36A), a mixture of 3.0 mM MgCl, and T-C), the nanopore protein (C.-hemolysin), DNA template 0.7 mMSrC1 (see current trace of FIG.36B), or just 3.0 mM (SimpleBell 83-mer) and the nanopore array chip used (i.e., MgCl, (see current trace of FIG. 37). The presence of the ~1x1 mm CMOS microchip with a 264 array of 5 um diam catalytic divalent Mn" or Mg" ions on the cis side, resulted eter silver electrodes in shallow wells, fabricated by Genia in the initiation of the catalytic processivity of the Phi29 DNA Technologies, Mountain View, Calif., USA) were the same as polymerase. The potential applied across the pore was also used in Example 16. The DNA polymerase used in this varied. A 160 mV potential was applied and maintained in the example, however, was the Phi29 polymerase and was experiments of FIGS. 36A and 36B, whereas a 100 mV poten attached to the C-hemolysin nanopore using the SpyCatcher tial was applied, and maintained in the experiment of FIG.37. approach described in Zakeri and Howarth (2010). The varying amounts of the non-catalytic Sr" on the cis 0336 Additionally, this sequencing example included the and/or trans sides of the membrane also affected the poly presence of all four different tagged nucleotides and the cata merase processivity and the resulting ion current level traces lytic metal ion salt MgCl, to allow for the complete poly (as shown in FIGS. 36A,36B, and 37). Changes inion current merase reaction to occur with incorporation of the comple levels across the nanopores in the array were measured for mentary tagged nucleotide into the extended primer Strand 3-10 minutes. and release of the tag. 0343 Results 0337 Preparation of C-HL-Phi29 Conjugates: 0344 As shown in FIG. 36, four distinct current levels 0338. In this approach, two fragments of the collagen below the open current level were transiently observed, indi adhesion domain (CnaB2) of the Streptococcus pyogenes cating the capture by the nanopore of four different the tags fibronectin-binding protein FbaB recognize each other and associated with each of the four different nucleotides. The Subsequently generate a peptide bond between the c-amino relative current level changes observed during this sequenc group of a lysine in one fragment (i.e., the "SpyCatcher) and ing experiment with all four tagged nucleotides present were the carboxyl side group of an aspartic acid in the other frag in agreement with those observed during nanopore array ment (i.e., the "SpyTag”). In the present example, the SpyTag measurements with only a single tag and non-catalytic Sr.'" fragment was attached via a short peptide linker to the N-ter divalent metal ions present (see e.g., Example 16). As minus of the C-HL monomer, and the SpyCatcher fragment expected, the ranking of lowest to highest residual currents was attached to N-terminus of the Phi29 DNA polymerase via (MO observed for the four different tagged nucleotides was a similar short peptide linker. C.-HL monomers with and consistent with the relative residual currents observed for without the SpyTag were mixed allowing assembly of hep these tagged nucleotides using the nanopore array chips of tameric nanopores, and those heptameric nanopores with Example 16: dA6P-Cy3-T-FldT-T-FldT-T-C (-0.15), only one SpyTag-modified C.-HL monomer were purified by

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 125

<21 Os SEQ ID NO 1 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthesized sequence 22 Os. FEATURE: <221s NAMEAKEY: n <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: T-biotin-streptavidin 22 Os. FEATURE: US 2015/036871.0 A1 Dec. 24, 2015 35

- Continued <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (13) <223> OTHER INFORMATION: abasic Site 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (17) . . (19) <223> OTHER INFORMATION: abasic Site

<4 OOs, SEQUENCE: 1 ttitt ttitt tt nint tithinnt tttitt tttitt 3 O

<210s, SEQ ID NO 2 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: T-biotin-streptavidin 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (17) . . (19) <223> OTHER INFORMATION: abasic Site

<4 OOs, SEQUENCE: 2 ttitt tttitt t t t t tittnint tttitt tttitt 3 O

<210 SEQ ID NO 3 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: T-biotin-streptavidin

<4 OOs, SEQUENCE: 3 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 4 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: T-biotin-streptavidin 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (18) ... (18) <223> OTHER INFORMATION: T-fluorescein

<4 OOs, SEQUENCE: 4 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 5 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic sequence 22 Os. FEATURE: US 2015/036871.0 A1 Dec. 24, 2015 36

- Continued <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: 5'-hexyne-phosphate-cyanine 3-phosphate-T

<4 OOs, SEQUENCE: 5 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 6 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: 5'-hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222. LOCATION; (7) . . (7) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (19) . . (19) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (21) ... (21) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (23) . . (23) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (25) ... (25) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (27) . . (27) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: US 2015/036871.0 A1 Dec. 24, 2015 37

- Continued <221 > NAMEAKEY: misc feature <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: thiophosphate diester bond to following T

<4 OOs, SEQUENCE: 6 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 7 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: 5'-hexyne-phosphate-T

<4 OO > SEQUENCE: 7 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 8 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222 LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (14) <223> OTHER INFORMATION: furan amidite

<4 OOs, SEQUENCE: 8 titt t t t t t t t t t t t t t t t t t tit 3 O

<210s, SEQ ID NO 9 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (16) <223> OTHER INFORMATION: thiophosphate diester bond to following T <4 OOs, SEQUENCE: 9 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 10 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: US 2015/036871.0 A1 Dec. 24, 2015 38

- Continued <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) . . (6) <223> OTHER INFORMATION: furan amidite

<4 OOs, SEQUENCE: 10 tttninnittitt ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 11 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (10 <223> OTHER INFORMATION: furan amidite

<4 OOs, SEQUENCE: 11 tttittt thinn ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 12 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (13) <223> OTHER INFORMATION: furan amidite

<4 OOs, SEQUENCE: 12 ttitt ttitt tt nintttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 13 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (14) . . (16) <223> OTHER INFORMATION: furan amidite

<4 OOs, SEQUENCE: 13 ttitt tttitt t t t thinnittitt tttitt tttitt 3 O

<210s, SEQ ID NO 14 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: US 2015/036871.0 A1 Dec. 24, 2015 39

- Continued <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223 is OTHER INFORMATION: hexanol-T

<4 OOs, SEQUENCE: 14 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 15 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223 is OTHER INFORMATION: hexanol

<4 OOs, SEQUENCE: 15 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210 SEQ ID NO 16 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (14) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223 is OTHER INFORMATION: hexanol-T

<4 OOs, SEQUENCE: 16 titt t t t t t t t t t t t t t t t tit 3 O

<210s, SEQ ID NO 17 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (6) <223> OTHER INFORMATION: nitropyrrole 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (12) <223> OTHER INFORMATION: nitropyrrole 22 Os. FEATURE: US 2015/036871.0 A1 Dec. 24, 2015 40

- Continued <221 > NAMEAKEY: misc feature <222s. LOCATION: (17) . . (18) <223> OTHER INFORMATION: nitropyrrole 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (23) . . (24) <223> OTHER INFORMATION: nitropyrrole 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: nitropyrrole 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: nitropyrrole-propanol

<4 OOs, SEQUENCE: 17 titt titt tt t t t t t t t tilt titt 3 O

<210s, SEQ ID NO 18 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222 LOCATION; (5) . . (6) <223> OTHER INFORMATION: nebularine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (12) <223> OTHER INFORMATION: nebularine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (17) . . (18) <223> OTHER INFORMATION: nebularine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (23) . . (24) <223> OTHER INFORMATION: nebularine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: nebularine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: nebularine-propanol

<4 OOs, SEQUENCE: 18 titt titt tt t t t t t t t tilt titt 3 O

<210s, SEQ ID NO 19 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) 223 OTHER INFORMATION: 18-atom PEG 22 Os. FEATURE: US 2015/036871.0 A1 Dec. 24, 2015 41

- Continued <221 > NAMEAKEY: misc feature <222s. LOCATION: (27) . . (27) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 19 ttittnt tittt ttt tttitt tt tttitt tt 27

<210s, SEQ ID NO 2 O &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (6) <223> OTHER INFORMATION: 18-atom PEG-phosphate 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (25) ... (25) <223> OTHER INFORMATION: T-propanol 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (28) ... (28) <223> OTHER INFORMATION: propanol-T

<4 OOs, SEQUENCE: 2O ttittninttitt ttt tttitt tt tttitt 25

<210s, SEQ ID NO 21 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (6) <223> OTHER INFORMATION: 9-atom PEG-phosphate 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (28) ... (28) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 21 ttittninttitt ttt tttitt tt tttitt titt 28

<210s, SEQ ID NO 22 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (12) <223> OTHER INFORMATION: heptylamine amidite 22 Os. FEATURE: US 2015/036871.0 A1 Dec. 24, 2015 42

- Continued <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 22 tttitt thinnin nint titt tttt tttitt tttitt 3 O

<210s, SEQ ID NO 23 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (12) <223> OTHER INFORMATION: pyrrollidine amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 23 tttitt thinnin nint titt tttt tttitt tttitt 3 O

<210 SEQ ID NO 24 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (12) <223> OTHER INFORMATION: aminoethyl dT amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 24 tttitt thinnin nint titt tttt tttitt tttitt 3 O

<210s, SEQ ID NO 25 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: spermine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (27) . . (27) <223> OTHER INFORMATION: T-propanol US 2015/036871.0 A1 Dec. 24, 2015 43

- Continued

<4 OOs, SEQUENCE: 25 ttittnt tittt ttt tttitt tt tttitt tt 27

<210s, SEQ ID NO 26 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: spermine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) ... (8) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (27) . . (27) 223 OTHER IN O R A. ON: T-propanol

<4 OOs, SEQUENCE: 26 ttt thinnitt ttt tttitt tt tttitt tt 27

<210 SEQ ID NO 27 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: spermine 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: fluorescein dT amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (27) . . (27) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 27 ttittninttitt ttt tttitt tt tttitt tt 27

<210s, SEQ ID NO 28 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol US 2015/036871.0 A1 Dec. 24, 2015 44

- Continued

SEQUENCE: 28 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

SEQ ID NO 29 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthesized oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (1) ... (1) OTHER INFORMATION: hexyne-phosphate-cyanine 3.5-T FEATURE: NAMEAKEY: misc feature LOCATION: (30) ... (30) OTHER INFORMATION: T-propanol

SEQUENCE: 29 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

SEQ ID NO 3 O LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthesized oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (1) . . (1) OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-cyanine-3- phosphate-T FEATURE: NAMEAKEY: misc feature LOCATION: (30) ... (30) OTHER INFORMATION: T-propanol SEQUENCE: 3 O ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

SEQ ID NO 31 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthesized oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (1) ... (1) OTHER INFORMATION: hexyne-phosphate-T FEATURE: NAMEAKEY: misc feature LOCATION: (7) . . (7) OTHER INFORMATION: cyanine 3.5-phosphate FEATURE: NAMEAKEY: misc feature LOCATION: (30) ... (30) OTHER INFORMATION: T-propanol

SEQUENCE: 31 ttitt ttnttt ttt tttitt tt tttitt tttitt 3 O

SEQ ID NO 32 LENGTH: 30 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthesized oligonucleotide US 2015/036871.0 A1 Dec. 24, 2015 45

- Continued

22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: cyanine 3-phosphate 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 32 ttitt ttitt tt htttitt tttt tttitt tttitt 3 O

<210s, SEQ ID NO 33 &211s LENGTH: 45 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (45) ... (45) <223> OTHER INFORMATION: T-propanol < 4 OO SEQUENCE: 33 ttitt cqgcgc gtaag.cgc.cg tttitttttitt tttittttittt tttitt 45

<210s, SEQ ID NO 34 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (14) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 34 titt t t t t t t t t t t t t t t t t t tit 3 O

<210s, SEQ ID NO 35 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: thiophosphate diester bond to following T US 2015/036871.0 A1 Dec. 24, 2015 46

- Continued

22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (17) . . (17) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE <221> NAME/KEY: misc-feature <222s. LOCATION: (19) . . (19) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (21) ... (21) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (23) . . (23) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (25) ... (25) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (27) . . (27) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: thiophosphate diester bond to following T 22 Os. FEATURE: <221 > NAMEAKEY: misc-feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 35 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 36 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T US 2015/036871.0 A1 Dec. 24, 2015 47

- Continued

22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (29) <223> OTHER INFORMATION: thiophosphate diester bond to following T

<4 OOs, SEQUENCE: 36 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 37 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (29) <223> OTHER INFORMATION: thiophosphate diester bond to next T <4 OO > SEQUENCE: 37 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 38 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 38 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 39 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 39 ttitt tttittt tttitt 15

<210s, SEQ ID NO 4 O &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide US 2015/036871.0 A1 Dec. 24, 2015 48

- Continued

22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 4 O ttitt tttitt t t t t t t t t titt 2O

<210s, SEQ ID NO 41 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (25) ... (25) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 41 ttitt tttitt t t t t t t t t t t t t t titt 25

<210s, SEQ ID NO 42 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3 - phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: 18-atom PEG-phosphate 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (25) ... (25) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 42 tthtttitt tt ttt tttitt tt tttitt 25

<210s, SEQ ID NO 43 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (12) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol US 2015/036871.0 A1 Dec. 24, 2015 49

- Continued

<4 OOs, SEQUENCE: 43 titt t t t t t t t t t t t t t t t t t tit 3 O

<210s, SEQ ID NO 44 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (12) <223> OTHER INFORMATION: aminoethyl dT amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 44 tttitt thinnin nint titt tttt tttitt tttitt 3 O

<210s, SEQ ID NO 45 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: 9 atom PEG-phosphate 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (25) ... (25) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 45 ttittnt tittt ttt tttitt tt tttitt titt 28

<210s, SEQ ID NO 46 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (4) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 46 thinnittitt tt ttt tttitt tt tttitt tttitt 3 O US 2015/036871.0 A1 Dec. 24, 2015 50

- Continued

<210s, SEQ ID NO 47 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (7) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 47 ttt thinnittt ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 48 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221> NAME/KEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (10 <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 48 tttittt thinn ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 49 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (13) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 49 ttitt ttitt tt nintttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 50 &211s LENGTH: 30 US 2015/036871.0 A1 Dec. 24, 2015 51

- Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (7) <223> OTHER INFORMATION: fluorescein dT amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 50 ttt thinnittt ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 51 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221> NAME/KEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: fluorescein-dT-amidite 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: fluorescein-dT-amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 51 ttittnthttt ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 52 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-spermine-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 52 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 53 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide US 2015/036871.0 A1 Dec. 24, 2015 52

- Continued

22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-cyanine 3-phosphate-propanol

<4 OOs, SEQUENCE: 53 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 54 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: spermine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: T-propanol < 4 OO SEQUENCE: 54 ttitt ttittnt titt tttitt tt tttitt ttitt 29

<210s, SEQ ID NO 55 &211s LENGTH: 44 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (44) ... (44) <223> OTHER INFORMATION: T-propanol <4 OO > SEQUENCE: 55 ttittggttgg tdtggttggit tttitttttitt tttittttittt ttitt 44

<210s, SEQ ID NO 56 &211s LENGTH: 57 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (57) . . (57) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 56 ttitt coggcg cqgcgcgtaa gogcc.gc.gcc ggtttitttitt tttitttittitt tttittitt f US 2015/036871.0 A1 Dec. 24, 2015 53

- Continued

<210s, SEQ ID NO 57 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) ... (8) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OO > SEQUENCE: 57 ttitt thinnitt ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 58 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221> NAME/KEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) ... (9) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 58 tttitt thinnt titt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 59 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (8) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OO > SEQUENCE: 59 ttt thinnitt ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 60 &211s LENGTH: 30 US 2015/036871.0 A1 Dec. 24, 2015 54

- Continued

TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthesized oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (1) ... (1) OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T FEATURE: NAMEAKEY: misc feature LOCATION: (5) ... (9) OTHER INFORMATION: furan amidite FEATURE: NAMEAKEY: misc feature LOCATION: (30) ... (30) OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 60 ttt thinnnnt titt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 61 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221> NAME/KEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: dodecyl amidite-phosphate 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (29).. (29) <223> OTHER INFORMATION: T-propanol 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 61 ttitt thittitt ttt tttitt tt tttitt ttitt 29

<210s, SEQ ID NO 62 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (6) <223> OTHER INFORMATION: hexyl amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 62 ttittninttitt ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 63 &211s LENGTH: 30 US 2015/036871.0 A1 Dec. 24, 2015 55

- Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (7) <223> OTHER INFORMATION: propyl amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 63 ttt thinnittt ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 64 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221> NAME/KEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 64 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 65 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) . . (10 <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol <4 OOs, SEQUENCE: 65 tt t t t t t t t t t t t t t t t t t t tit 3 O

<210s, SEQ ID NO 66 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T US 2015/036871.0 A1 Dec. 24, 2015 56

- Continued

22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (31) ... (33) <223> OTHER INFORMATION: propyl amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (34) . . (34) <223> OTHER INFORMATION: propyl amidite-propanol

<4 OOs, SEQUENCE: 66 ttitt tttitt t t t t t t t t t t t t t t t t t t t t t nnnn 34

<210s, SEQ ID NO 67 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-phosphate <4 OO > SEQUENCE: 67 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 68 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propylamine <4 OOs, SEQUENCE: 68 ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 69 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) . . (10 <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 69 tt t t t t t t t t t t t t t t t t t t tit 3 O US 2015/036871.0 A1 Dec. 24, 2015 57

- Continued

<210s, SEQ ID NO 70 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-cyanine 3-phosphate-propylamine-phosphate -proparyl-propionamide

<4 OO > SEQUENCE: 7 O ttitt tttitt t t t t t t t t t t t t t t t t titt tt 3 O

<210s, SEQ ID NO 71 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: phosphate-T & 22 O FEATURE; <221 > NAMEAKEY: misc feature <222s. LOCATION: (25) ... (27) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-cyanine 3-phosphate-propylamine-phosphate proparyl-propionamide

<4 OOs, SEQUENCE: 71 ttitt tttitt t t t t t t t t t t t t t t thinnittit 3 O

<210s, SEQ ID NO 72 &211s LENGTH: 45 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (45) ... (45) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 72 ttitt cqgcgc gtaag.cgc.cg tttitttttitt tttittttittt tttitt 45

<210s, SEQ ID NO 73 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature US 2015/036871.0 A1 Dec. 24, 2015 58

- Continued

LOCATION: (1) ... (1) OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-C FEATURE: NAMEAKEY: misc feature LOCATION: (30) ... (30) OTHER INFORMATION: C-propanol <4 OO > SEQUENCE: 73 cc cc cc cc cc cococc cc cc cocco cocco 3 O

<210s, SEQ ID NO 74 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (10 <223> OTHER INFORMATION: 2'-deoxyinosine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OOs, SEQUENCE: 74 tettt tettettettettt tetettettettett 3 O

<210s, SEQ ID NO 75 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (10 <223> OTHER INFORMATION: 5-nitroindole 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OO > SEQUENCE: 75 ttt thinnnnn ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 76 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OO > SEQUENCE: 76 US 2015/036871.0 A1 Dec. 24, 2015 59

- Continued titt.ccc.ccct ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 77 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (10 <223> OTHER INFORMATION: 5-iodo deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OO > SEQUENCE: 77 ttt thinnnnn ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 78 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (10 <223> OTHER INFORMATION: 5-pyrene-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: T-propanol

<4 OO > SEQUENCE: 78 ttt thinnnnn ttt tttitt tt tttitt tttitt 3 O

<210s, SEQ ID NO 79 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: hexyne-phosphate-cyanine 3-phosphate-T 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3 O <223> OTHER INFORMATION: T-propanol 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: furan amidite 22 Os. FEATURE: <221 > NAMEAKEY: misc feature