US 2011 0160O82A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0160082 A1 WOO et al. (43) Pub. Date: Jun. 30, 2011

(54) MOBILITY-MODIFIED NUCLEOBASE Publication Classification POLYMERS AND METHODS OF USING (51) Int. Cl. SAME C40B 30/04 (2006.01) C7H 2L/00 (2006.01) C7H 2L/02 (2006.01) (75) Inventors: Sam L. WOO, Redwood City, CA C7H 2L/04 (2006.01) (US); Ron GRAHAM, Carlsbad, C40B 40/06 (2006.01) CA (US); Jing TIAN, Mountain C07F 9/24 (2006.01) View, CA (US) (52) U.S. Cl...... 506/9:536/23.1; 506/16; 558/185 (57) ABSTRACT (73) Assignee: LIFE TECHNOLOGIES CORPORATION, Carlsbad, CA The present invention relates generally to nucleobase poly merfunctionalizing reagents, to mobility-modified sequence (US) specific nucleobase polymers, to compositions comprising a plurality of mobility-modified sequence-specific nucleobase (21) Appl. No.: 13/009,827 polymers, and to the use of Such polymers and compositions in a variety of assays, such as, for example, for the detection of a plurality of selected nucleotide sequences within one or (22) Filed: Jan. 19, 2011 more target nucleic acids. The mobility-modifying polymers of the present invention include phosphoramidite reagents Related U.S. Application Data which can be joined to other mobility-modifying monomers and to sequence-specific oligonucleobase polymers via (60) Continuation of application No. 1 1/456,836, filed on uncharged phosphate triester linkages. Addition of the mobil Jul. 11, 2006, now Pat. No. 7,897,338, which is a ity-modifying phosphoramidite reagents of the present inven division of application No. 10/856,754, filed on Jun. 1, tion to oligonucleobase polymers results in unexpectedly 2004, now Pat. No. 7,235,927, which is a division of large effects the mobility of those modified oligonucleobase application No. 09/836,704, filed on Apr. 16, 2001, polymers, especially upon capillary electrophoresis in non now Pat. No. 6,743,905. sieving media. US 2011/0160082 A1 Jun. 30, 2011

MOBILITY-MODIFIED NUCLEOBASE In the illustration, DMT represents dimethoxytrity1 and iPr POLYMERS AND METHODS OF USING represents isopropyl. When x=5, each PEO reagent added to SAME the nucleobase polymer imparts the nucleobase polymer with an electrophoretic retardation of approximately 2 nucleotides 1. CROSS REFERENCE OF RELATED as compared to the unmodified nucleobase polymer under APPLICATIONS both sieving and nonsieving electrophoretic conditions. Due to limitations of DNA chemistry, no more than about 40 PEO 0001. This application is a divisional of pending U.S. reagents can be coupled to a nucleobase polymer and result in application Ser. No. 10/856,752, filed May 28, 2004, which is homogenous product. Accordingly, the greatest electro a divisional of U.S. application Ser. No. 09/836,704, filed phoretic mobility retardation that can be achieved using these Apr. 16, 2001 (now U.S. Pat. No. 6,743,905), all of which are standard PEO modifying reagents is about 80 nucleotides. incorporated herein by reference. 0006. However, in light of the increasing need to simulta neously analyze vast numbers of nucleotide sequences in a 2. FIELD OF THE INVENTION single experiment, e.g., the 200 identified alleles associated 0002 The present invention relates generally to nucleo with cystic fibrosis, there remains a need in the art for new base polymer functionalizing reagents, to mobility-modified mobility-modifying polymers that have different charge to sequence-specific nucleobase polymers, to compositions translational frictional drag ratios than currently available comprising a plurality of mobility-modified sequence-spe mobility-modifying polymers, and that can impart electro phoretic mobility retardations of greater than the 80 nucle cific nucleobase polymers, and to the use of Such polymers otides achievable with available PEO modifying reagents. and compositions in a variety of assays, such as, for example, The availability of such new mobility-modifying polymers for the detection of a plurality of selected nucleotide would greatly increase the repertoire of available mobility sequences within one or more target nucleic acids. modifications, thereby enabling the ability to perform 3. BACKGROUND OF THE INVENTION extremely complex sequence analyses in simple, preferably automated, formats. 0003 Methods used to detect selected nucleotide sequences within target nucleic acids underlie an extensive 4. SUMMARY OF THE INVENTION array of practical applications including, but not limited to, 0007. In one aspect, the present invention provides mobil paternity testing, forensic analysis, organ donor recipient ity-modifying phosphoramidite functionalizing reagents matching, disease diagnosis, prognosis and treatment, and comprising a polymeric portion and a phosphoramidite moi prenatal counseling. ety. The phosphoramidite moiety comprises an oxygen-pro 0004. There exists a need in the art for materials and meth tecting group that, quite unlike the B-cyanoethyl oxygen pro ods that permit pluralities of selected nucleotide sequences to tecting group used in conventional phosphoramidite reagents, be simultaneously detected and analyzed, under uniform is stable to basic conditions such as the conditions and experimental conditions, preferably in a single, automated, reagents used in conventional phosphoramidite oligonucle assay reaction. One approach towards meeting this need has otide synthesis and deprotection. As a consequence of this been the development of mobility-modifying polymers that stable oxygen protecting group, the bond formed between the can be attached to sequence-specific nucleobase polymers functionalizing reagent and the compound functionalized is that act to increase the effective size of the modified nucleo an uncharged phosphate triester. The mobility-modifying base polymers. Where the charge to translational frictional phosphoramidite reagents of the invention may be used to draw ratio of the mobility-modifying polymer differs from functionalize a wide variety of Substances and materials to that of the nucleobase polymer to which it is attached, the add mass and size to the Substance or material without Sub resulting modified nucleobase polymer will have an electro stantially altering its overall net charge. phoretic mobility that differs from that of the unmodified 0008. The mobility-modifying phosphoramidite reagents nucleobase polymer. This alteration of the charge to transla of the invention are compatible with standard phosphoramid tional frictional drag ratio may be employed in various appli ite synthetic schemes and can be used with commercially cations to effect electrophoretic separation of similarly-sized available nucleobase polymer synthesis instruments. Thus, nucleobase polymers under both sieving and non-sieving the mobility-modifying phosphoramidite reagents of the conditions. present invention are particularly convenient for mobility 0005. The most commonly employed mobility-modifying modifying synthetic sequence-specific nucleobase polymers polymers are polyethylene oxides (PEO) that are attached to Such as, for example, 2'-deoxyoligonucleotides. They may be a nucleobase polymer using standard DNA chemistry (Gross readily attached to the 5'-terminus of the nucleobase polymer, man et al. (1994) Nucleic Acids Research 22 (21): 4527–34). to the 3'-terminus, or to both the 5’- and 3'-termini, depending An exemplary standard PEO phosphoramidite reagent (“PEO on the particular application and/or desired degree of mobil reagent’) that can be added to a nucleobase polymer using ity modification. standard DNA chemistry is illustrated below: 0009. The terminus of the polymeric portion that is distal to the phosphoramidite moiety may be protected with a group that is selectively removable under the desired synthesis con 1N1 CN ditions, or it may comprise a group that is essentially non reactive, Such as, for example an alkyl, aryl, arylakyl, etc. pro-ha-'l-no o K group. In the former embodiment, the protecting group may N--iPr) be selectively removed for sequential condensation of one or more additional phosphoramidite reagents. Suitable selec tively removable protecting groups will depend upon the US 2011/0160082 A1 Jun. 30, 2011 identity the group being protected and will be apparent to the invention permit the formation of mobility-modified sub those of skill in the art. Selectively removable groups suitable stances comprising branched or dendritic polymer segments. for protecting hydroxyl groups include, by way of example 0013. In one convenient embodiment, the polymer portion and not limitation, any of the acid-labile groups that are of the mobility-modifying phosphoramidite reagents of the commonly used to protect the 5'-hydroxyl of conventional nucleoside phosphoramidites oligonucleotide synthesis present invention is a polyalkylene oxide, an illustrative reagents. Such as acid-labile trityl groups (e.g., monomethox embodiment of which is depicted as Formula (I) below: ytrityl, dimethoxytrityl, etc.). 0010. The mobility-modifying phosphoramidite reagents of the invention may be used alone to mobility-modify sub stances such as nucleobase polymers to add mass and size to the nucleobase polymer without altering its overall net charge. Alternatively, they may be used in conjunction with conventional mobility-modifying reagents, such as the PEO reagent illustrated above, to mobility-modify Substances Such as nucleobase polymers. Because the mobility-modifying 0014 wherein: phosphoramidite reagents of the invention may be used to add I0015 R is selected from the group consisting of hydro mass and size to a Substance Such as a nucleobase polymer gen, protecting group, reporter molecule, and ligand; without altering its overall charge, when used in conjunction 0016 X is selected from the group consisting of O, S, NH, with conventional reagents such as PEO reagents, they vastly increase the repertoire of available mobility-modifications and NH C(O); that can be added to Substances such as nucleobase polymers. 0017 each a is, independently, an integer from 1 to 6: 0.011 The polymeric portion composing the reagent may (0018 b is an integer from 0 to 40; be any of a variety of polymers that are soluble under the 0019 RandR are each independently selected from the desired conditions of use and that either include, or can be group consisting of C1-C alkyl, Cs-Co cycloalkyl, Co-Co modified to include, a functional group, Such as, for example, aryl, and Co-C, arylalkyl, and a primary hydroxyl group, that can be conveniently converted to a phosphoramidite moiety, typically using standard art (0020 R is selected from the group consisting of alkyl known chemistries. Typical polymers include, but are not comprising at least two carbon atoms, aryl, (R)-Si- where limited to, polyoxides, polyamides, polyimines and polysac each R is independently selected from the group consisting charides. The polymers may be used singly or in combina of linear and branched chain alkyl and aryl, base-stable pro tions, such as in the form of copolymers or block polymers. tecting groups, and R X-(CH), Ola (CH), . Exemplary polymers include linear or branched polyalkylene 0021. In another aspect, the present invention provides oxides, or derivatives thereof, comprising from about 2 to 10 sequence-specific nucleobase polymers that have been monomer units. Typical derivatives include, for example, mobility-modified with the mobility-modifying phosphora those in which the terminal hydroxyl is replaced with a sul midite reagents of the invention, either alone or in combina fanyl group or an amino group. A useful polyalkylene oxide tion with conventional mobility-modified reagents. Such polymer is polyethylene glycol. The polymer may optionally sequence-specific mobility-modified nucleobase polymers include a label or other reporter group or molecule, a protect ing group for protecting the mobility-modified nucleobase generally comprise a mobility-modifying polymeric segment polymer during Subsequent synthesis reactions, or other and a sequence-specific nucleobase segment. groups for, e.g., binding the mobility-modified nucleobase 0022. The nucleobase polymer segment is typically an polymer to other moieties or chemical species, such as, e.g., oligonucleotide such as a DNA oligomer oran RNA oligo ligands, etc. mer, but may also be any of a number of different analogs or 0012. As discussed above, the group protecting the phos derivatives of DNA and/or RNA, as will be described in more phoramidite oxygen atom of the reagents of the invention is detail in a later section. The nucleobase polymer has a stable to the basic conditions used to deprotect and/or cleave sequence of nucleobases that is at least partially complemen synthetic nucleobase polymers such as oligonucleotides. tary to a desired nucleotide sequence of a target nucleic acid Thus, the oxygen protecting group should generally be stable Such that the nucleobase polymer segment specifically binds to treatment with ammonium hydroxide at a temperature of the target sequence, under specified conditions. 55° C. for a period of about 18 hrs. Of course, if milder 0023 The mobility-modifying polymer segment has a deprotection and/or cleavage conditions are used, the oxygen ratio of charge-to-translational frictional drag that is different protecting group need only be stable to these milder condi from that of the nucleobase polymer segment in a given tions. Groups stable to Such basic conditions that can be used electrophoretic medium. Consequently by virtue of the to protect the oxygen atom of the phosphoramidite reagents mobility-modifying polymer segment, the mobility-modified of the invention will be apparent to those of skill in the art, and include by way of example and not limitation, alkyls com sequence-specific nucleobase polymers of the invention have prising at least two carbon atoms, aryls and (R),Si- where electrophoretic mobilities that are retarded as compared with each R is independently selected from the group consisting those of the corresponding unmodified nucleobase polymer. of linear and branched chain alkyl and aryl. Alternatively, the 0024. According to one illustrative embodiment of the oxygen protecting group may be a polymer segment, option invention, the mobility-modified sequence-specific nucleo ally having a selectively removable terminal protecting group base polymer is a compound according to structural formula as described above. In this latter embodiment, the reagents of (II): US 2011/0160082 A1 Jun. 30, 2011

of structural formulae (III), at least some and/or Rare other than hydrogen. Where R'' and R are other than hydrogen, (II) R'' and/or Rare R. O 0036. In another embodiment, the present invention pro vides compositions comprising a plurality of mobility-modi R-X--(CH2) -O-(CH2) O-P-O-OLIGO fied sequence-specific nucleobase polymers of the invention, wherein, in certain embodiments, each said mobility-modi OR2 fied nucleobase polymer has a structure independently selected from the group consisting of structural formulae (II) 0025 or a salt thereof, wherein: and (III). At least two of the mobility-modified nucleobase I0026 R. R. X., a, and b are as in Formula (I); and polymers of the plurality has a distinctive ratio of charge to 0027 OLIGO is a sequence-specific nucleobase poly translational frictional drag Such that each of the two or more mer comprising at least five nucleobases. mobility-modified nucleobase polymers has a distinct elec 0028. The mobility-modifying polymer can be attached to trophoretic mobility. The distinctive ratios of charge to trans the 5'-end, the 3'-end, or both the 5'-end and the 3'-end of the lational frictional drag may be due to differences in the OLIGO. The mobility-modifying polymer can also be lengths (i.e., the number of monomer units) of the mobility attached to the 5'-end of a first nucleobase polymer and to the modifying polymer segment of the molecule, differences in 3'-end of a second nucleobase polymer, thereby providing a the number of mobility-modifying polymer segments mobility-modified oligonucleobase polymer having the attached to the nucleobase polymer (i.e., differences in: Vari mobility-modifying polymer segment linking two nucleo abled instructural formulae (II) and (III)), the charges linking base polymer segments. multiple mobility-modifying polymer segments, the number 0029. By virtue of substituent R in the mobility-modified of charged versus uncharged subunits, the length and charge nucleobase polymers according to Formula (II), the illus of the nucleobase polymer, or a combination of these features. trated phosphate triester is uncharged at physiological pH. 0037. In yet another aspect, the present invention provides The illustrated mobility-modifying polymeric group, there a method of detecting a plurality of nucleotide sequences fore, would add only mass, and not charge, to the OLIGO. within one or more target nucleic acids. According to the 0030 The mobility-modified nucleobase polymer of the method, a plurality of mobility-modified sequence-specific invention are not limited to those including only uncharged nucleobase polymer probes, each of which optionally has a phosphate triester linkages. By judiciously selecting combi structure independently selected from the group consisting of nations of uncharged phosphate triester linkages and charged structural formula (II) and (III), is contacted with one or more phosphate diester linkages, the repertoire of available mobil target nucleic acids, generally under conditions that distin ity-modifications can be dramatically increased. Accord guish those mobility-modified probes that hybridize to the ingly, in a second illustrative embodiment, the mobility target nucleic acid in a base-specific manner from those that modified sequence-specific nucleobase polymer is a do not. The mobility-modified nucleobase polymer probes compound according to structural formula (III): that hybridize to the target are then fractionated by electro phoresis. The presence of selected sequence(s) in the target nucleic, acid is detected according to the observed electro (III) phoretic migration rates of the mobility-modified nucleobase O polymer probes, or, optionally, according to the identity of a label or by a combination thereof. R3 X-(CH-OH,-(CH-O--O OLIGO 0038. The mobility-modified nucleobase polymer probes OR O d may be either labeled or unlabeled. Alternatively, they may be modified to include a label during the method, as well be described more fully below. When unlabeled, the electro 0031 or a salt thereof, wherein: phoretic migration rates of the mobility-modified probes may be monitored by conventional means, for example by absor bance spectroscopy. When labeled, for example with a fluo O rophore, the electrophoretic migration rates may be moni tored by detecting the label. R3 is R x-cha-ol-cha-0--0 0039. In one embodiment of the present invention, in order OR4 d to facilitate detection of the mobility-modified probes in a multiplex assay, the mobility-modified probes are labeled are labeled with different fluorescent labels such as, but not lim 0032. R. R., a, b, X, and OLIGO are as previously ited to, 5-carboxyfluorescein (5-FAM), 6-carboxy-fluores defined in Formulae (I) and (II): cein (6-FAM), 2,7-dimethoxy-4',5'-dichloro-6-carboxyfluo 0033 each d is independently an integer from 1 to 200; rescein (JOE), N.N.N' N-tetramethyl-6-carboxy and rhodamine (TAMPA), 6-carboxy-X-rhodamine (ROX), 4.7, 0034 each R and each R' is independently selected 2',4',5',7-hexachloro-6-carboxy-fluorescein (HEX-1), 4.7.2", from the group consisting of hydrogen and R, with the 4.5',7-hexachloro-5-carboxy-fluorescein (HEX-2), 2',4',5'. proviso that at least one R' or at least one R is other 7'-tetrachloro-5-carboxy-fluorescein (ZOE), 4.7.2.7'- than hydrogen. tetrachloro-6-carboxy-fluorescein (TET-1), 1',2,7,8'- 0035. In the compounds of structural formula (III), eacha, dibenzo-4,7-dichloro-5-carboxyfluorescein (NAN-2), and b, d, X, R'' and R may, independently of one another, be the 1,2,7,8-dibenzo-4,7-dichloro-6-carboxyfluorescein. Guid same or different. In certain embodiments of the compounds ance for selecting appropriate fluorescent labels can be found US 2011/0160082 A1 Jun. 30, 2011 in Smithetal. (1987) Meth. Enzymol. 155:260-301, Kargeret 0043. This cycle of hybridization, joining, and denatur al. (1991) Nucl. Acids Res. 19:4955-4962, Haugland (1989) ation, may be repeated in order to amplify the concentration Handbook of Fluorescent Probes and Research Chemicals of the ligation product formed. In this instance, the joining (Molecular Probes, Inc., Eugene, Ore.). Exemplary fluores may be accomplished by means of a thermostable ligase. cent labels include fluorescein and derivatives thereof, such Furthermore, additional nucleobase polymer probes, which as those disclosed in U.S. Pat. No. 4,318,846 to Khanna et al. together are Sufficiently complementary to the ligated prod and Lee et al. (1989) Cytometry 10:151-164, and 6-FAM, uct to hybridize thereto and be covalently joined to one JOE, TAMA, ROX, HEX-1, HEX-2, ZOE, TET-1 or NAN-2, another as above, are also included, thereby affording geo as described above, and the like. When a plurality of fluores metric amplification of the ligated product, i.e., a ligase chain cent dyes are employed, they should, in many cases be spec reaction. The product of such aligase chain reaction therefore trally resolvable. is a double stranded molecule consisting of two strands, each 0040. In one convenient embodiment of the method, the of which is the product of the joining of at least two sequence target nucleic acid(s) are immobilized on a Solid Support. specific nucleobase polymer probes. Accordingly, in yet Following hybridization, unhybridized probes are removed, another aspect of the present invention, at least one of the typically by washing, and the hybridized probes are recov sequence-specific nucleobase polymers incorporated within ered, typically by denaturing the hybrids, and those recovered the ligase chain reaction product comprises a detectable label, hybridized probes are fractionated by electrophoresis as and at one of the sequence-specific nucleobase polymers is a described above. mobility-modified sequence-specific nucleobase polymer 0041. In another embodiment of the method, mobility selected from the group consisting of structural formula (II) modified probes that specifically hybridize to the target and (III) such that at least one strand of the ligase chain nucleic acid are modified to incorporate a label. The modifi reaction product has a distinctive ratio of charge to transla cation may be accomplished in numerous different ways, see, tional frictional drag. for example, U.S. Pat. Nos. 5,807,682, 5,703.222, and 5,470, 0044. In a second general method, the modification is 705, which disclose methods and compositions useful for the achieved via a template-directed fill-in reaction or via PCR. In selective modification of probes when bound to a target this aspect, a target nucleic acid, which may be in Solution or nucleic acid in a base-specific manner. In one general method immobilized, is contacted with a plurality of sequence-spe a second sequence-specific nucleobase polymer probe which cific nucleobase polymers probes, two of which hybridize to includes a detectable label is covalently joined to the hybrid opposite ends of complementary Strands flanking a nucle ized mobility-modified probe. This second labeled probe may otide sequence of interest within the target nucleic acid. also optionally include a mobility-modifying polymer. The Repeated cycles of extension of the hybridized sequence two probes can be covalently joined to one another when they specific probes, optionally by a thermostable polymerase, both adjacently hybridize to the same target nucleic acid thermal denaturation and dissociation of the extended prod molecule (i.e., the probes have confronting terminal nucleo uct, and annealing, provide a geometric amplification of the base residues that basepair with adjacent bases of the target region bracketed by the two nucleobase polymer probes. The nucleic acid). The covalent joining may be accomplished by product of Such a polymerase chain reaction therefore is a chemical means or biological means, such as by a DNA or double stranded molecule consisting of two strands, each of RNA ligase. which comprises a sequence-specific nucleobase polymer 0042. Thus, according to this aspect of the invention, the probe. In this aspect of the present invention, at least one of target nucleic acid(s), which may be in Solution or immobi the sequence-specific nucleobase polymer probes is a mobil lized, are contacted with a first plurality of mobility-modified ity-modified sequence-specific nucleobase polymer probe sequence-specific nucleobase polymer probes according to according to the invention, optionally a probe selected from the invention, each of which has a distinctive ratio of charge the group consisting of structural formula (II) and (III). Such to translational frictional drag and is, optionally, selected that the double stranded polymerase chain reaction product from the group consisting of structural formula (II) and (III), has a distinctive ratio of charge to translational frictional drag. and a second, labeled sequence-specific nucleobase polymer The polymerase chain reaction product formed in this aspect probe, generally under conditions that distinguish those of the invention may further comprise a label, which may be probes that hybridize to the target in a sequence-specific incorporated within either of the sequence-specific nucleo manner. Probes that adjacently hybridize to the same target base polymer probes used as primers, or it may be incorpo nucleic acid molecule are then covalently joined together rated within the substrate deoxyribonucleoside triphosphates (ligated) to form a mobility-modified labeled ligation prod used by the polymerizing enzyme. In other instances, the uct. Each labeled ligation product has a distinctive ratio of polymerase chain reaction product may be labeled by inter charge to translational frictional drag. In a further aspect, calation with an intercalating dye or by other non-covalent three or more nucleobase polymer probes are hybridized to association with a detectable indicator molecule. In yet adjacent sequences of a target nucleic acid in Such a manner another aspect, the polymerase chain reaction product formed that at least three probes can be covalently joined to form a is analyzed under denaturing conditions, providing separated ligation product, wherein at least one of the probes so joined single Stranded products. In this aspect, at least one of the comprises a detectable label, and at least one of the probes so single stranded products comprises both a label and a mobil joined is a mobility-modified sequence-specific nucleobase ity-modified sequence-specific nucleobase polymer of the polymer probe, optionally selected from the group consisting invention, optionally selected from the group consisting of of structural formula (II) and (III) such that the ligation prod structural formula (II) and (III) such that the single stranded uct bears a label and has a distinctive ratio of charge transla product derived from double stranded polymerase chain reac tional frictional drag. The labeled ligation products, which are tion product has a distinctive ratio of charge to translational hybridized to the target nucleic acid, are recovered and frac frictional drag. As is well known in the art, such a single tioned by electrophoresis, as described above. Stranded product may also be generated by carrying out the US 2011/0160082 A1 Jun. 30, 2011

PCR reaction with limiting amounts of one of the two 0049. In a further aspect of the invader assay, for example, sequence-specific nucleobase polymer probes used as a the downstream nucleobase polymer, which carries a label primer. and a first mobility-modifying polymer of the present inven 0045. In a third general embodiment, bound mobility tion attached to the 5'-terminus, further comprises a second mobility-modifying polymer attached to the 3'-terminus. The modified probes are reacted with reporter-labeled nucleotide presence of the second mobility-modifying polymer triphosphate molecules, in the presence of a DNA polymerase increases the sensitivity of the invader assay by enhancing the to attach reporter groups, which include but are not limited to difference between the electrophoretic mobility of the flap radioactive and fluorescent moieties, to the 3' end of the endonuclease generated product, comprising the 5'-terminus, probes. label, and first mobility-modifying polymer, and the electro 0046. In a fourth general embodiment, each mobility phoretic mobility of the uncleaved downstream nucleobase modified probe includes a sequence that may be enzymati polymer. Accordingly, the second mobility-modifying poly cally cleaved when the probe is bound to a target nucleic acid. mer has a molecular weight of at least 2000. In other embodi The cleavage reaction may remove a portion of the nucleo ments, the second mobility-modifying polymer has a molecu base polymer segment to modify the probe's ratio of charge? lar weight of at least 5,000, at least 10,000, at least 20,000, translational frictional drag, or may separate a reporter label and at least 100,000. In one embodiment, the second mobil carried at one end of the probe from a polymer chain carried ity-modifying polymer is a mobility-modifying polymer of at the other end of the probe to modify the charge/transla the present invention, while in other embodiments, the second tional frictional drag of the portion carrying the reporter label. mobility-modifying polymer is a mobility-modifying poly One method for detecting Such events relies upon a process, mer of the art, which is, in one illustrative, non-limiting referred to as fluorescence energy resonance transfer (FRET), example, an uncharged mono methyl polyethyleneglycol in which energy is passed between a fluorophore donorandan polymer. Moreover, the second mobility-modifying polymer acceptor molecule. Therefore, in one aspect of this embodi may comprise a mixture of species of different molecular ment, the mobility-modified probe comprises two moieties, weight, provided that those species do not interfere substan separated by the cleavage site, which serve as photon donor tially with detection of the signal product, i.e., the flap endo and acceptor. Where the acceptor molecule is not a fluoro nuclease generated product, comprising the 5'-terminus, phore, the effect is the quenching of donor fluorescence. label, and first mobility-modifying polymer see Example 5, Cleavage of the bound mobility-modified probe bound to the below). target nucleic acid physically separates the donor and accep 0050. In a fifth general embodiment, bound mobility tor moieties and restores fluorescence by the donor moiety, modified probes are contacted with reporter molecules, which is readily, and sensitively detected. including but not limited to intercalating dyes, that bind in a 0047. In still another aspect of the fourth general embodi non-covalent manner to the duplex DNA structure formed ment, each mobility-modified probe, which includes a between the probe and target nucleic acids. Such reporter sequence that may be cleaved when the probe is bound to a molecules may form fluorescent complexes when bound to target nucleic acid, comprises a first mobility-modifying duplex DNA structures, or the non-covalently bound reporter polymer attached to the labeled terminus of the probe, which molecule may comprise, for example, a radioactive moiety or can be either the 5'-end or the 3'-end of the probe, and a second other detectable moiety, or a chemical group forming one mobility-modifying polymer attached to the unlabeled termi member of a cognate binding pair, thereby modifying those nus of the probe. This aspect of the present invention is mobility-modified probes that have bound to a target nucleic illustrated, in a non-limiting manner, by the use of the mobil acid. ity-modifying polymers of the present invention in “invader 0051. In yet another aspect, the present invention provides assays, which are SNP-identifying procedures based upon a method for separating target nucleic acid molecules, which flap endonuclease cleavage of structures formed by two over may comprise different numbers of nucleotide residues, but lapping nucleobase polymers that hybridize to a target nucleic nevertheless have substantially the same ratio of charge to acid (see e.g. Cooksey et al., 2000, Antimicrobial Agents and translational frictional drag. This method comprises contact Chemotherapy 44: 1296-1301). Such cleavage reactions ing a mixture of target nucleic acid molecules with a mobility release products corresponding to the 5'-terminal nucleobase modified sequence-specific nucleobase polymer, optionally (s) of the “downstream nucleobase polymer. Where those having a structure selected from the group consisting of struc cleavage products are labeled and can be separated from the tural formula (II) and (III); attaching the mobility-modified uncleaved nucleobase polymer, an invader assay can be used sequence-specific nucleobase polymer to Substantially all the to discriminate single base differences in, for example, target nucleic acids forming mobility-modified target nucleic genomic sequences or PCR-amplified genomic sequences. acids, thereby providing each target nucleic acid molecule 0048. Attachment of the mobility-modifying polymers of having the same number of nucleotide residues with a dis the present invention to the labeled 5'-terminus of the down tinctive ratio of charge to translation frictional drag, and stream nucleobase polymer used in an invader assay provides separating the mobility-modified target molecules. Gener cleavage products with distinctive charge to translational fric ally, the mobility-modified target molecules so formed are tional drag ratios. Accordingly, a plurality of SNP's are ana separated by electrophoresis, e.g. by capillary electrophore lyzed simultaneously using a plurality of sequence-specific sis, or by capillary electrophoresis in a non-sieving medium. downstream nucleobase polymers, wherein the sequence 0.052 A mixture of such target molecules is generated, in specific downstream nucleobase polymers comprise a mobil one embodiment, by chain termination or chemical cleavage ity-modifying polymer of the present invention attached to sequencing reactions, in which case the target nucleic acids the labeled 5'-terminus, such that the product generated by generally comprise at least one detectable label. Where the flap endonuclease cleavage at each SNP has a distinctive target molecules are generated by chain termination reac charge to translational frictional drag ratio. tions, those target molecules are primer extension products. US 2011/0160082 A1 Jun. 30, 2011

In this aspect, the primer extension products and the nucleobase polymers which is filled with a DNA polymerase sequence-specific mobility-modified nucleobase polymer are in the presence of at least one deoxyribonucleoside triphos further contacted under suitable conditions with a template phate Substrate, thereby forming an extended mobility-modi nucleic acid. The template nucleic acid has a 3'-region com fied sequence-specific nucleobase polymer. Upon filling of prising at least 4 nucleotide residues and a 5'-region compris this gap, the extended mobility-modified sequence-specific ing at least 4 nucleotide residues, wherein the 3'-region is nucleobase polymer is ligated to the 5'-terminal nucleotide complementary to 3'-terminal residues of the mobility-modi residue of the primer extension product to form a mobility fied sequence-specific nucleobase polymer, and wherein the modified primer extension product having a distinctive ratio 5'-region is complementary to 5'-terminal residues of each of of charge to translation frictional drag. The resulting product the primer extension products, such that the 3'-terminal resi may also contain a detectable label, which may be incorpo due of the mobility-modified sequence-specific nucleobase rated into one or more of the mobility-modified sequence polymer abuts the 5'-terminal residue of a primer extension specific primer, deoxyribonucleotide substrate(s). The product, when the mobility-modified nucleobase polymer detectable label, may be, but is not limited to, a radioactive and the primer extension product are hybridized to the tem label or a fluorescent label. plate nucleic acid. Therefore, in this embodiment, the tem 0054 The invention also includes kits useful for carrying plate nucleic acid is used to align the mobility-modified out the methods of the present invention. Kits of the invention sequence-specific nucleobase polymer and primer extension comprise one or more mobility-modified sequence-specific products under appropriate conditions, so that the hybridized, nucleobase polymers. The kits may also comprise a second aligned nucleobase polymers can be covalently joined to one nucleobase polymer, typically an oligonucleotide, which is another, optionally by enzymatic ligation. The product optionally mobility-modified, where the intended assay formed thereby comprises a mobility-modified sequence requires a second oligonucleotide; for example, kits for oli specific nucleobase polymer and a primer extension product, gonucleotide ligation assays and PCR analysis. Similarly, kits providing a mobility-modified primer extension product hav designed for ligase chain reaction amplification will further ing a distinctive ratio of charge to translational frictional drag. comprise at least two additional nucleobase polymers, which Generally, in this aspect of the invention the primer extension together are complementary to a diagnostic ligase reaction product will further comprise at least one detectable label. product. The kits further may also comprise treating reagents The detectable label, which may be incorporated into one or Such as restriction enzymes, DNA polymerases, RNases, mis more of the mobility-modified sequence-specific primer, match binding proteins, ligases, and exonucleases. Primer deoxyribonucleotide substrate(s) or dideoxyribonucleotide extension kits appropriate for sequencing or oligonucleotide Substrate(s), may be, as non-limiting examples, a radioactive extension assays for detecting single nucleotide polymor label or a fluorescent label. In one embodiment, the chain phisms, may further comprise nucleoside triphosphates and/ termination sequencing reaction comprises each of the four or chain terminating nucleotides. Therefore, components of dideoxyribonucleotide substrates, wherein each is labeled the kits of the present invention include one more sequence with a different fluorescent moiety and wherein each of the specific nucleobase polymers, one or more mobility-modified four different fluorescent moieties are spectrally resolvable sequence-specific nucleobase polymers, and/or one or more from one another. In other aspects of this embodiment, four nucleoside triphosphates and/or chain terminating nucle separate chain termination sequencing reactions are carried otides, wherein one or more of these components may com out, wherein each of those four reactions comprises a single prise a reporter label. The kit may also comprise reaction dideoxyribonucleotide substrate and a mobility-modified buffers for carrying out hybridizations and enzymatic treat sequence specific primer carrying a single, spectrally-resolv mentS. able fluorescent moiety. In this instance, the sequencing reac 0055. In another embodiment, the invention includes kits tions are terminated and combined to provide a mixture of comprising one or more of the mobility-modifying phos primer extension products each of which is terminated with a phoramidite reagents of present invention. One or more of the dideoxyribonucleotide residue and is labeled with a spec mobility-modifying phosphoramidite reagents, in Such kits, trally-resolvable fluorescent moiety that corresponds to that may further comprise one or more protecting groups, reporter dideoxyribonucleotide residue. molecules, or ligands. Such kits may also comprise one or 0053. In another aspect of this method, the template more solvents, reagents, or solid Surface-bound nucleobase nucleic acid employed has a 3'-region comprising at least 4 monomer for use in the synthesis of mobility-modified nucleotide residues, a 5'-region comprising at least 4 nucle sequence specific nucleobase polymers. otide residues, and a central region disposed between the 0056. The mobility-modified nucleobase polymers of the 3'-region and the 5'-region, wherein the 3'-region of the tem present invention provide one or more advantages over cur plate nucleic acid is complementary to 3'-terminal residues of rently available modified oligonucleotides, as follows. For the mobility-modified sequence-specific nucleobase poly example, synthesis of the mobility-modified nucleobase mer, and wherein the 5'-region of the template nucleic acid is polymers of the present invention is compatible with reagents complementary to 5'-terminal residues of each of the primer and methods employed in conventional automated instru extension products. In this instance, hybridization of the ments for DNA synthesis. Furthermore, when the mobility sequence-specific mobility-modified nucleobase polymer modified nucleobase polymers include only uncharged phos and a primer extension product to the template nucleic acid phate triester linkages. Such as nucleobase polymers molecule, provides a structure in which the 3'-terminal resi according to structural formulae (II) and (III), Substantially due of the mobility-modified sequence-specific nucleobase greater alterations of electrophoretic mobilities can be polymer is separated from the 5'-terminal nucleotide residue achieved as compared with the charged PEO modifiers in of a primer extension product by a nucleobase sequence cor current use. As illustrated in the working examples provided responding to the central region of the template nucleic acid. infra, there is a large difference in the electrophoretic mobili In this instance, a gap remains between the hybridized ties between mobility-modified nucleobase polymers of the US 2011/0160082 A1 Jun. 30, 2011

invention which differ by only a single mobility-modifying 0065 “Spectrally resolvable:” means, in reference to a set monomeric unit. As a consequence, the invention permits for of fluorescent dyes, that the fluorescence emission bands of greater mobility modifications than can be achieved using the respective dyes are sufficiently distinct, i.e., sufficiently conventional PEO modifiers. Significantly, electrophoretic non-overlapping, that the dyes, either alone or when conju mobility retardations of greater than 100 nucleotides can be gated to other molecules or Substances, are distinguishable readily achieved using standard DNA and RNA chemistries. from one another on the basis of their fluorescence signals 0057 Moreover, use of charged phosphate diester link using standard photodetection systems such as photodetec ages in combination with uncharged phosphate triester link tors employing a series of band pass filters and photomulti ages, such as the mobility-modified nucleobase polymers plier tubes, charged coupled devices (CCD), spectrographs, according to structural formulae (II) and (III), greatly etc., as exemplified by the systems described in U.S. Pat. Nos. increases the repertoire of available, resolvable mobility 4,230,558 and 4,811,218 or in Wheeless et al., 1985, Flow modifications. Thus, the present invention enables the ability Cytometry: Instrumentation and Data Analysis, pp. 21-76, to simultaneously analyze for greater numbers of target Academic Press, New York. Generally, all of the dyes com nucleic acid sequences than can be analyzed using currently prising a spectrally resolvable set of dyes are excitable by a available PEO modifiers. single light source. 0.066 “Nucleobase:” refers to a substituted or unsubsti 5. DETAILED DESCRIPTION OF THE tuted nitrogen-containing parent heteroaromatic ring of a PREFERRED EMBODIMENTS type that is commonly found in nucleic acids. Typically, but not necessarily, the nucleobase is capable of forming Watson 0058. The present invention provides nucleobase polymer Crick and/or Hoogsteen hydrogen bonds with an appropri functionalizing reagents and methods for the synthesis of ately complementary nucleobase. The nucleobases may be nucleobase polymer functionalizing reagents, as well as pro naturally occurring, such as the naturally-occurring encoding cedures for the polymerization of Such functionalizing nucleobases A, G, C, T and U, or they may be modified or reagents and for their attachment to nucleobase polymers. synthetic. Common modified or synthetic nucleobases 0059. The present invention also relates to novel mobility include 3-methyluracil, 5,6-dihydrouracil, 4-thiouracil, modified sequence-specific nucleobase polymers that com 5-bromouracil, 5-chlorouracil, 5-iodouracil, 6-dimethyl prise at least one mobility-modifying polymer attached to a amino purine, 6-methyl amino purine, 2-amino purine, 2.6- nucleobase polymer through one or more mobility-modify diamino purine, 6-amino-8-bromo purine, inosine, 5-methyl ing polymer Subunits connected through uncharged linkages. cytosine, 7-deazaadenine, and 7-deazaguanosine. Additional The mobility-modified sequence-specific nucleic acids and non-limiting examples of modified or synthetic nucleobases nucleobase polymers provide improved ratios of charge to of which the target nucleic acid may be composed can be translational frictional drag, allowing more effective electro found in Fasman, CRC PRACTICAL HANDBOOK OF BIOCHEM phoretic separation of individual nucleobase polymers within ISTRY AND McOLECULAR BIOLOGY, 1985, pp. 385-392: Beil a larger population of nucleic acids, in both sieving and non stein's Handbuch der Organischen Chemie, Springer Verlag, sieving electrophoretic media. Berlin and Chemical Abstracts, all of which provide refer 0060. The present invention also provides methods for the ences to publications describing the structures, properties and detection of nucleotide sequences within one or more target preparation of Such nucleobases. nucleic acids using the mobility-modified, sequence-specific 0067. As will be recognized by those of skill in the art, nucleobase polymers disclosed herein. many of the above-described modified or synthetic nucleo bases are capable of forming Watson-Crick base pairing inter 5.1 Abbreviations and Conventions actions with the naturally occurring encoding nucleobases A, 0061 The abbreviations used throughout the specification T. C. G and U. However, in certain embodiments of the and in the FIGS. to refer to the naturally occurring encoding invention, it may be desirable to include in a nucleobase nucleobases are conventional and areas follows: adenine (A), polymer synthetic nucleobases which are not capable of guanine (G), cytosine (C), thymine (T) and uracil (U). forming Watson-Crick base pairs with either the naturally 0062 Unless specified otherwise, nucleobase polymer occurring encoding nucleobases A, T, C, G, and U and/or sequences and/or target nucleic acid sequences that are rep common analogs thereof, but that are capable of forming resented as a series of one-letter abbreviations are presented non-standard (i.e., non-Watson-Crick) base pairs with one in the 5'-->3' direction. another. Nucleobases having these properties are referred to herein as “non-standard synthetic nucleobases. Examples of 5.2 Definitions Such non-standard synthetic nucleobases include, but are not limited to, iso-guanine (iso-G), iso-cytosine (iso-C), Xanthine 0063 As used herein, the following terms are intended to (X), kappa (K), nucleobase H, nucleobase J, nucleobase M have the following meanings: and nucleobase N (see U.S. Pat. No. 6,001,983). These non 0064) “Reporter, label,” “reporter label” “label” or “tag” standard synthetic nucleobases base-pair with one another to refers to a fluorophore, chromophore, radioisotope, chemilu form the following non-standard base pairs: iso-Ciso-G, minescent, spin label, or an enzyme, which causes a detect KX, HJ and MN. Each of these non-standard base pairs has able event or which allows direct detection of a labeled three hydrogen bonds. Additional non-standard synthetic nucleobase polymer probe by a Suitable detector, or a ligand nucleobases, as well as methods for their synthesis and meth or other first member of a cognate binding pair that can bind ods for incorporating them into nucleobase polymers are specifically and with high affinity to a detectable anti-ligand, found in U.S. Pat. Nos. 5,432,272, 5,965,364 and 6,001,983, anti-hapten, or other second member of a cognate binding the disclosures of which are incorporated herein by reference. pair, Such as, but not limited to, reporter-labeled avidin or a 0068 “Nucleobase polymer:” refers to a series of nucleo reporter-labeled antibody. bases that are connected to one another by linkages that US 2011/0160082 A1 Jun. 30, 2011 permit the linked polymer to hybridize by standard Watson group derived by the removal of one hydrogen atom from a Crick base pairs or non-standard base pairs to a target nucleic single carbon atom of a parent alkane, alkene or alkyne. acid having the complementary sequence of nucleobases, or Typical alkyl groups include, but are not limited to, methyl; that can hybridize to a duplex target nucleic acid to form a ethyls such as ethanyl, ethenyl, ethynyl; propyls such as pro triplex structure via Hoogsteen base pairing rules. A variety of pan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, nucleobase polymers capable of hybridizing to a complemen prop-1-en-2-yl, prop-2-en-1-yl, cycloprop-1-en-1-yl; cyclo tary nucleic acid are described in the art. All of these nucleo prop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls base polymers are within the scope of the invention. Such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-me Examples of such nucleobase polymers include native DNAS thyl-propan-2-yl, cyclobutan-1-yl, but-1-en-2-yl, 2-methyl and RNAs, as well as analogs of DNAs and RNAs. Common prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien analogs include, but are not limited, to DNAs and RNAs in which the respective 2'-deoxyribo- or ribo-nucleosides are 1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en connected by phosphonate linkages, phosphoramidate link 3-yl, cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl, but-1-yn-3-yl, ages, phosphorothioate linkages, phosphate triester linkages. but-3-yn-1-yl, etc.; and the like. Where specific levels of Nucleobase polymers also include molecules having posi saturation are intended, the nomenclature “alkanyl.” “alk tively charged Sugar-guanidyl interlinkages, such as those enyl and/or “alkynyl' is used. In certain embodiments, the described in U.S. Pat. No. 6,013,785 and U.S. Pat. No. 5,696, alkyl groups are (C-C) linear alkyl groups. Furthermore, as 253 (see also, Dagani, 1995, Chem. & Eng. News 4-5:1153; used herein, the term “lower alkyl refers to alkyl groups that Dempey et al., 1995, J. Am. Chem. Soc. 117:6140-6141). consist of from one to six carbon atoms, and, in certain Sugar-guanidyl analogs in which the Sugar is 2'-deoxyribose embodiments, lower alkyl groups are (C-C) linear alkyl. are referred to as "DNGs, whereas those in which the sugar 0074 Aryl:” refers to a monovalent aromatic hydrocar is ribose are referred to as “RNGs. bon group derived by the removal of one hydrogenatom from 0069. Examples of nucleobase polymers having a posi a single carbonatom of a parent aromatic ring system. Typical tively charged polyamide backbone with alkylamine side aryl groups include, but are not limited to, groups derived chains are described in U.S. Pat. No. 5,786,461; U.S. Pat. No. from aceanthrylene, acenaphthylene, acephenanthrylene, 5,766,855; U.S. Pat. No. 5,719,262; U.S. Pat. No. 5,539,082 anthracene, aZulene, benzene, chrysene, coronene, fluoran and WO 98/03542 (see also, Haaima et al., 1996, Angewandte thene, fluorene, hexacene, hexaphene, hexylene, as-indacene, Chemie Int’l Ed. in English 35:1939-1942: Lesnik et al., S-indacene, indane, indene, naphthalene, octacene, 1997, Nucleosid. Nucleotid. 16:1775-1779; D’Costa et al., octaphene, octalene, ovalene, penta-2,4-diene, pentacene, 1999, Org. Lett. 1:1513-1516 see also Nielson, 1999, Curr. pentalene, pentaphene, perylene, phenalene, phenanthrene, Opin. Biotechnol. 10:71-75). picene, pleiadene, pyrene, pyranthrene, rubicene, triph 0070 Nucleobase polymers having uncharged backbones enylene, trinaphthalene, and the like. In certain embodiments, have also been described in the art. For example, nucleobase polymers having uncharged polyamide backbones are the aryl group is (C-C) aryl, and, in certain embodiments described in WO 92/20702 and U.S. Pat. No. 5,539,082. (Cs-Co). In further embodiments, aryls are cyclopentadi Nucleobase polymers having uncharged morpholino-phos enyl, phenyl and naphthyl. phoramidate backbones are described in U.S. Pat. No. 5,698, 0075 "Capillary electrophoresis:” means electrophoresis 685, U.S. Pat. No. 5,470,974, U.S. Pat. No. 5,378,841 and in a capillary tube or channel, where the largest inner dimen U.S. Pat. No. 5,185,144 (see also, Wages et al., 1997, Bio sion of the channel cross-section, which need not be a circle, Techniques 23:1116-1121). is between about 25-500 microns, allowing efficient heat 0071. Additional nucleobase interlinkages which may dissipation throughout the separation medium, with conse comprise a nucleobase polymer include, but are not limited quently low thermal convection within the medium. to, peptide-based nucleic acid mimetic interlinkages (see, 0076 “Sieving matrix or sieving medium': refers to an e.g., U.S. Pat. No. 5,698,685), carbamate interlinkages (see, electrophoresis medium containing crosslinks or non e.g., Stirchak &, Summerton, 1987, J. Org. Chem. 52:4202), crosslinked polymers which create a network effective to amide interlinkages (see, e.g., Lebreton, 1994, Synlett. Feb retard migration of charged species in an electric field. ruary, 1994:137), methylhydroxylamine interlinkages (see, Examples of a sieving matrix are those based on cross-linked e.g., Vasseur et al., 1992, J. Am. Chem. Soc. 114:4006), polyacrylamide or agarose. A sieving medium may also com 3'-thioformacetal interlinkages (see, e.g., Jones et al., 1993, J. prise, as disclosed in U.S. Pat. No. 5,567,292, polylactams Org. Chem. 58:2983), sulfamate interlinkages (see, e.g., U.S. such as polyvinylpyrrolidone, N,N-disubstituted polyacryla Pat. No. 5,470.967), and linkages including locked nucleo mides and N-substituted polyacrylamides. side analogs (LNA), which include bicyclic and tricyclic 0077. “Non-sieving matrix or non-sieving medium: nucleoside and nucleotide analogs that may be incorporated refers to a liquid medium which is substantially free of a mesh into nucleobase polymers that are capable of forming or network of polymers which are effective to retard the sequence-specific duplex and triplex structures with single mobility of analytes. stranded and double stranded nucleic acids (see, e.g., WO (0078 “Distinctive electrophoretic mobility:” refers to the 99/14226). rate at which an analyte migrates in an electric field in a 0072 The nucleobase polymers may be composed wholly particular electrophoretic medium. A distinctive mobility of a single type of interlinkage, or may comprise combina refers to a distinctive electrophoretic mobility of the analyte, tions of different interlinkages. In certain embodiments, the as compared with electrophoretic mobility of all other detect nucleobase polymer will be a native DNA or RNA, or a able analytes present in the sample tested. common analog thereof. 0079. “Polymorphism or polymorphic sequences:” refers 0073. “Alkyl:” refers to a saturated or unsaturated, to a sequence present in a population which shows variation branched, straight-chain or cyclic monovalent hydrocarbon between members of the population. For example, the poly US 2011/0160082 A1 Jun. 30, 2011 morphisms may relate to single nucleotide differences (single I0088 each a is independently an integer from 1 to 6; and nucleotide polymorphisms: SNP) or differences is the num I0089 b is an integer from 0 to 40; ber of repeat sequences. 0090 RandR are each independently selected from the 0080 “Endonuclease:” refers to any enzyme which group consisting of C1-C alkyl, Cs-Co cycloalkyl, Co-Co aryl, and Co-C27 arylalkyl, and cleaves a nucleic acid internally. An endonuclease may act on 0091 R is selected from the group consisting of alkyl either single stranded or double Stranded nucleic acids. comprising at least two carbon atoms, aryl, (R)-Si- where 0081. “Short tandem nucleotide repeats:” refers to the col each R is independently selected from the group consisting lection of different, simple tandem repeats that are present of linear and branched chain alkyl and aryl, base-stable pro throughout the genome of many organisms. In a non-limiting tecting groups, and R X-(CH2), Ola-(CH2), . example, one set of repeated sequences has been character 0092. In certain embodiments, Rand Rare both isopro ized as those sequences having the general formula (AG pyl. Where dimers and/or polymers of the mobility-modify TC), where A, G, T, and C represent the four nucleotides ing phosphoramidite reagent are desired, R is, interalia, Hor and w, x, y, and Z represent number from 0 to 7, wherein the other reactive moiety. Moreover, such dimers and/or poly Sum ofw-X+y+Z ranges from 3 to 7 and n is the repeat number mers can comprise the reagents of the present invention either (Edwards, A. et al., DNA typing and genetic mapping with alone or with other mobility-modifying phosphoramidite trimeric and tetrameric tandem repeats, Am. J. Hum. Genet. reagents of the art. In many embodiments, R is not hydrogen, (1991) 49(4): 746-56; Caskey, C. T. et al., U.S. Pat. No. and in many embodiments R is a protecting group. When R 5,364,759). is a protecting group, for example, dimers or polymers of the 0082 “Ligand:” refers to a chemical moiety or structure mobility-modifying phosphoramidite reagent are formed by corresponding to one member of a cognate binding pair that is sequential addition of mobility-modifying phosphoramidite specifically recognized and bound in a stable complex by a reagent monomers using standard phosphoramidite synthesis second member of the cognate binding pair. Examples of such chemistry. As used herein, the phrase protecting group cognate binding pairs include, but are not limited to, biotin encompasses not only the conventional, Versatile, selectively avidin, and biotin-streptavidin. Other examples include phe cleavable protecting groups well known and widely used in nylboronic acid reagents and phenylboronic acid complex phosphoramidite chemistry and as disclosed below, but also ing reagents derived from aminosalicylic acid (see e.g., U.S. those alternative protecting groups that are not readily or Pat. No. 5,594,151). Therefore, as used herein, the term selectively removed by the procedures and conditions of ligand encompasses the term hapten, which refers to a chemi phosphoramidite chemistry. Such alternative protecting cal moiety or structure, for example digoxigenin, as one groups, particularly those groups resistant to removal under member of a cognate binding pair, where the second member basic conditions, are used in certain embodiments of the of the cognate binding pair is an element of the immune present invention. For example, in certain embodiments, an system, including but not limited to an intact antibody, a alternative protecting group, R is alkyl or other non-readily single chain antibody, or an antibody fragment. or non-selectively cleavable moiety, including, as non-liming 0083. “Translational frictional drag.:” refers to the mea examples, compounds of the formula CH-(CH2). O— Sure of a polymer's frictional drag as it moves through a —(CH2). O——(CH2). O—, in which a, and b are as defined, sieving or non-sieving medium. defined in Formula (I) above, and d is an integer in the range 0084) “Distinctive ratio of charge to translational fric of 0 to 5. tional drag:” refers to the distinctive electrophoretic mobility 0093. The mobility-modifying phosphoramidite reagents of a mobility-modified nucleobase polymer as compared to of the present invention are generally synthesized according other detectable nucleic acids or nucleobase polymers, both to Scheme I, using methods and reagents well known to those modified and unmodified, that are present within the sample skilled in the art, which provides, for example the illustrative analyzed. mobility-modifying phosphoramidite reagent of the inven tion (7). In Scheme (I), DMT represents dimethoxytrityl, iPr 5.3 Mobility-Modifying Phosphoramidite Reagents represents isopropyl and R and b are as previously defined for structural Formula (I). 0085. In one embodiment, the present invention provides mobility-modifying phosphoramidite reagents that are nucleobase polymer functionalizing reagents having a struc Scheme (I ture according to Formula (I): C NH(iPr) R2-OH P (I) P-Cl -- (iPr),N1 YNGP). He R6 1 3

3 -R R-X-(CH3), O-(CH), O V P O-R2 (iPr)N 1 n N(iPr) 5 wherein: I0086) R is selected from the group consisting of hydro DMTO -ha- N-n OH gen, protecting group, reporter molecule, and ligand; 6 0087 X is selected from the group consisting of O, S, NH, and NH C(O); US 2011/0160082 A1 Jun. 30, 2011 10

with (7) to provide a mobility-modified nucleobase polymer -continued with two molecules of (7) joined to the 5'-end of the mobility modified nucleobase polymer. Therefore, repetition of this pro-fa- O)-n OPCOR2 condensation reaction with (7) through n cycles of reagent N(iPr) addition, provides a mobility-modified nucleobase polymer 7 with n molecules of the mobility-modifying phosphoramidite reagent of the present invention attached to the 5'-end of the 0094) Referring to Scheme (I), a DMT protected polyeth nucleobase polymer. ylene oxide phosphoramidite reagent 7 that can be used in 0099. In another embodiment, the mobility-modifying connection with standard phosphoramidite DNA chemistry is phosphoramidite reagents of the present invention are added prepared. to the 3'-end of a nucleobase polymer, rather than, or in 0095. Initially, phosphorous trichloride 1 is reacted with addition to, the 5'-end of the nucleobase polymer to be modi diisopropylamine 2 in a solvent, for example toluene, to form fied. In one illustrative example of this embodiment, depicted bis(diisopropylamino)chlorophosphine ester 3. Subse in Scheme V, infra. In this synthetic scheme, a mobility quently, the tetraisopropylaminophosphine 3 is reacted with modifying phosphoramidite reagent of the present invention the hydroxyl group on alcohol 4 thereby generating R-bis (7), is condensed with the 5'-hydroxyl moiety of a nucleobase (diisopropylamino)phosphite ester 5. Addition of an appro residue, thymidine, which is attached to a solid Support, via, priately protected mobility-modifying polymer having a free the 3'-hydroxyl moiety of the nucleobase residue. The con hydroxyl, such as DMT protected polyethylene oxide 6, densation product (23) obtained is then oxidized to the phos along with an activator, for example tetrazole, gives rise to the phate triester (24), deprotected with mild acid to provide the phosphoramidite, reagent 7, an R-diisopropylaminophos free hydroxyl moiety of (25). The phosphate triesters synthe phite ester wherein the polymer is attached to the phospho sized are chiral compounds and both the Rand Senantiomers rous atom through an ester bond. are formed when synthesized according to the methods dis 0096. The phosphoramidite reagent 7 is suitable for use in closed herein. The racemic mixture of the phosphate triesters DNA synthesizers to couple the mobility-modifying polymer synthesized is used without separation into enantiometrically to a nascent nucleobase polymer. As described in Example 2 pure R and S forms. Condensation of (25) with a 5'-protected and illustrated in Scheme (II), infra. nucleotide phosphoramidite reagent (26), yields a dinucleo 0097. The mobility-modifying phosphoramidite reagents base intermediate having a mobility-modifying phosphora of the present invention are compatible with standard phos midite reagent of the present invention positioned between phoramidite synthetic schemes, and, therefore can be used the two nucleobase monomers (27). Repeated cycles of con with commercially available instruments for nucleobase densation with 5'-protected nucleotide phosphoramidite, fol polymer synthesis. In addition the mobility-modifying phos lowed by deprotection and cleavage from the Solid Support, phoramidite reagents of the present invention are readily provides a nucleobase polymer carrying a mobility-modify attached to either the 5'-end or the 3'-end of a nucleobase ing polymer at the 3'-end of that nucleobase polymer (29). As polymer. noted Supra, one or more of the mobility-modifying phos 0098. In one embodiment of the present invention, the phoramidite reagents of the present invention can be added to mobility-modifying phosphoramidite reagent of the present the nucleobase polymer either alone or in combination with invention is added to the 5'-end of a nucleobase polymer one or more mobility-modifying phosphoramidite reagents attached to a solid support (8), as depicted in Scheme II, infra. known in the art. Accordingly, one or more of the linking In this instance the illustrative mobility-modifying phos groups formed between mobility-modifying phosphoramid phoramidite reagent of the present invention (7) is condensed ite reagents or between a mobility-modifying phosphoramid with the free 5'-hydroxyl moiety of the surface-bound nucleo ite reagent and the nucleobase polymer can be a charged base polymer to yield the intermediate structure (9), which is phosphate diester bond. Furthermore, one or more of the then sequentially deprotected to remove base-labile and acid mobility-modifying reagents employed can be a branched labile protecting groups and to cleave the product from the compound, e.g. as in structure (21), provided it is not the Solid Support, yielding the mobility-modified nucleobase 5'-proximal moiety with which a 5'-protected nucleotide polymer (12). In this embodiment, the esterified moiety R is phosphoramidite is to be condensed. stable to each step in the above reaction scheme, and, there 0100. In another embodiment a mobility-modifying phos fore the phosphate triesterlinkage is uncharged. Moreover, as phoramidite reagent of the present invention is attached to a would be apparent to those skilled in the art, the surface readily-cleaved chemical link that is bonded to a solid support bound intermediate (10), can be treated with mild acid, Such as controlled pore glass or polystyrene. An example of dichloroacetic acid (DCA), to remove the DMT protecting Such a readily-cleaved chemical link bonded to a solid Sup group to provide a free hydroxyl moiety and then condensed port is compound 33:

33 O DMT- o-ha- O)-n4 --> N N1 Nopcs or Polystyrene Support O US 2011/0160082 A1 Jun. 30, 2011 11 the synthesis of 33 is described below:

Scheme (WI

DMT-C + to-ha- S-N4 OH a DMT-O -knu-S-n4 OH 30 31

O

O O

DMT-O -knu-S-n4 O OH O

O 32 |xc + H2N-CPG or polystyrene O H

N DMT - O -N-)-n4 O N1 Nepg or Polystyrene

33

0101. In this manner, nucleobase polymers carrying PEO phosphoramidite reagent depicted supra, which com mobility-modifying reagents at the 3'-end are assembled prises an esterified cyanoethyl moiety. As depicted in Scheme III, infra, the surface-bound nucleobase polymer intermediate without the additional nucleobase residue at the 3'-end of the (10), which comprises one molecule of a mobility-modifying molecule, as depicted in Scheme V and compound (29). How phosphoramidite reagent of the present invention, can be ever, the advantage of the general method depicted in Scheme treated with mild acid, e.g. 3% DCA, to remove the DMT V, is that nucleobase-bound solid Supports not only are com protecting group to provide the Surface-bound compound mercially available, but they are also provided in the form of (13) carrying a free hydroxyl moiety. Condensation of the pre-assembled cartridges, which are filled with a nucleotide PEO phosphoramidite reagent, (14), with surface-bound (13) bound Solid Support, that are compatible with automated provides a nucleobase polymer with two mobility-modifying nucleobase polymer synthesizing machines. reagents joined to the 5'-end of the nucleobase polymer. In this scheme, deprotection and cleavage of the product from 0102 The mobility-modifying phosphoramidite reagents the solid surface also results in removal of the esterified of the present invention yield uncharged phosphate triester cyanoethyl group of reagent (14). Therefore, in this embodi linkages when joined to either or both of the 5'-end and/or the ment, the linkage formed between the second mobility-modi 3'-end of a nucleobase polymer, as well as when joined to fying reagent (14) added and the mobility-modified nucleo another mobility-modifying phosphoramidite reagent of the base polymer, is a charged phosphate diester bond. present invention. Accordingly the esterifiedmoiety, i.e. Rof 0105. In a further embodiment, the mobility-modifying Formula I, and, in certain embodiments, R'' and/or R of phosphoramidite reagents of present invention encompass dendritic reagents comprising two esterified mobility-modi Formulae II and III, infra, will be stable to all steps of con fying groups attached to each phosphorous atom. An example ventional phosphoramidite chemistry. That is the esterified of Such a compound (21) and a general scheme for its syn moiety should be not be removed during, interalia, the depro thesis are depicted in Scheme IV, infra. As would be apparent tection steps, depicted, for example in Schemes III and V. to those skilled in the art, repeated cycles of condensation More specifically, the esterified moiety should be stable to the using reagent (21) would provide a geometric increase in the procedures and conditions required for deprotection of pro number of mobility-modifying phosphoramidite reagents tected amines and cleavage of the mobility-modified nucleo attached to a surface-bound nucleobase polymer, according base polymer from the Solid Support, Such that the linkage to the synthetic method depicted in Scheme IV, infra. It would between the mobility-modifying monomerunits and between also be apparent to one of skill in the art that a nucleobase a mobility-modifying monomer unit and the 3'-end or 5'-end polymer can be modified with one or more of the mobility of the nucleobase polymer is an uncharged phosphate ester. modifying phosphoramidite reagents (7), (14), and (21), gen (0103) Accordingly, when R of Formula I, and R'' and/or erally according to Scheme II, III, or V, infra, to provide a R" of Formulae II and III, infra, are alkyl, R', and R'' and/or series of mobility-modified nucleobases polymers, having Rare selected from the group consisting of alkyl comprising one or more branched and/or unbranched, charged and/or at least two carbon atoms, e.g. C-C linear alkyl. uncharged mobility-moth mg moieties. 0104. The mobility-modifying phosphoramidite reagents 5.4 Compositions of Mobility-Modified Nucleobase of the present invention may also be used in conjunction with Polymers other mobility-modifying phosphoramidite reagents that are 0106. In one aspect of the present invention, mobility known in the art, including, for example, the representative modifying polymer chains are attached to sequence-specific US 2011/0160082 A1 Jun. 30, 2011 nucleobase polymers by a linking group. Various polymers dimethoxytrityl (DMT), or with base labile group such as adaptable to the embodiment include, among others, polyox fluorenyl methyl chloroformate (Fmoc). ides, polyamides, polyimines, and polysaccharides. The com 0110 Protecting groups useful in the present invention, positions also embody polymer chains in the form of copoly encompass not only the conventional, Versatile, selectively mers or block polymers, such as polyethylene oxide and cleavable protecting groups well known and widely used in polyamine (see e.g. Vinogradov, S.V. (1998) Self assembly of phosphoramidite chemistry and as disclosed above, but also polyamine-poly(ethylene glycol) copolymers with phospho those alternative protecting groups that are not readily or rothioate nucleobase polymers, Bioconjugate Chem., 9: 805 selectively removed by the procedures and conditions of 12), having one or more uncharged linkers between monomer phosphoramidite chemistry. Such alternative protecting units. groups, particularly those groups resistant to removal under 0107. In one embodiment, mobility-modifying polymers basic conditions, are used in certain embodiments of the are polyoxides or polyethers. In this context, the term poly present invention. For example, in certain embodiments, as an oxide is used to denote polymers with oxygen atoms in the alternative protecting group, R is alkyl or other non-readily main chain, particularly those with monomer units of the type or non-selectively cleavable moiety, including, as non-liming O—(CH), where n is an integer selected from the range of examples, compounds of the formula CH-(CH2). O— 1 to 15, in certain embodiments n is selected from the range of —(CH), O——(CH), O—, in which a, and b are as 2 to 6, and in other embodiments, n=2, together with their defined in Formula (I) above, and c is an integer in the range derivatives. Linear polyoxides applicable to the composition of 0 to 5. include, for example, poly(methylene oxide), poly(ethylene 0111. Another aspect of the invention involves linking oxide), poly(trimethylene oxide) and poly(tetramethylene groups that attach the mobility-modifying polymer to the oxide). Branched polyoxides provide additional bases for sequence-specific nucleobase polymer. In a general embodi mobility-modification by, in Some cases, imparting to the ment, the group attaching the mobility-modifying polymer mobility-modified nucleobase polymer a translational fric chain to the nucleobase polymer comprises phosphate tri tional drag that is different than that provided by a linear ester, phosphonate, phosphoamidate, phosphothioester or polymer chain. Branched polymers, for example poly(propy phosphodithioate linkage. Phosphonate and phosphate tri lene oxide) which are appreciably soluble in aqueous sol ester linkages permit attachment of other chemical constitu vents, are used in certain embodiments. Other applicable ents to the phosphorous atom to effect further differences in branched polymers include poly(acetaldehyde), and poly the ratio of charge to translational frictional drag between (but-1-ene oxide). mobility-modified nucleobase polymers. Thus, one embodi 0108. In another embodiment, the mobility-modifying ment includes alkylphosphonate linkages, such a methyl polymer is a monodisperse linear polyoxide of polyethyl phosphonate. eneoxide (PEO) because of its high degree of solubility in a 0112. In a further embodiment, the linkage is a phosphate variety of aqueous and organic solvents. Moreover, chemistry triester, wherein the free ester has attached various chemical of poly(ethylene oxide) and methods of use for modifying groups so as to render the linker uncharged. Such as alkyls, chemical and biological compounds are well known in the art, functionalized alkyls, or polymers. When the chemical group (see e.g. Grossman, P. D. et al., U.S. Pat. No. 5,777,096: is an alkyl, the compound may be a linear or branched alkyl, Muller, W. et al. (1981) Polyethylene glycol derivatives of generally a lower alkyl group. Linear alkyls include, but are base and sequence-specific DNA ligands: DNA interaction not limited to, methyl, ethyl, propyl, or butyl groups, while and application for base specific separation of DNA frag branched alkyls include, but are not limited to, isopropyl or ments by gel electrophoresis, Nucleic Acids Res. 9: 95-119): tertbutyl groups. However the chemical groups attached to Maskos, U., (1992) Oligonucleotide hybridization on glass the free ester are generally limited to those groups that are Supports: a novel linker for oligonucleotide synthesis and stable to all steps of conventional phosphoramidite chemistry, hybridization properties of oligonucleotides synthesized in including deprotection steps and especially to the procedures situ, Nucleic Acids Res. 20:1679-84). Accordingly, those and conditions required for the deprotection of protected skilled in the art can readily vary the number of polyethylene amines, such that the resulting linkage is an uncharged phos units in the mobility-modifying polymer to impart distinctive phate triester. Therefore, when Such groups are alkyl, the ratio of charge to translational frictional drag to the mobility group is generally an alkyl other than methyl, for example, modified sequence-specific nucleobase polymer. C-C linear alkyl, since mono methyl phosphate triesters 0109. In addition, the mobility-modifying polymers of the tend to be less stable than higher-order alkyl phosphate tri embodiment may further comprise functional groups, such as esters. The alkyl group may also have attached functional a hydroxyl, Sulfhydral, amino or amide group. These func moieties, such as reporters, ligands or biotin molecules. tional groups permit attachment of various reporter mol Reporter molecules include but are not limited to fluorescent, ecules, ligands, or other polymer chains. Protecting groups chemiluminescent or bioluminescent molecules, while may be present on the functional group when the mobility ligands include, but are not limited to, molecules such as modifying polymer is being coupled to the sequence-specific cholesteryl, digoxigenin, 2.4 dinitrophenol, and biotin. When nucleobase polymer, or during reaction of other functional the chemical group is a polymer, the same types of polymers groups with the sequence-specific mobility-modified nucleo set forth above, including but not limited to polyoxides, base polymer. Groups suitable for protecting specific func polyamides, polyimines, polysaccharides, and polyure tional groups, including methods for removal, are well known thanes, function as Suitable Substituents. in the art Such that the art provides ample guidance for select 0113. The sequence-specific nucleobase polymers in one ing the appropriate protecting reagents (Fee e.g. Greene and embodiment of the invention are natural or synthetic. Natural Wuts, Protective Groups in Organic Synthesis, 2nd ed., John nucleic acids and oligonucleotides are obtained by cloning Wiley & Sons, Inc., New York, 1991). For example, hydroxyl the desired fragment in a cloning vector and isolating the groups are protectable with acid labile groups such as desired nucleic acid fragment by restriction enzyme diges US 2011/0160082 A1 Jun. 30, 2011 tion. Alternatively, they are made using oligonucleotide tem (PNA): Oligonucleotide analogues with an achiral peptide plates and enzymatic synthesis, Such as polymerase chain backbone, J. Am. Chem. Soc. 114: 1895-1876; Hanvey, J. C., reaction (Sambrook, J. et al., Molecular Cloning: A Labora (1992) Antisense and antigene properties of peptide nucleic tory Manual, Cold Spring Harbor Laboratory, New York, acids, Science 258: 1481-5; Nielson, P. E. et al., (1993) 2000). Sequence-specific inhibition of DNA restriction enzyme 0114. In another embodiment, the sequence-specific cleavage by PNA nucleic acids, Nucleic Acids Res. 21: 197 nucleobase polymers are synthetic nucleobase polymers. 200). Synthetic sequence-specific nucleobase polymers comprise deoxyribonucleic acids (DNA), ribonucleic acids (RNA) or 0119 Oligonucleotide and nucleobase polymer analogs composites of DNA and RNA. Further modification of the with phosphorothioate orphosphorodithioate internucleotide nucleobases of the deoxy or ribonucleic acids are possible. linkages have sulfur atom in place of the oxygen as the non Modified bases include inosine, deoxynapthosine, etheno bridging ligands bound to the phosphorous (Eckstein, F., adenosine and cytidine, and bromodeoxyuridine. Other (1985), Nucleoside phosphorthioates, Ann. Rev. Biochem., modified bases readily used in nucleobase polymer synthesis 54:317-402). Phosphorothioate diesters are chiral at the are 7-deaza purines, N° methyl adenosine, O' methyl gua phosphorous atom (Rp and Sp) and have utility as nucleobase nosine, and 2-aminopurine. polymer probes and antisense molecules because of their 0115. In another embodiment, the sequence-specific resistance to nucleases (Zon, G., Phosphorothioate oligo nucleic acids and nucleobase polymers are analogs, for nucleotides. In Oligonucleotides and Analogues: A practical example phosphonate nucleobase polymer, nucleobase poly approach, (Eckstein, F. ed) IRL Press, Oxford, pg. 87-108). mers comprising one or more locked nucleoside analogues, Phosphorodithioates have both the non-bridging ligands peptide nucleic acids (PNA), phosphorothioate nucleobase bound to the phosphorous as sulfur atoms (Caruthers, M. H. et polymers, phosphate triester nucleobase polymers, or nucleo al., (1992), Chemical synthesis of Deoxyoligonucleotides base polymers having chain terminating nucleosides. and Deoxyoligonucleotide analogs, Meth. Enzy. 211: 3-20). 0116 Phosphonate nucleobase polymers have a backbone 0.120. In another embodiment of the invention, the comprising phosphonate internucleotide linkages. The phos sequence-specific nucleobase polymer analogs comprise dif phonates known in the art of nucleobase polymer synthesis ferent combinations of internucleotide linkages. Thus a include H-phosphonate, alkylphosphonate (e.g. methylphos nucleobase polymer may comprise methylphosphonate, phonate), 2-aminoethylphosphonate, and benzylphosphonate phosphate diester, and N-(2-aminoethyl)-glycine internucle (Samstag, W., (1996) Synthesis and properties of new anti otide linkages (Miller, P. S. et al., (1999). A psoralen-conju sense nucleobase polymers containing benzylphosphonate gated triplex forming oligodeoxyribonucleotide containing linkages, Antisense Nucleic Acid Drug Dev. 6: 153-56: Fathi, alternating methylphosphonate phosphate diester linkages: R., (1994) nucleobase polymers with novel, cationic back synthesis and interactions with DNA, Bioconjugate Chem. bone constituents: aminoethylphosphonates, Nucleic Acids 10:572-577; Gildea, B. D. et al., U.S. Pat. No. 6,6063,569). Res. 22:5416-24: Seliger, H., (1990). Simultaneous synthesis Other sequence-specific nucleobase polymer analogs may of multiple nucleobase polymers using nucleoside-H-phos comprise a combination of phosphorothioate-phosphate phonate intermediates, DNA Cell Biol. 9: 691-6. Zhou, Y. diester internucleotide linkages (see e.g. Ghosh M. K. (1993), (1996) Solid-phase synthesis of oligo-2-pyrimidinone-2'- Phosphorothioate-phosphate diester oligonucleotide deoxyribonucleotides and oligo-2-pyrimidione-2'deoxyribo co-polymers: assessment for antisense, Anticancer Drug Des, side methylphosphonates, Nucleic Acids Res. 24; 2652 8(1): 15-32). Combinations of different internucleotide link 2659). The advantages of nucleobase polymers having ages offer nucleobase polymers with different hybridization phosphonate linkages are their property of readily forming characteristics and nuclease resistance. stable triple helix structures and their higher resistance to I0121. In another embodiment of the present invention, the nuclease action. mobility-modified sequence-specific nucleobase polymer 0117 Locked nucleoside analogues (LNA) include bicy comprises at least one non-negatively charged internucle clic and tricyclic nucleoside and nucleotide analogues that otide linkage. In one non-limiting example, at least one inter may be incorporated into nucleobase polymers that are nucleotide linkage of the mobility-modified sequence-spe capable of forming sequence-specific duplex and triplex cific nucleobase polymer is an uncharged mono alkyl structures with single stranded and double stranded nucleic phosphate triester linkage, while in another non-limiting acids. Those duplex and triplex structures that comprise example, the internucleotide linkage is a positively charged LNA-containing, sequence specific nucleobase polymers are amide linkage comprising an alkylamine side chain. In this more thermostable than the corresponding structures formed embodiment, the non-negatively charged internucleotide with non-analogue-containing nucleobase polymer mol linkage further alters the charge to translational frictional ecules (see e.g. WO99/14226). drag ratio of the mobility-modified sequence-specific nucleo 0118 Peptide nucleic acids (PNA) are synthetic polya base polymer of the present invention. In another non-limit mides which comprise repeating units of the amino acid, ing example, at least one internucleotide linkage of the mobil N-(2-aminoethyl)-glycine to which bases such as adenine, ity-modified sequence-specific nucleobase polymer is a cytosine, guanine, thymine are attached via the methylene phosphoramidate linkage, which is another non-negatively carbonyl group. Other bases including pseudo isocytosine, 5 charged internucleotide linkage that will alter the charge to methyl cytosine, pseudouracil, hypoxanthine are Suitable for translational frictional drag ratio of the mobility-modified incorporation into PNAS. The resistance of PNA nucleobase sequence-specific nucleobase polymer of the present inven polymers to nucleases and the high stability of PNA-DNA tion. Synthesis of oligonucleotides comprising a phrophora hybrids make them desirable probes for identifying target midate internucleotide linkage is described in U.S. Pat. No. polynucleotides, except in methods requiring nuclease treat 5,476.925, which is hereby incorporated by reference in its ments (Egholm, M., et al. (1992) Peptide nucleic acids entirety. US 2011/0160082 A1 Jun. 30, 2011

0122. In another embodiment the mobility-modified I0127 each R" is independently selected from the group sequence-specific nucleobase polymers are conjugated to consisting of hydrogen and R: various reporters, ligands and polymer molecules. All com ponents of the mobility-modified sequence-specific nucleic 0.128 each b is independently an integer from 0 to 40; acid or nucleobase polymer are amenable to conjugation, 0.129 each d is independently an integer from 1 to 200; including at internal or terminal nucleotide residues, the and phosphate triester group linking the polymer to the nucleo 0.130 OLIGO is a sequence-specific nucleobase poly base polymer, and the mobility-modifying polymer. Those mer, typically comprising at least 5 nucleobases, with skilled in the art are well versed in generating the appropriate modifications to form the desired conjugate (see e.g. Oligo the proviso that at least one R' or at least one R is other nucleotides and Analogs, F. Eckstein, ed., Chapter 8-9, IRL than hydrogen. Press, 1990; Agrawal, S. (1986), Efficient methods for attach I0131. Amongst the various mobility-modified nucleobase ing non-radioactive labels to the 5' ends of synthetic oligode polymers of structural formulae (II) and (III) are those com oxyribonucleotides, Nucleic Acids Res., 14: 6227-45; Nelson, pounds in which each X is O., eacha is the same (generally 2), (1992), Nucleic Acids Res., 20 (23): 6253-6259). When the each b is in the range of 1 to 15 and, and in certain embodi conjugation is to the polymer of the mobility-modified ments, b is in the range of 1 to 6, and the OLIGO is a DNA, nucleobase polymer, the polymer has appropriate functional groups such as hydroxyl, Sulfhydral, amide or amino groups RNA, and/or an analog of DNA or RNA, oligomer, each d is that permit attachment of one or more reporter, and ligand in the range of 1 to 200, in certain embodiments in the range molecules. When the conjugation is to a phosphate triester of 1 to 100, and further embodiments in the range of 1 to 50. linking group, the free ester provides the site of attachment. 0.132. In the compounds of structural formula (III) each 0123 Conjugating various reporter and ligand moieties R" and/or R' may, independently of one another, be a hydro permits detection, modification, or immobilization of the gen atom. When R'' and/or R are hydrogen, the resultant sequence-specific mobility-modified nucleobase polymer. phosphate ester group typically has a pKa in the range of 0 to They also permit further mobility-modification of the 1. Thus, when the pH of the assay conditions is above the pKa, sequence-specific mobility-modified nucleobase polymer. as is usually the case in biological assays, the hydrogenatom Reporter molecules include, but are not limited to fluorescent exchanges with solvent and at least, a net fraction of the compounds, such as fluoresceine, rhodamine, Texas red, cya phosphate ester groups are negatively charged. Due to the nine dyes, and acridine dyes. Ligands include, but are not ionizability of the phosphate ester group, those of skill in the limited to, 2,4 dinitrophenol, digoxigenin, and cholesterin, as art will appreciate that for purposes of defining the invention, well as enzymes, or enzyme Substrates that could be attached selecting R'' and/or R to be hydrogen includes within its to the sequence-specific mobility-modified nucleobase poly Scope both the unionized form and the ionized (i.e., nega mer. Conjugated ligands, including but not limited to biotin, tively charged) form of the resultant phosphate ester group. permit isolation, detection, or immobilization of the mobility 0.133 Similarly, other groups within the illustrated or modified nucleobase polymer by binding to avidin or strepta described compounds may be ionizable. Moreover, many of vidin. the compounds may include chiral centers or existin different 0124. In view of the embodiments set forth above, certain tautomeric or geometric isomeric forms. As any structural embodiments of sequence-specific mobility-modified drawings may represent only a single ionizable, tautomeric, nucleic acids or nucleobase polymers have a structure accord enantiomeric or geometric isometric forms, it will be under ing to structural formula (II) or (III): stood that the structural drawings are not intended to be limiting, and any non-illustrated, ionizable, tautomeric, enan tiomeric or geometric isomeric forms of the compounds are (II) O intended to be within the scope of the present invention. I0134. In the mobility-modified nucleobase polymers of R*-x-ECH-O-(CH-O--O-OLIGO structural formulae (II) and (III), the mobility-modifying seg ment of the molecule may be branched or linear. When linear, OR2 each R'' and R is either hydrogen (where possible), alkyl (III) comprising at least two carbon atoms, or aryl. When branched, at least one R' or R is R X(CH), O R3 X-(CH-OH,-(CH-O--O OLIGO (CH2)—. In these embodiments where an uncharged phos O phate esterlinkage is desired, the chemical groups attached to OR d the free ester are generally limited to those groups that are stable to all steps of conventional phosphoramidite chemistry, or a salt thereof, wherein: including deprotection steps and especially the procedures and basic conditions required for the deprotection of pro tected amines. Therefore, when such groups are alkyl, the O group is an alkyl other than methyl, for example, C-C linear alkyl, since methylphophotriesters tend to be less stable than R is R x-cha-ol-cha-0--0 higher-order alkyl phosphate triesters. 4 0.135 Identical or different mobility-modifying polymer OR d combinations can be used to provide the mobility-modified sequence-specific nucleobase polymer with distinctive, pre (0.125 R. R. X., a, and b are as in Formula (I); dictable mobility alterations. The sequence-specific nucleo I0126 each R' is independently selected from the base polymers comprise DNA, RNA, or analogs thereof, as group consisting of hydrogen and R: described above. The OLIGO may be labeled or unlabeled. US 2011/0160082 A1 Jun. 30, 2011

Those skilled in the art are well versed in devising nucleobase with acetic anhydride and 1-methylimidazole, to terminate polymer sequences useful for detecting a selected nucleic any nucleotide chains that failed to react. The internucleotide sequence within one or more target nucleic acids by the phosphite linkage is oxidized, with iodine as the preferred various methods described below. oxidizing agent, to the more stable phosphate triester. Fol lowing oxidation, the hydroxyl protecting group is removed 5.5 Methods of Synthesis with a weak acid, such as trichloroacetic and the cycle of 0136. The methods for synthesizing the nucleobase poly reactions is repeated until formation of a nucleobase polymer mer-functionalizing reagents of the present invention and for of the desired length and sequence is complete. Base treat synthesizing the various compositions of mobility-modified ment cleaves the nucleobase polymer from the Solid Support sequence-specific nucleobase polymers comprising those and also removes any phosphate protecting groups, such as functionalizing reagents, follow variations of known reaction B-cyanoethyl. Complete deprotection of the exocyclic amino schemes used for sequence-specific nucleobase polymer Syn groups on the nucleoside bases is accomplished by treating thesis and modification. As regards the mobility-modifying the nucleobase polymer in base at elevated temperatures, for polymers of the embodiments, methods of synthesizing Vari example 55° C. in concentrated ammonium hydroxide. The ous polymers are well known. Polymers suitable as mobility remaining protecting groups, usually dimethoxytrityl (DMT) modifiers include, among others, polyoxide, polyamide, groups on the 5'-hydroxyl, are removed during synthesis or, polyimine, and polysaccharides (see e.g. Molyneux, P. alternatively, may be left on if reverse phase HPLC is the Water-Soluble Synthetic Polymers. Properties and Behavior, purification method of choice. After synthesis, the nucleobase CRC Press, 1984; Gravert, D.J., (1977) Synthesis on soluble polymer is amenable to labeling at the 5' terminus (see e.g. polymers: new reactions and the construction of small mol Oligonucleotides and Analogs, F. Eckstein (1990), Ed. Chap ecules, Curr Opin Chem Biol. 1 (1): 107-13). Long chain ter 8, IRL Press; Orgel et al., (1983) Nucleic Acids Research alcohols, such as lipohilic Calcohols used as spacer arms to 11:6513: U.S. Pat. No. 5,118,800), the phosphate diester conjugate functional moieties, are also suitable as mobility internucleotide linkages (see e.g., Orgel et al., Supra at Chap modifiers. Functional groups, such as the hydroxyls on poly ter 9), or the 3 terminus (see e.g. Nelson, (1993), Nucleic oxides, are protected by an appropriate protecting agent, Such Acids Research 20:6253-6259). as 4,4'-dimethoxytrityl chloride. Groups suitable for protect 0140. In another embodiment, the nucleobase polymers ing specific functional groups, as well as methods for are analogs having modifications of the base, the Sugar, or the removal, are well known and will be apparent to those skilled backbone. Modifications in the backbone, include, but are not in the art. Guidance for selecting protecting reagents can be limited to polyamide (i.e. peptide nucleic acids), phospho found, for example, in Greene and Wuts, Protective Groups in nate, phosphorothioate, phosphodithioester, and phosphoa Organic Synthesis, 2nd ed., John Wiley & Sons, Inc., New midate internucleotide linkages. Methods of synthesizing York, 1991) sequence-specific nucleobase polymeranalogs with modified 0.137 The nucleobase polymers attached to the mobility internucleotide linkages are well known to those skilled in the modifying polymer chains are natural or synthetic nucleic art (see e.g. Oligonucleotides and Analogs, A Practical acids having defined nucleotide sequences. In one embodi Approach, Eckstein, F. Ed., IRL Press, (1990); Agrawal, S., ment the sequence-specific nucleobase polymer is a natural (1993), Protocols for Oligonucleotides and Analogs, Meth. DNA or RNA. Natural nucleic acids are readily prepared by Mol. Biol. Vol 20, Humana Press). cloning the desired fragment in a cloning vector and isolating 0141 Sequence-specific nucleobase polymers with polya the desired nucleic acid fragment by restriction enzyme mide backbones between nucleobases are also known as pep digestion of the recombinant molecule. Alternatively, they are tide nucleic acids (PNA), one example of which is a polymer made from nucleic acid templates by enzymatic synthesis, having repeating units of N-(2-aminoethyl)-glycine to which Such as polymerase chain reaction (Sambrook, J. et al., nucleobases are attached through a methylene carbony group Supra). (Nielson, P. E. (1991), Sequence-selective recognition of 0.138. In one embodiment, the nucleobase polymers are DNA by strand displacement with a thymine-substituted chemically synthesized using the phosphoramidite or phos polyamide. Science 254: 14971500). phate triester methods, either in solution or on a suitable inert 0142. As used herein, “PNA” refers to a polymer of solid support. Methods for nucleobase polymer synthesis are nucleobases linked together via an uncharged polyamide well know to those skilled in the art (see e.g. Caruthers et al. backbone. The PNA backbone may be any backbone of acy (1982) Genetic Engineering 4:1-17: Users Manual Model clic, achiral and neutral polyamide linkages to which nucleo 392 and 394 Polynucleotide Synthesizers, (1990), pages 6-1 bases can be attached and that satisfies the criteria discussed through 6-22, Applied Biosystems, Part No. 901237). supra. PNAS useful in the present methods are described, for 0.139. The phosphoramidite method is one method of syn example, in U.S. Pat. No. 5,539,082 and WO92/20702, the thesis for sequence-specific oligodeoxyribonucleic and oli disclosures of which are incorporated herein by reference. goribonucleic acids. In general, a protected nucleoside is The amino acids which form the polyamide backbone may be conjugated to a solid Support and then treated with acid, for identical or different, but are generally identical. In certain example trichloroacetic acid, to remove the 5'-hydroxyl pro embodiments, PNAS are those in which the nucleobases are tecting group, thus generating a free hydroxyl group for the attached to an N-(2-aminoethyl)-glycine backbone, i.e., a Subsequent coupling reaction. A protected nucleoside phos peptide-like, amide-linked unit (see, e.g., U.S. Pat. No. 5,719, phoramidite monomer and an activating reagent Such as tet 262; Buchardt et al., 1992, WO 92/20702; Nielsen et al., razole are reacted with the nucleoside bound to the solid 1991, Science 254: 1497-1500). Support. The activating agent protonates the nitrogen of the 0.143 Various strategies for PNA synthesis are available. phosphoramidite, permitting nucleophilic attack of the phos In one method, PNA monomers used for synthesis have the phorous atom by the exposed hydroxyl group. Following exocyclic amino groups protected by benzyloxycabonyl (Z) nucleoside addition, the growing chain is capped, generally while the amine is protected with tertbutyl oxycarbonyl US 2011/0160082 A1 Jun. 30, 2011

(tBoc). Protected monomer bound to a solid substrate is ylphosphonates utilizing the tert butylphenoxyacetyl group deprotected using a strong acid, such a trifluoroacetic acid for exocyclic amine protection, Nucleic Acids. Res. 22 (15): (TFA), to generate a free amino group for the Subsequent 3119-23; Hogrefe, R.I. etal. (1993), Animproved method for synthesis and deprotection of methylphonate oligonucle coupling to the next PNA monomer. A PNA monomer acti otides, Meth. Mol. Biol. 20: 143-63; Zhou, Y. (1996), Solid vated, for example by carbodiimides or O-(7azabenzotriazol phase synthesis of oligo-2-pyrimidone-2"deoxyribonucle 1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate otides and oligo-2-pyrimidone-2'-dexoriboside methylphos (HATU), reacts with the free amino group of the solid support phonates, Nucleic Acids. Res., 24:2652-2659). Alkyphospho bound PNA, to form the amide bond. Capping of the PNA namidites nucleoside reagents are prepared by chain is accomplished with acetic anhydride in presence of phosphitylation, which involves reacting alkyldichlorophos N-methylpyrrolidone and pyridine (see e.g. Koch, T., et al. phine. Such a methyldichlorophosphine, and diisopropy (1997) Improvements in automated PNA synthesis using lamine with the appropriately protected nucleoside, thereby Boc/Z monomers, J. Peptide Res. 4.2:80-88: Maison, W. forming the 5'-protected nucleoside diisopropylmethylphos (1991) Modified PNAS: A simple method for the synthesis of phonamidite. In the nucleobase polymer synthetic cycle, monomeric building blocks, Bioorg. Med. Chem. Lett., 9: monomer hound to the solid substrate is deprotected (i.e. 581-584). The completed nucleobase polymer is removed depixylated or detritylated) using acid, for example dichlo from the Solid Support with a strong acid, for example hydrof roacetic acid, and reacted with the alkylphosphonamidite luoric acid or trifluoromethane sulfonic acid. Other strategies nucleoside reagent in presence of an activating agent, such a for synthesizing PNAS include use of 9-fluorenylmethoxy tetrazole. The free OH group attacks the phosphorous atom carbonyl (Fmoc) or monomethoxytrityl protecting groups generating a methylphosphointe intermediate. Subsequent (Breipohl, G., et al., (1996), Synthesis of polyamide nucleic oxidation, which precedes the capping step, results in conver sion to the methylphosphonate linkage. Capping is accom acids (PNAS) using a novel Fmoc/Mmt protecting group plished with acetic anhydride and dimethylaminopyridine. combination, Bioorg. Med. Chem. Lett. 6:665-670: Will, D. Continued cycles of deprotection, coupling, oxidation and W. et al., The synthesis of polyamide nucleic acids using a capping generate the desired oligonucleotide. Release from novel monomethoxytrityl protecting group strategy, Tetrahe the solid support is affected by gentle deprotection, for dron, 51:12069-12082). Also available are PNA synthesis example by treatment with ethylenediamine or hybrazine methods that use acid labile backbone protecting groups and hydrate (see e.g. Hogrefe, R. I. An improved method for base labile protecting groups for the exocyclic amines on the synthesis and deprotection of methylphosphonate oligo nucleobases (Gildea, B. D., U.S. Pat. No. 6,063,569). nucleotides. In Protocols for Oligonucleotides and Analogs, 0144 Phosphonate nucleobase polymer analogs have Meth. Mol. Biol. Vol 20, Sudhir Agrawal, Ed., Humana Press, H-phosphonate or alkylphosphonate internucleotide link Inc. 1993). Other phosphonates, such as benzylphosphonates ages. Nucleoside hydrogen phosphonate monomers used for and aminoethylphosphonates, may also serve as internucle the synthetic cycle is Suitably protected by protecting groups otide linkages (Samstag, W. et al., Supra; Fathi, R., et al., normally used in the phosphoramidite synthetic scheme. The Supra). 5'-OH protecting group on the protected nucleoside bound to 0146 Phosphorothioate and phosphorodithioate sequence the Solid Support is removed with acid, Such as dichloroacetic nucleobase polymer analogs have Sulphur Substituted for acid. In the coupling step, the nucleoside phosphonate mono oxygen as the non-bridging ligands bonded to the phospho rous atom. Synthesis of sequence-specific nucleobase poly meris activated, for example by pivaloyl chloride, 1-adaman mers having phosphorothioate linkages is accomplished by a tane carbonyl chloride, or dipentafluororophenyl carbonate, variety of methods, including H-phosphonate or phosphora resulting in formation of an anyhydride that reacts with the midite chemistry. One exemplary method uses phosphora free hydroxyl group on the Solid Support bound nucleoside. midite route to synthesis which permits introduction of the Capping is dependent on the type of activating reagent: use of phosphorothioate linkage at any point in the nucleobase poly unhinderdactivating reagent, Such as paivaloyl chloride, may mer synthetic scheme. In this method, the Solid Support bound not require capping. Otherwise, capping is carried out with nucleoside, which is protected at the 5’ OH group with DMT, agents such as cyanoethyl-H-phosphonate or isopropyl-H- is deprotected and coupled to the incoming nucleoside phos phosphonate. Unlike the phosphoramidite method set forth phoramidite reagent in the presence of an activating agent, above, there is no oxidation of the phosphonate internucle Such as tetrazole. The 3-cyanoethyl phosphite linkage is con otide linkage. Subsequent base treatment results in release of Verted to the phosphorothioate with a sulfurization reagent, the completed nucleobase polymer from the solid Support and such as tetraethylthiuram disulfide (TETD), 3H-1,2-benzot removal of exocyclic protecting groups (see e.g. Seliger, H. dithiole-3-one-1,1-dioxide (Beacauge reagent), or dibenzoyl (1990), Simultaneous synthesis of multiple oligonucleotides tetrasulfide. Capping and release from Solid Support follows using nucleoside H-phosphonate intermediates, DNA Cell normal phosphoramidite chemistry procedures. Biol. 9 (9) 691-96. 0147 Dithioate analogs are prepared similarly to phos phorothioate except that a nucleoside 3' phosphorothioamid 0145 Nucleobase polymer analogs with alkylphospho ite is used. These synthons are generated, for example using a nate internucleotide linkages are readily synthesized by sev Suitably protected nucleoside and tris(pyrrolidino)phosphine eral methods (Oligonucleotides and Analogues, A Practical with a tetrazole activating agent. The resulting nucleoside Approach, Eckstein, F. Ed., IRE, Press, pp 137-154). By way diamidite is converted to the phosphorthioamidite with addi of example, one exemplary method uses alkylphosphonamid tion of 2,4 dichlorobenzylmercaptain (see e.g. Caruthers, M. ite chemistry for the synthesis reactions. In this method, the H., et al. (1992) Chemical Synthesis of Deoxyoligonucle nucleoside monomers used for the synthetic reactions have otides and Deoxyoligonucleotide analogs, Meth. Enzy. 211: the exocyclic amines protected with Suitable protecting 3-20). Reaction with deprotected, solid support bound groups, for example benzoyl, isobutyryl, or tert-butylphe nucleoside results in a nucleobase polymer with a thiophos noxyacetyl (t-BPA) groups, while the 5’ hydroxyls are pro phite triester linkage having the 2.4 dichlorobenzylmercap tected with a group Such as a pixyl or dimethoxytrity1. (Sinha, tan. Introduction of elemental sulfur yields the phospho N. D. et al. (1994), Synthesis of oligodeoxynucleoside meth rodithioate derivative. Deprotection is accomplished with US 2011/0160082 A1 Jun. 30, 2011 thiophenol, which removes the 2,4 dichlorobenzylmercaptan, 0150. In another aspect, the mobility-modified sequence while treatment with base releases the oligonucleotide from specific nucleobase polymers of the present invention com the Solid Support. prise modified bases, a plethora of which have been described in the literature. Methods for synthesizing protected, modi 0148 Another nucleobase polymer analog embodied in fied bases and their incorporation into nucleobase polymers the invention include nucleobase polymers with phosphate are well known in the art (see e.g. Connolly, B.A., Oligode triester linkages. Synthesis of phosphate triester analogs use oxynucleotides containing modified basis. In Oligonucle Suitably protected (for example, benzoyl, isobutyryl, or iso otide Analogs: A practical approach, Supra). Base analogs propoxyacetyl protecting groups) diisopropylphosphoramid incorporable into nucleic acids, include among others, deox ites. Alkyl groups include, but are not limited to, methyl, ynapthosine, etheno adenosine and cytidine, 6-thiogua ethyl, trifluoroethyl, isopropyl, and neopentyl. In these nosine, 4-thiothymidine, 7-deazapurines, N-methyladenos embodiments, the chemical groups attached to the free ester ine, O'-methylguanosine, and 2-aminopurine. 0151. In another embodiment of the present invention, the are generally limited to those groups that are stable to all steps linkages used to attach the mobility-modifying polymerchain of conventional phosphoramidite chemistry, including depro to the sequence-specific nucleobase polymer comprise vari tection steps and especially the procedures and conditions ous phosphoester analogs, other than phosphate diester link required for deprotection of protected amines, such that the ages, including, but not limited to phosphate triester, alky resulting linkage is an uncharged phophotriester. Therefore, lphosphonate, and phosphorothiate groups, or other Suitable when Such groups are alkyl, the group is, in certain embodi linkages. As set forth below, synthetic methods to generate ments, an alkyl other than methyl, for example, C-C linear various linkages can adopt synthetic strategies used in alkyl, since methylphophotriesters tend to be less stable than nucleobase polymer synthesis. higher-order alkyl phosphate triesters. Synthesis follows gen 0152. In one aspect, the linkage between the sequence eral phosphoramidite chemistry with variations in linkage to specific nucleobase polymer and the mobility-modifying polymer chain is an alkylphosphonate linkage. In one syn Solid Supports (e.g. oxalyl linker) and release from Solid Sup thetic scheme, methyldichlorophosphine is reacted with port following synthesis (e.g., 25% aq NH). Other nucleo diisopropylamine to form methylcholoro-N,N-diisopropy base polymer analogs embodied in the invention include laminophosphine. Addition of Suitably protected polymer, boranophosphates, and phosphofluoridate linkages. Methods such as DMT pentaethylene oxide, in the presence of diiso for their synthesis are described in Protocols for Oligonucle propylamine generates methylphosophonamidite derivative otides and Analogs, Meth. Mol. Biol. Vol 20, Chapters 11 and with the attached polymer. Coupling of methylphosphoam 12, S. Agrawal, Ed., 1993, Humana Press, Inc., which is idite derivative to a free hydroxyl group of a protected, solid hereby incorporated by reference. Support bound oligonucleotide occurs in the presence of acti 014.9 The sequence-specific nucleobase polymers Vating agent, such as tetrazole, resulting in a methylphospho embodied in the invention are not limited those with homo nite linkage. Subsequent oxidation, for example with iodine, geneous internucleotide linkages; sequence-specific nucleo results in conversion of the methylphosphonite to the meth base polymers comprising more than one type of internucle ylphosphonate. Other phosphonate linking groups, such as otide linkage are also encompassed by the present invention. benzylphosphonate, may be synthesized according to similar Thus, mobility-modified sequence-specific nucleobase poly chemistry used to generate benzylphosphonate oligonucle mers with combinations of phosphate diester, phosphate tri otides. ester, phosphorothioate, and alkylphosphonate internucleo 0153. In one embodiment, linking of the mobility-modi side linkages are all encompassed within the scope of the fying polymer to the sequence-specific nucleobase polymeris invention. Synthesis of sequence-specific nucleobase poly accomplished through a phosphate triester linkage. Various mers having various combinations of internucleoside link routes of synthesis are available to form phosphate triester ages are well known in the art, and references describing their linkages between the mobility-modifying polymer and synthesis are incorporated by reference herein (see e.g. Zhou, sequence-specific nucleobase polymer. In one embodiment, L. (1994), Synthesis of phosphorothioate-methylphospho attaching the polymer chain to the nucleobase polymer fol nate oligonucleotide co-polymers, Nucleic Acids Res. lows phosphoramidite chemistry, as described in detail in 22:453-456; Miller, P. S. (1999), Psoralen conjugated triplex Examples 1 and 2 and as illustrate Schemes (II) and (III), forming oligodeoxyribonucleotide containing alternating below with ethylene oxide mobility-modifying polymers. methylphosphonate-phosphate diester linkages: synthesis 0154) In Schemes (II) and (III), DMT represents and interactions with DNA, Bioconjugate Chem. dimethoxytrityl, iPr represents isopropyl and R and b are as 10:572-577) previously defined for structural formula (I).

Scheme (II

O 1nue-nu-N, ODMT ()-ATGCATGC-OH - 7I - o–AIGCAIGC-o-KOR2 8 US 2011/0160082 A1 Jun. 30, 2011

-continued O O Ho-AIGCAIGC-0--01-k O n1, opMr O-AIGCAIGC-0--01) 'N1,NoDMT OR2 OR2 11 10 " DCA O Ho-AIGCAIGC-0--01-k O N1,Nort OR2 12

(O155 Referring to Scheme (II), the R’ N,N-diisopropy higher-order mono alkyl phosphate triesters. The embodi laminophosphite 7 is reacted with the free 5’ hydroxyl of a ments of the invention also envision attachment of various nascent nucleobase polymer 8, thereby generating a nucleo modified alkyls having functional moieties, such as reporters, base polymer with a mobility-modifying polymer attached to and ligand molecules. For example, B-cyanoethoxy phos the 5'-terminus by an R-phosphite triester linkage. Subse phoramidites with conjugated functional moieties, including quent oxidation with iodine and deprotection with base con but not limited to biotin, psoralen, acridine dye, cholesterol, verts the phosphite linkage to the Rf-phosphate triester link fluoresceine, rhodamine, 2.4 dinitrophenol, tris-(2,2'-bipyri age. The resultant mobility-modified sequence-specific dine rutheium (II) chelate (TBR), and histidines are well nucleobase polymer 10 is cleaved off the solid support with known in the art (see e.g. Smith, T. H. (1999), Bifunctional base (e.g., NHOH, 55°C., 4 hr) to yield 11. Oligonucleotide Phosphoramidite Reagents for the Introduction of Histidyl 11 may be purified, for example by reverse-phase HPLC, and and Dihistidyl Residues into Oligonucleotides, Bioconjugate Chem., 10:647-652; Kenten, J. H. (1992), Improved electro the remaining trityl group removed with weak acid (e.g., 3% chemiluminescent label for DNA probe assays: rapid quanti dichloroacetic acid in CHCl) to yield, mobility-modified tative assays of HIV-1 polymerase chain reaction products, nucleobase polymer 12, which may be further purified, for Clin. Chem, 38:873-9). In general, these functional moieties example by chromatography on a PD 10 column. Alterna are coupled to the phosphoramidite by a hydroxyl group tively, the trityl group may be removed prior to cleavage from present on polyoxide or aliphatic spacer arms conjugated to the solid support and the resultant mobility-modified nucleo the derivatized moiety. Accordingly, the cognate alkyl phos base polymer purified by conventional means. phoramidite reagent having a derivatized, functional moiety 0156. As will be appreciated by those skilled in the art, and an attached mobility-modifying polymer can be readily when R is an alkyl, the alkyl phosphoramidite reagent used synthesized by those skilled in the art by adapting the Syn to couple the mobility-modifying polymer segment to the thetic scheme provided in the instant application. nucleobase polymer may have linear or branched alkyls of 0157. In some embodiments of the invention, multiple various lengths. Where the phosphate ester is to remain mobility-modifying polymers are attached to the nucleobase polymer. In one embodiment, the multiple mobility-modify uncharged, the chemical groups attached to the free ester are ing polymers are attached linearly to one another, either by generally limited to those groups that are stable to all steps of way of uncharged linkages Such as phosphate triesterlinkages conventional phosphoramidite chemistry, including depro (exemplified by the compounds of structural formula (II)), or tection steps and especially to the procedures and conditions by way of charged linkages such as negatively charged phos required for the deprotection of protected amines, such that phate ester linkages (exemplified by the compounds of struc the resulting linkage is an uncharged phosphate triester. tural formula (III)). A method for synthesizing these types of Therefore, when Such groups are alkyl, the group is an alkyl mobility-modified nucleobase polymer polymers using stan other than methyl, for example, C-C linear alkyl, since dard phosphoramidite DNA chemistry is illustrated in mono methyl phosphate triesters tend to be less stable than Scheme (III), below.

Scheme (III O O 1n-ka-1N 3%. DCA 1a-e-u1N AIGCAIGco--o OH -- AIGCAcco--o ODMT OR2 OR2 13 10

pro-fa- N-n OPC O N-1NN N(iPr) 14 o US 2011/0160082 A1 Jun. 30, 2011 19

-continued O 1-S-1N 1-S-1N Arcacco--o i o--o ODMT OR2 O N-1NcN 15 Itoi s DCA O O to Icaco--o 1nues-NO-P-O 1-S-NOH OR2 be 16

In Scheme (III), the various abbreviations and substituents are as defined for Schemes (I) and (II). 0158 Referring to Scheme (III), support-bound nascent Scheme (IV mobility-modified nucleobase polymer 10, which is synthe NH(iPr) C sized as illustrated in Scheme (II), is treated with a weak acid, 2 6 in this case 3% dichloroacetic acid in dichloromethane, to P-Cl remove the DMT protecting group, yielding support-bound (iPr)N N(iPr) compound 13. Support-bound compound 13 is coupled with 3 DMT-protected polyethylene oxide B-cyanoethyl phosphora midite reagent 14 in the presence of an activator Such as -knu- O)-n ODMT tetrazole (typically 0.5 M in acetonitrile). Phosphoramidite P reagent 14 may be prepared using standard syntheses. For (iPr),N1) NGP). example, phosphoramidite reagent 14 may be prepared according to Scheme (I) by Substituting 2-cyanoethan-1-ol for compound 4. Oxidation, such as by reaction with a solu tion of iodine in tetrahydrofuran, 2.6-lutidine and water, yields compound 15. Cleavage from the resin, removal of any groups protecting the nucleobases and removal of the DMT protecting group yields mobility-modified nucleobase poly mer 16. Additional polymers may be added by removing the DMT group from compound 15 and reacting it with phos phoramidite reagent 7 or 14. 0159. When the polymers are added sequentially, as for example in an automated DNA synthesis instrument, addition 0.161 Scheme (IV) is similar to Scheme (I), except that a of additional mobility-modifying polymer chains is accom suitably protected polymer, such as DMT protected poly plished by deprotecting the OH protecting group on the ter (alkylene oxide) 6 substitutes for alcohol 4, thereby forming minus of the mobility-modifying polymer chain and repeat bis(diisopropylamino)phosphite 20 having an attached poly ing the cycles of coupling and oxidation as set forth above. mer. Subsequent coupling of the second mobility-modifying Different mobility-modifying polymer chains may be added polymer 6 generates a phosphoramidite reagent 21 having at each cycle to provide mobility-modified sequence-specific two mobility-modifying polymer chains. Coupling to the free nucleobase polymers with distinctive ratios of charge to hydroxyl group of a Solid Support bound sequence-specific translational frictional drag. nucleobase polymer and Subsequent oxidation produces a 0160 Alternatively, the invention also contemplates branched mobility-modified sequence-specific nucleobase mobility-modified nucleobase polymers in which the poly polymer. meric segment is a branched or dendritic structure. An exem 0162. Further addition of mobility-modified polymers are plary method for synthesizing a phosphoramidite reagent that possible. The protecting group, for example DMT, on the can be used in conjunction with standard DNA synthesis mobility-modified polymers linked to the sequence specific chemistry to synthesize such branched and/or dendridic nucleobase polymer are removed with a weak acid, thus gen mobility-modified nucleobase polymers is illustrated in erating free hydroxyl groups. Reactions with derivatized Scheme (IV), below. In Scheme (IV), the various abbrevia N,N-diisopropylaminophosphite having an attached mobil tions and substituents are as defined for Schemes (I) and (II). ity-modifying polymer results in coupling of the additional US 2011/0160082 A1 Jun. 30, 2011 20 mobility-modifying polymer units to the free hydroxyl tional polymer units to the polymer protected with DMT groups. Coupling of any additional mobility-modifying poly (Iwai, S. (1988), 5'-Levulinyl and 2'-tetrahydrofuranyl pro merunits may be limited to only one of the sequence-specific tection for the synthesis of oligoribonucleotides, Nucleic nucleobase polymer linked mobility-modifying polymers if Acids Res, 16:9443-56). This orthogonal strategy removes the the other linked mobility-modifying polymer has a nonreac protecting groups under mutually exclusive conditions. tive terminus. Alternatively, protecting the hydroxyl group of 0163 The mobility-modifying phosphoramidite reagent one nucleobase polymer linked polymer with protecting of the present invention is also added to the 3'-end of a group that is stable to reagents used to remove DMT protect nucleobase polymer, according to Scheme V, which is ing groups, such as levulinyl, restricts coupling of any addi depicted below:

Scheme (W p-(CH),--O-(CH),--O-DMT 8-T-OH - - o-r-o-K OR2 23 o O Q-T-O--O-(CH)--O-(CH),--O-DMT OR2

24 | DCA

O

OR2

25

DMT-O DMT - O T

O O O

P NCCH2CH 1NNk-4- o-i-o-i-o-cis-to-cha-i-o- -N 27 26 o DMT-O T

O O o-i-o-i-o-cha-to-cha-0--0

28 US 2011/0160082 A1 Jun. 30, 2011

0164. According to Scheme V, the mobility-modifying 0.168. Therefore, the present invention also relates to a phosphoramidite reagent of the present invention is con mobility-modified sequence-specific nucleobase polymer densed with the free 5'-hydroxyl of a nucleobase monomer comprising a mobility-modifying polymer linked to the residue that is bound to a solid support (indicated in Scheme 3'-end of a first sequence-specific nucleobase polymer and to V as O), rather than the free 5'-hydroxyl of a nucleobase the 5'-end of a second sequence-specific nucleobase polymer polymer bound to a solid Support, as depicted in Scheme II. according to Structural formula (IV): The first step in Scheme V generates a mobility-modified phosphoramidite polymer bound to the Solid Support via a nucleobase monomer linker (24). Mild acid treatment of 24 (IV) cleaves the DMT protecting group, thereby providing a free hydroxyl moiety upon which a nucleobase polymer is assembled via repeated cycles of condensation of activated, OLIGO X-ECH-OH,-(CH-O--O OLIGO protected phosphoramidite nucleobase monomers using stan dard phosphoramidite chemistry, generally using an auto OR d mated instrument. For example, eight further cycles are car ried out, in which the following monomers are added, in (0169 or a salt thereof, wherein: order, A, T, G, C, A, T, G, and C. (0170 each R' is independently selected from the 0.165. After addition of the desired specific nucleobase group consisting of hydrogen, alkyl comprising at least polymer sequence to the Surface-bound mobility-modifying two carbon atoms, aryl, (R) Si where each R is polymer of the present invention, the mobility-modified independently selected from the group consisting of lin sequence specific nucleobase polymer, in which the mobility ear and branched chain alkyl and aryl, base-stable pro modifying polymer segment is carried at the 3'-end of the tecting groups, R X-(CH2), O-(CH2) , nucleobase polymer, is deprotected and cleaved from the protecting group, reporter molecule, and ligand, with the Solid Support, yielding, continuing with the above illustrative proviso that at least one R' is not hydrogen; synthesis, the product according to (29): 0171 each X is independently selected from the group consisting of O, S, NH and NH CO); 0172 each a is independently an integer from 1 to 6: 29 0173 each b is independently an integer from 0 to 40; (0174 d is an integer from 1 to 200; O (0175 OLIGO" is a first sequence-specific nucleobase polymer, and s-Ho-CGIACGIAT+(CH-0+(CH-0--0-1-3OR2 (0176) OLIGO is a second sequence-specific nucleo base polymer. (0177 OLIGO' and OLIGO are sequence-specific 0166 Alternatively, the substrate-bound molecule having nucleobase polymers, typically comprising at least 5 nucleo a mobility-modifying polymer attached to the 3'-end of a bases. Moreover, where desired, mobility-modifying poly nucleobase polymer, e.g. structure (28) of Scheme V, is sub mers of the present invention are also added to the 5'-end of jected only to a mild acid treatment, removing the 5'-DMT the first nucleobase polymer and/or to the 3'-end of the second moiety, and providing a free hydroxyl group on a Substrate nucleobase polymer generally according to Schemes V and II, bound nucleobase polymer structure, which would be analo respectively, in addition to the mobility-modifying polymer gous with (8) of Scheme II. Subsequent condensation with a positioned between the first and second nucleobase polymers, mobility-modifying phosphoramidite reagent of the present according to Structural formula V. invention would provide a sequence-specific nucleobase 0178. In yet another embodiment, the multiple mobility polymer having mobility-modifying polymer segments modifying polymerchains may be synthesized independently attached at both the 3'-end and the 5'-end. and then attached in a single step to the sequence-specific 0167. In another embodiment, a mobility-modified nucleobase polymer. Various schemes may be devised for nucleobase polymer is synthesized that comprises a mobility synthesizing the multiple mobility-modifying polymer modifying polymer attached to the 5'-end of a first nucleobase chains independently of the sequence-specific nucleobase polymer as well as to the 3'-end of a second nucleobase polymer. In one method, a mobility-modifying polymerchain polymer; that is the mobility-modifying polymer is “inserted such as pentaethylene oxide is protected with two different within a nucleobase polymer. In one non-limiting illustrative protecting groups, which are removable under mutually approach, a nucleobase polymer comprising a mobility exclusive deprotecting conditions. One such orthogonal strat modifying polymer is synthesized according to Scheme II to egy is to protect the polymer with dimethoxytrity1 at the provide intermediate (10), which comprises a first nucleobase hydroxyl group at one terminus and with levulinyl at the polymer attached to a solid Substrate and carrying a mobility hydroxyl group of the other terminus. Alternatively, the poly modifying polymer on the 5'-end of the first nucleobase poly mer is attached to a solid Support, analogous to attachment of mer. Treatment of (10) with mild acid (3% DCA) to remove nucleosides to Solid Supports for sequence-specific nucleo the DMT protecting group provides a structure, analogous to base polymer synthesis, through a bond that is resistant to compound (25), onto which a second nucleobase polymer is conditions of DMT deprotection. In either case, treatment added according to Scheme V, thereby providing a mobility with weak acid preferentially removes the DMT protecting modified nucleobase polymer that comprises a first and a group, thereby exposing a free OH, while the other hydroxyl second nucleobase polymer in which a mobility-modifying group remains protected. In the presence of an activating polymer is attached to the 5'-end of the first nucleobase poly agent, such as tetrazole, a derivatized phosphoramidite meras well as to the 3'-end of the second nucleobase polymer. reagent having an attached mobility-modifying polymer US 2011/0160082 A1 Jun. 30, 2011 22 reacts with the hydroxyl group, resulting in coupling of the 0182. In another aspect of this method, the target nucleic two polymers. Subsequent oxidation of the intermediate acid, which may be immobilized, is contacted with a plurality phosphite generates the 3-cyanoethylphosphate orphosphate of sequence-specific nucleobase polymer probes whereby triester linkage depending on the type of phosphoramidite two nucleobase polymer probes hybridize to adjacent reagent used for the coupling. Repeating the cycles of depro sequences of the target nucleic acid such that the 5'-end of one tection, coupling and oxidation generates linked mobility nucleobase polymer probe, which generally will carry a modifying polymer chains, wherein the multiple mobility 5'-phosphate moiety, abuts the 3'-end of the second nucleo modifying polymer chain is protected with DMT at one end base polymer probe, so that the two nucleobase polymer and the other end with levulinyl. Deprotection by removal of probes can be covalently joined to one another, in certain the levuliny with hydrazine, or cleavage from the solid sup embodiments, with a DNA chemical or enzymatic ligating port bound polymer, exposes a free hydroxyl, which is then activity, to form a ligated product. In this aspect of the available for synthesizing the derivatized phosphoramidite method, the ligated product is formed by the joining of two reagent, for example an alkyl-N,N-diisopropylaminophos nucleobase polymer probes, at least one of which comprises phite having an attached multiple mobility-modifying poly a detectable label and at least one of which is a mobility mer chain. Coupling of the derivatized phosphoramidite modified sequence-specific nucleobase polymer selected reagent to a suitably protected nucleobase polymer results in from the group consisting of Structural formulae (II) and (III), attachment of the multiple mobility-modifying polymer Such that the ligated product has a distinctive ratio of charge chain to the sequence specific nucleobase polymer. Subse to translational frictional drag. In a further aspect, three or quent oxidation with iodine followed by treatment with base more nucleobase polymer probes are hybridized to adjacent removes any protecting groups on the exocylic amines and sequences of a target nucleic acid in Such a manner that at B-cyanoethyl groups on the phosphates, with concomitant least three nucleobase polymer probes can be covalently release of the mobility-modified sequence specific nucleic joined to form a ligated product, wherein at least one of the acid or nucleobase polymer from the Solid Support. Treatment nucleobase polymer probes so joined comprises a detectable with weak acid removes any remaining MAT protecting label, and at one of the nucleobase polymer probes so joined group on the mobility-modifying polymer. is a mobility-modified sequence-specific nucleobase polymer selected from the group consisting of Structural formulae (II) 5.6 Methods of Use and (III) such that the ligated product has a distinctive ratio of 0179 The present invention further encompasses methods charge to translational frictional drag. Generally, the ligated of using mobility-modified sequence-specific nucleobase product, which is hybridized to the target nucleic acid, is polymers, as well as compositions comprising a plurality of released by denaturation, and the single stranded ligated mobility-modified sequence-specific nucleobase polymers, product having a distinctive ratio of charge to translational wherein each said mobility-modified nucleobase polymer frictional drag, is detected and analyzed, to provide informa optionally has a structure independently selected from the tion about the selected nucleotide sequence within the target group consisting of Structural formulae (II) and (III), and nucleic acid. wherein each mobility-modified nucleobase polymer has a 0183 This cycle of hybridization, joining, and denatur distinctive ratio of charge to translational frictional drag, to ation, may be repeated in order to amplify the concentration detect and characterize one or more selected nucleotide of the ligated product formed. In this instance, the joining is sequences within one or more target nucleic acids. optionally accomplished by means of a thermostable ligating 0180. In one aspect, the present invention provides a enzyme. These reactions are conveniently carried out in ther method of detecting a plurality of sequences within one or mal cycling machines with thermally stable ligases. (Barany, more target nucleic acids, comprising contacting a plurality F. (1991), Genetic disease detection and DNA amplification of mobility-modified sequence-specific nucleobase poly using cloned thermostable ligase, Proc. Natl. Acad. Sci. USA mers, wherein each mobility-modified nucleobase polymer 88 (1): 189-193; Housby J. N. et al. (2000), Optimised Liga has a structure independently selected from the group con tion of oligonucleotides by thermal ligases: comparison of T. sisting of Structural formulae (II) and (III), with one or more scOtoductus and Rhotothermus marinus DNA ligases to other target nucleic acids, generally under conditions that distin thermophilic ligases, Nuc. Acids Res. 28 (3): E10). guish those mobility-modified sequence-specific nucleobase 0.184 Furthermore, additional nucleobase polymers, polymers that hybridize to the target nucleic acid, and detect which together are sufficiently complementary to the ligated ing those mobility-modified sequence-specific nucleobase product to hybridize thereto and be covalently joined to one polymers which have hybridized to the target nucleic acid. another as above, are also included, thereby affording geo 0181. In one aspect of this method, the target nucleic acids metric amplification of the ligated product, i.e., a ligase chain are immobilized. In this aspect, the immobilized target reaction (Wu, D. Y. and Wallace B. (1989), The ligation nucleic acids are contacted with mobility-modified sequence amplification reaction (LAR)-Amplification of Specific DNA specific nucleobase polymer probes, which further comprise sequences using sequential Rounds of Template Dependent a detectable label, under conditions that distinguish those Ligation, Genomics 4:560-569; Barany, (1991), Proc. Natl. probes having Sufficient homology to hybridize to the target Acad. Sci. USA, 8:189; Barany, (1991), PCR Methods and nucleic acid. Non-hybridized probes are washed away and Applic. 1:5). To suppress unwanted ligation of blunted ended hybridized probes, which are bound to the target nucleic acid hybrids formed between complementary pairs of the mobil immobilized on the membrane, are detected. Alternatively, ity-modified and second oligonucleotides and the second pair the non-hybridized probes are washed away and hybridized of oligonucleotides, conditions and agents inhibiting blunted probes are recovered as single-stranded products after dena ended ligation, for example 200 mMNaCl and phosphate, are turation of the base-paired structure formed between the included in the ligation reaction. mobility-modified sequence-specific nucleobase polymer 0185. The product of such aligase chain reaction therefore probe and the immobilized target nucleic acid. is a double stranded molecule consisting of two strands, each US 2011/0160082 A1 Jun. 30, 2011 of which is the product of the joining of at least two sequence within a target nucleic acid that is immediately adjacent to the specific nucleobase polymer probes. Accordingly, in yet site of interest. A second sequence-specific nucleobase poly another aspect of the present invention, at least one of the mer is hybridized to the selected region within the target sequence-specific nucleobase polymers incorporated within nucleic acid such that the hybridized oligonucleotides are the ligase chain reaction product comprises a detectable label, separated by a gap of at least one nucleotide residue. In and at one of the sequence-specific nucleobase polymers is a another embodiment, the length of the gap is a single nucle mobility-modified sequence-specific nucleobase polymer otide residue representing a single polynucleotide polymor selected from the group consisting of Structural formulae (II) phism in the target nucleic acid. Following hybridization, the and (III) such that the ligase chain reaction product has a complex, which consists of the two nucleobase polymers distinctive ratio of charge to translational frictional drag. hybridized to the target nucleic acid, is treated with a nucleic 0186. In another aspect of the oligonucleotide ligase acid polymerase in the presence of at least one deoxyribo assays described above, mismatches, i.e. non-complementary nucleoside triphosphate. If the deoxyribonucleoside triphos nucleobases, existing between selected nucleotide sequences phate(s) provided are complementary to the target polynucle within the target nucleic acid and either or both of the mobil otide's nucleotide residues which define the gap, the ity-modified sequence-specific nucleobase polymer and the polymerase fills the gap between the two hybridized nucleo second oligonucleotide interfere with the ligation of the two base polymers. Subsequent treatment with ligase joins the nucleobase polymers either by preventing hybrid formation two hybridized oligonucleotides to form a ligated, mobility or preventing proper joining of the adjacent terminal nucle modified product, which can, in one embodiment, be sepa otide residues. Thus, when the binding conditions are chosen rated from the template by thermal dissociation, thereby pro to permit hybridization of both nucleobase polymers despite viding a diagnostic product having a distinctive ratio of at least one mismatch, the formation of a mobility-modified charge to translational frictional drag. This diagnostic prod ligated product reveals the sequence of the selected nucle uct will generally comprise a reporter molecule, which may otide sequence as it exists within the target nucleic acid, at be included within either of the ligated nucleobase polymers, least with respect to the terminal, adjacent residues of the two be attached to the one or more nucleobases added by the nucleobase polymers. Those skilled in the art are well versed polymerizing activity, or be added Subsequent to the covalent in selecting appropriate binding conditions, such as cation joining of the nucleobase polymers, using methods disclosed concentration, temperature, pH, and oligonucleotide compo infra. By treating with polymerase in the presence of fewer sition to selectively hybridize the nucleobase polymers to the than four nucleoside triphosphates, the nucleotide residues selected nucleotide sequences within the target nucleic acid. comprising the gap may be determined. Further amplification 0187. Since the base pairing of terminal adjacent residues of ligated mobility-modified product is achieved by repeated affects ligation, in one embodiment the nucleobase polymer cycles of denaturation, annealing, nucleic acid polymerase providing the 3 terminal nucleobase involved in the joining gap filling, and ligation in the presence of at least one of the reaction is designed to be perfectly complementary to the nucleoside triphosphates. target sequence while the nucleobase polymer providing the 0190. If the treatment with nucleic acid polymerase occurs 5' terminal nucleobase residue involved in the joining reac in the presence of one labeled nucleoside triphosphate or a tion is designed to be perfectly complementary in all but the mixture containing one labeled and 3 unlabeled nucleoside 5' terminal nucleobase. In another embodiment, the nucleo triphosphates, ligated mobility-modified products compris base polymers are designed Such that the nucleobase polymer ing at least one incorporated, labeled nucleoside are readily providing the 3' terminal nucleobase is perfectly complemen detected upon electrophoretic separation of the labeled tary except for the 3' terminal nucleobase residue while the mobility-modified ligated products. Modification of the oligonucleotide providing the 5' terminal nucleobase is per nucleotide mixture to one having one labeled nucleoside fectly complementary (Wu, D. Y. and Wallace, B., (1989), triphosphate and three chain terminating nucleoside triphos Specificity of nick-closing activity of bacteriophage T4DNA phates Suppresses unwanted ligation of oligonucleotides with ligase, Gene 76: 245-254; Landegren, U. etal. (1988) Aligase incorrectly incorporated nucleotide residues. mediated gene detection technique, Science 2241: 1077 0191 Mobility-modified sequence-specific nucleobase 1080). polymers of the present invention are also useful as primers 0188 In a modification of the method set forth above, the for nucleic acid sequence analysis by the chain termination mobility-modified sequence-specific nucleobase polymer method, a method well known to those skilled in the art. In probe comprises a nucleobase sequence that is complemen one embodiment, mobility-modified nucleobase polymers tary to the target sequence, but comprises a non-terminal are hybridized to target nucleic acid and extended by a nucleic mismatch with respect to non-target sequences. In this aspect acid polymerase in the presence of a mixture of nucleoside of the invention, the composition of die mobility-modified triphosphates and a chain terminating nucleoside triphos sequence-specific nucleobase polymer probe and the nature phate. The polymerase reaction generates a plurality of chain of the experimental conditions are such that the probe will terminated mobility-modified nucleic acids fragments, which only hybridize to the target sequence. In this embodiment for are separated, for example by capillary electrophoresis. example, a second nucleobase polymer that hybridizes to the Chain termination by the incorporated chain terminating target nucleobase, either upstream or downstream of the nucleoside triphosphate identifies the 3 terminal residue of hybridized mobility-modified sequence-specific nucleobase the terminated nucleic acid fragment. polymer probe, may be ligated to that probe to form the 0.192 For the purposes of detecting the chain terminated ligated, mobility-modified product that is diagnostic of the species, various substituents of the mobility-modified nucleic presence of the target nucleotide sequence. acid fragments are amenable to conjugation with detectable 0189 In a further modification of the embodiment set reporter molecules. These include the functional groups on forth above, the mobility-modified sequence-specific nucleo the mobility-modifying polymer, the phosphate triester group base polymer is hybridized to a selected nucleotide sequence linking the mobility-modifying polymer to the sequence-spe US 2011/0160082 A1 Jun. 30, 2011 24 cific nucleobase polymer, or nucleoside triphosphate precur nucleobase polymer provides the identity of the residue sors, including the chain terminators, incorporated into the immediately adjacent to the hybridized mobility-modified nucleic acid. Detectable reporter molecules may be radioac sequence-specific nucleobase polymer primer. In this tive, chemiluminescent, bioluminescent, fluorescent, or embodiment, the use of a plurality of different mobility ligand molecules. In one embodiment, the detectable label is modified sequence-specific nucleobase polymers permits the a fluorescent molecule, for example fluorescein isothiocyan simultaneous detection and analysis of a plurality of target ate, Texas red, rhodamine, and cyanine dyes and derivatives sequences in a single separation. thereof. In another embodiment, the fluorescent dyes are 0.195 Detecting the extended primer is accomplished by mobility-modified to reduce the variations in electrophoretic including a reporter molecule conjugated to the extended, mobility of nucleic acids caused by the fluorescent label (see mobility-modified sequence-specific nucleobase polymer are e.g. Ju, J. et al. (1995). Design and synthesis of fluorescence used as primers in the same manner as described above for energy transfer dye-labeled primers and their application for standard sequencing reactions. Thus, in one embodiment, the DNA sequencing and analysis, Anal. Biochem. 231: 131-40; chain terminating nucleoside triphosphate is labeled with one Metzker, et al. (1996) Electrophoretically uniform fluores of four spectrally resolvable fluorescent molecules such that cent dyes for automated DNA sequencing, Science 211: the fluorescent label uniquely identifies the chain terminating 1420-22; Hung, S. C. et al. (1997) Comparison of fluores nucleotide. The composition of the residue immediately adja cence energy transfer primers with different donor-acceptor cent to the hybridized oligonucleotide primer is then readily dye combinations, Anal. Biochem. 252: 77-88; Tu, O. et al. ascertained from the colors of the extended oligonucleotide (1998) The influence of fluorescent dye structure on the elec primer. As will be apparent to those skilled in the art, this trophoretic mobility of end-labeled DNA, Nucleic Acids Res. modified sequencing format is adaptable to other mixtures of 26:2797-2802) fluorescently labeled chain terminating nucleoside triphos 0193 In one embodiment, the mobility-modified phates. Thus the embodiments encompass nucleotide combi sequence-specific nucleobase polymers of the present inven nations having two or four chain terminating nucleoside tion are used within a format for sequencing selected regions triphosphates wherein only one chain terminator is labeled within a target polynucleotide wherein one of four spectrally with one of four resolvable reporter labels. Mixing the prod resolvable fluorescent molecules is used to label the nucleic ucts of the extension reactions, followed by separation and acid fragments in reactions having one of four chain termi detection of the extended products in a single gel lane or nating nucleoside triphosphates. In another aspect of this capillary provides the ability to determine all possible embodiment, the mobility-modified sequence-specific sequence variations at the nucleotide residue adjacent to the nucleobase polymers of the present invention are used for hybridized primer. Further increase in sensitivity of the meth sequencing selected regions within a target polynucleotide ods are possible by using Substantially exonuclease-resistant wherein one of four spectrally resolvable fluorescent mol chain terminators, such as those which form thio-ester inter ecules is used to label an oligonucleotide primers in a reaction nucleotide linkages, to reduce removal of incorporated chain containing one of four chain terminating nucleoside triphos terminators by polymerase associated exonuclease. phates. Thus, in both aspects of this embodiment, detecting 0196. In another embodiment, mobility-modified the fluorescent color of the chain terminated nucleic acid sequence-specific nucleobase polymers are used in poly fragment identifies the 3' terminal nucleotide residue. Sepa merase chain reactions (PCR) to detect and amplify selected ration of the chain-terminated products by electrophoresis, nucleotides within one or more target nucleic acids (Mullis, typically in a single gel lane or capillary, along with simulta K., U.S. Pat. No. 4,683.202; Saiki, R. K., et al., Enzymatic neous on-line detection of four spectrally resolvable fluores Amplification off-Globin Genomic Sequences and Restric cent molecules allows rapid sequence determination from the tion Site Analysis for Diagnosis of Sickle Cell Anemia, In colors of the separated nucleic acid fragments (Prober, J. M. PCR. A practical approach, M.J. McPherson, P. Quirke, and et al. (1985), A System for Rapid DNA Sequencing with G. R. Taylor, Eds., Oxford University Press, 1991). In this Fluorescent Chain Terminating Dideoxynucleotides, Science aspect of the present invention, the detection method involves 238: 336-341; Karger, A. E. et al., (1991), Multiwavelength PCR amplification of nucleotide sequences within the target Fluorescence Detection for DNA Sequencing Using Capil nucleic acid. In this aspect, a target nucleic acid, which may lary Electrophoresis, Nucleic Acids Res. 19 (18):4955-62). be immobilized, is contacted with a plurality of sequence 0194 When the nucleotide sequence of interest is a small specific nucleobase polymers, two of which hybridize to region of the target nucleic acid, for example a site including complementary strands, and at opposite ends, of a nucleotide single nucleotide polymorphism, modified sequencing for sequence within the target nucleic acid. Repeated cycles of mats, optionally, are used. In one such embodiment, a mobil extension of the hybridized sequence-specific oligonucle ity-modified sequence-specific nucleobase polymer is otides, optionally by a thermo-tolerant polymerase, thermal hybridized in a sequence-specific manner Such that the 3'-ter denaturation and dissociation of the extended product, and minal nucleotide residue of the mobility-modified sequence annealing, provide a geometric expansion of the region specific nucleobase polymer is immediately adjacent to the bracketed by the two nucleobase polymers. The product of site of interest. The hybridized nucleobase polymer is Such a polymerase chain reaction therefore is a double extended by a nucleic acid polymerase in the presence of at Stranded molecule consisting of two strands, each of which least one chain terminating nucleoside triphosphate extends comprises a sequence-specific nucleobase polymer probe. In the oligonucleotide by one nucleotide if the chain terminating this aspect of the present invention, at least one of the nucleotide is complementary to the target nucleic residue sequence-specific oligonucleotides is a mobility-modified immediately downstream of the 3'-terminus of the hybridized sequence-specific nucleobase polymer selected from the mobility-modified sequence-specific nucleobase polymer. group consisting of Structural formulae (II) and (III) such that Separation and detection of the extended, mobility-modified the double stranded polymerase chain reaction product has a sequence-specific mobility-modified sequence-specific distinctive ratio of charge to translational frictional drag. The US 2011/0160082 A1 Jun. 30, 2011 polymerase chain reaction product formed in this aspect of employing multiplex amplification of short tandem repeat the invention further comprises a label, which may be incor loci, PCR Methods Appl., 3(1): 13-22). If the polymorphisms porated within either of the sequence-specific nucleobase relate to variations in VNTR or STR sequences, direct analy polymer probes used as primers, or it may be incorporated sis of PCR products without further treatment suffices for within the substrate deoxyribonucleoside triphosphates used detecting polymorphisms since the products differ in nucle by the polymerizing enzyme. In yet another aspect, the poly otide length. The presence of mobility-modified PCR prod merase chain reaction product formed is analyzed under ucts, however, expands the capability of the PCR analysis to denaturing conditions, providing separated single stranded detect multiple polymorphic loci in a single reaction. products. In this aspect, at least one of the single stranded 0199. If the polymorphisms relate to single nucleotide products comprises both a label and a mobility-modified differences, the variations are detectable by conducting PCR sequence-specific nucleobase polymer primer selected from reactions using nucleobase polymer primers designed to have the group consisting of Structural formulae (II) and olio Such mismatches with the selected nucleic acid sequence within a that the single-stranded product derived from double stranded target nucleic acid. The presence of intentional mismatches polymerase chain reaction product has a distinctive ratio of within the duplex formed by hybridization of the nucleobase charge to translational frictional drag. As is well known in the polymer primer and the selected nucleic acid sequence within art, Such a single-stranded product may also be generated by the target nucleic acid affects the thermal stability of those carrying out the PCR reaction with limiting amounts of one of duplex molecules, which is reflected in the T of those struc the two sequence-specific nucleobase polymer probes used as tures and thus, under selected conditions, results in preferen a primer. By using distinctive mobility-modified sequence tial amplification of one target segment as compared to specific nucleic acids or nucleobase polymers as primers, the another. Such allele-specific polymerase chain reactions per PCR reaction can detect many selected regions within one or mit identification of mutations in single cells, or tissues con more target polynucleotides in a single assay by allowing taining a low copy number of one selected nucleotide separation of one PCR product from another. Moreover, those sequences amongst a high background of other nucleotide skilled in the art will recognize that using various combina sequences within one or more target nucleic acids (Cha, R.S., tions of nucleobase polymer primers provides additional (1993), Mismatch amplification mutation assay (MAMA): ways to generate distinctive mobility-modified PCR prod application to the c-H-ras gene, PCR Methods Appl., 201) ucts. For example, a combination of a mobility-modified 14-20; Glaab, W. E. et al., (1999). A novel assay for allelic nucleobase polymerand a second nucleobase polymer primer discrimination that combines fluorogenic 5' nuclease poly pair in the PCR reaction generates a PCR product with a merase chain (TaqMan(R) and mismatch amplification muta single mobility-modified strand. On the other hand, a combi tion, Mutat. Res. 430: 1-12). nation of a mobility-modified nucleobase polymer and a sec 0200. In yet another aspect, single nucleotide differences ond nucleobase polymer, which is also mobility-modified, are distinguished through analysis of higher order conforma generates a PCR product having both strands that are mobil tions of single stranded nucleic acids that form in a sequence ity-modified, thus distinguishing itself from the PCR product dependent manner. In this embodiment, single stranded with one mobility-modified strand. Thus, by varying the type nucleic acids are generated by dissociating the PCR products of mobility-modifying group and the nucleic acid strands that into single strands, or by preferentially amplifying one strand are mobility-modified, the embodiments enlarge the capacity by using limiting amounts of one primer in the PCR reaction to detect multiple target segments. (i.e. single-sided PCR). Under selected conditions, the single (0197) Detection of the PCR products may be accom Stranded nucleic acids are allowed to form higher order struc plished either during electrophoretic separation or after an tures by intramolecular hydrogen bonding of the single electrophoretic separation. Intercalating dyes such as stranded nucleic acid. Those skilled in the art are well versed ethidium bromide, ethidium bromide dimers, SYBRR) Green, in defining such permissive conditions (i.e. temperature, or cyanine dye dimers such as TOTO, YOYO and BOBO re denaturant concentration, pH, cation concentration etc.) for available for post separation detection (Haugland, R. P. Hand forming the higher order structures. These conformations, book of Fluorescent Probes and Research Chemicals, 6" ed. which are sequence dependent and which therefore can be Molecular Probes, Inc., 1996). Alternatively, the PCR prod extremely sensitive to single nucleotide changes, affect the ucts further comprise reporter molecules, including but not electrophoretic mobility of the nucleic acid, and thus reveal limited to radioactive, chemiluminescent, bioluminescent, variation in a selected nucleotide sequence within a target fluorescent, or ligand molecules that permit detection either nucleic acid by their unique electrophoretic mobility profiles. during or Subsequent to an electrophoretic separation. Meth To enhance formation of higher order structures modifica ods for labeling the mobility-modified PCR products follow tions are introduced into the nucleobase polymer primers the general Schemes presented for labeling in other methods used for the PCR reactions. For example, a nucleobase poly described infra. mer primer is engineered with additional bases complemen 0198 Detecting a selected nucleotide sequence within a tary to a part of the selected nucleotide sequence within a target nucleic acid by PCR amplification also encompasses target nucleic acid containing the sequence variation, Such identifying sequence variations within segments of the target that higher order conformations form when the additional nucleic acid. These variations include, among others, single bases on the nucleobase polymer primer "snapback” or re nucleotide polymorphisms and polymorphisms in variable anneal to the normal sequence but not to variant sequences nucleotide tandem repeats (VNTR) and short tandem repeats (Wilton, S. D. (1998), Snapback SSCP analysis: engineered (STR), such as those defined by sequence tag sites (STS). conformation changes for the rapid typing of known muta Identifying polymorphic loci are of particular interest tions, Hum. Mutat. 11(3): 252-8). Since the reliability of because they are often genetic markers for disease Suscepti detecting single nucleotide variations is affected by size of the bility (see e.g. Gastier, J. M., (1995), Hum Mol Genet, 4(10): single stranded nucleobase polymer, conformation analysis 1829-36: Kimpton, C. P., (1993), Automated DNA profiling using mobility-modified sequence-specific nucleobase poly US 2011/0160082 A1 Jun. 30, 2011 26 mers, with each having a distinctive ratio of charge to trans as S1 nuclease from Aspergillus Oryzue, P1 from Penicillum lational frictional drag, permits detection of a plurality of citrinum, and mung bean nuclease (Shenk, et al., (1975) Proc. selected nucleotide sequences within a target nucleic acid Natl. Acad. Sci. USA 72 989-93). Although these nucleases while maintaining the optimal length needed for forming are less reactive towards single nucleotide mismatches, they higher order structures (Sheffield, V.C., (1993). The sensitiv can digest unpaired residues created by longer insertions and ity of single Stranded conformation polymorphism analysis deletions (Dodgson, J. B. et al., (1977), Action of single for the detection of single base substitutions, Genomics 16 stranded specific nucleases on model DNA heteroduplexes of (2): 325-32). defined size and sequence, Biochemistry, 16:2374-49). Cel 1 0201 In yet another aspect of the invention, mobility and SP endonucleases show activity toward unpaired nucle modified sequence-specific nucleobase polymers are cleaved otide residues resulting from nucleotides sequence variations to detect selected nucleotide sequences within one or more comprising deletions, insertions, and missense mutations, target nucleic acids. In one such embodiment, mobility-modi fied sequence-specific nucleobase polymers are hybridized to within selected nucleotide sequences of target nucleic acids. selected nucleotide sequences within one or more target (Oleykowski, C.A., (1998) Mutation detection using a novel nucleic acids. In another embodiment, mobility-modified plantendonuclease, Nucleic Acids. Res. 26:4597-602;Yeung, PCR products comprising at least one mobility-modified A. T., U.S. Pat. No. 5,869.245). Resolvases from various sequence-specific nucleobase polymer serve as Substrates for Sources, such as bacteriophage and yeast, represent yet sequence-specific enzymes, such as restriction enzymes. another class of cleaving enzymes useful in this embodiment Digestion of the substrates by the enzymes creates cleaved of the invention. Representative examples of resolvases products having a distinctive ratio of charge to translational includebut are not limited to phage encoded T4 endonuclease frictional drag, which provides information about sequence VII and T7 endonuclease 1, both of which cleave at mis composition of the target polynucleotides. This form of matches (Cotton, R. G. H., U.S. Pat. No. 5,958,692: Solaro, et restriction fragment length polymorphism (RFLP) analysis is al., (1993), Endonuclease VII of Phage T4 Triggers Mismatch well known to those skilled in the art (see e.g., Kidd, I. M., Correction in vitro.J. Mol. Biol. 230: 868-877; (Chang, D.Y. (1998). A multiplex PCR assay for the simultaneous detection et al., (1991), Base mismatch specific endonuclease activity of human herpesvirus 6 and human herpesvirus 7, will typing extracts from Saccharomyces cerevisiae; Nucleic Acids of HHV-6 by enzyme cleavage of PCR products, J. Virol. Research 19 (17): 4761-66). Methods 70 (1): 29-36; Gelernter, J., (1991), Sequence tagged 0205. In another aspect, of the present invention encom sites (STS) Taq I RFLP at dopamine beta-hydroxylase, passes methods that prevent cleavage at unpaired residues. Nucleic Acids. Res. 19 (8): 1957). Proteins, including but not limited to the MutS protein of E. 0202 In another aspect, mobility-modified nucleobase coli, bind to sites of single nucleotide mismatches in duplex polymers hybridized to selected nucleotide sequences within nucleic acid structures (Su, S. S. et al., (1986), Escherichia a target nucleic acid, wherein there is at least one nucleobase coli mutS encoded protein binds to mismatched DNA base not complementary to the corresponding nucleobase in the pairs, Proc. Natl Acad. Sci. USA 83: 5057-5061). The MutS target nucleic acid, are treated with agents that specifically protein is part of the methylation directed E. coli. Muth/S/L cleave the non-base-paired nucleotide residues. Generally, mismatch repair system, homologs of which are present in the unpaired residue occurs on the hybridized mobility-modi other bacteria, yeast and mammals (Eisen, J. A., (1998), A fied nucleobase polymer (Bhattacharya, et al., (1989), phylogenetic study of the MutS family of proteins, Nucleic Nucleic Acids. Res. 17, 6821-6840). Although chromosomal Acids. Res. 26:4291-300; Alain, E. (1996), The Saccharomy DNA may serve as the target nucleic acids, target nucleic ces cerevisiae Msh2 and Mshé proteins form a complex, that acids are cloned DNA fragments comprising selected nucle specifically binds to duplex oligonucleotides containing mis otide sequences of a target nucleic acid, or PCR amplification matched DNA base pairs, Mol. Cell Biol. 16: 5604-15; products comprising selected nucleotide sequences of a target Modrich, P. et al., (1996), Mismatch repair in replication nucleic acid. fidelity, genetic recombination and cancer biology, Annu. 0203 Cleavage may be accomplished with either chemi Rev. Biochem. 65: 101-33). Therefore in one embodiment of calorenzymatic reagents. In chemical cleavage reactions, the the invention, duplex structures comprising at least one non hybrids containing at least one non-complementary nucleo base-paired nucleobase unit formed by hybridization of a base, are treated with chemicals which specifically modify mobility-modified sequence-specific nucleobase polymer the unpaired residue, rendering the internucleotide linkage of with a selected nucleotide sequence within a target nucleic the modified nucleoside susceptible to hydrolysis. Suitable acid, are treated with mismatch binding proteins such as chemical agents include but are not limited to carbodiimide, MutS and then exposed to one or more exonucleases which osmium tetraoxide, hydroxylamine or potassium permanga degrade the duplex Strands in a unidirectional fashion. A nate/tetraethylammonium chloride (Ellis. T. P. et al., (1998), bound mismatch binding protein inhibits further action of the Chemical cleavage of mismatch: a new look at an established exonuclease on the strand containing the mismatch, thereby method, Hum. Mutat. 11:345-53: Roberts, E., (1997), Potas providing nucleic acid products of defined length and which sium permanganate and tetraethylammonium chloride are possess a distinctive ratio of charge to translational frictional safe and effective substitute for osmium tetraoxide in solid drag (Ellis, L.A., (1994), MutS binding protects heteroduplex phase fluorescent chemical cleavage mismatch, Nucleic DNA from exonuclease digestion in vitro: a simple method Acids. Res. 25: 3377-78). The use of potassium permangan for detecting mutations, Nucleic Acids Res. 22 (13):2710-1: ate?tetraethylammonium chloride rather than osmium tet Taylor, G. R., U.S. Pat. No. 5,919,623). Unidirectional exo raoxide enhances cleavage at T/G mismatched pairs. nucleases Suitable for use in this assay include, but are not 0204 Enzymatic cleaving reagents encompass a variety of limited to exonuclease III, bacteriophage 2 exonuclease, and nucleases which recognize unpaired regions. These include the 3' to 5’ exonucleases of 17 DNA polymerase, T4 DNA but are not limited to single stranded specific nucleases Such polymerase, and Vent(R DNA polymerase. US 2011/0160082 A1 Jun. 30, 2011 27

0206. In yet another aspect, a mobility-modified polynucleotide Such that the second nucleobase polymer dis sequence-specific nucleobase polymer is used in a cleavage places the 5' portion of the hybridized mobility-modified based method of detecting selected nucleotide sequences sequence-specific nucleobase polymer, whereas the 3' portion within a target nucleic, acid may be a DNA-RNA-DNA of the mobility-modified sequence-specific nucleobase poly nucleobase polymer, where an internal RNA segment is mer and the 5' portion of the second nucleobase polymer flanked by DNA segments. This tripartite mobility-modified remain annealed to the selected nucleotide sequence con sequence-specific nucleobase polymer is hybridized to a tained within a target nucleic acid. The displaced Strand, selected nucleotide sequence within a target nucleic acid at a which is a single Stranded segment that is not base-paired temperature below the T of the overall, i.e. tripartite nucleo corresponds to the 5'-end of the mobility-modified sequence base polymer. Digestion of this duplex structure with an specific nucleobase polymer then serves as a Substrate for appropriate RNase, hydrolyzes only the RNA portion of the cleavage nucleases, thus producing discrete mobility-modi DNA-RNA-DNA nucleobase polymer when hybridized to a fied digestion products having distinct ratios of charge to DNA template. In one embodiment, the RNase is a thermo translational frictional drag that reflect presence of specific stable RNase H (Bekkaoui, F., (1996), Cycling probe tech sequences on the target polynucleotide. nology with RNase H attached to an oligonucleotide, Bio 0208 Cleaving enzymes recognizing displaced Strands techniques, 20 (2): 240-8). If the temperature of the reaction are either naturally occurring nucleases or modified maintained above the T of the flanking DNA segments nucleases. Naturally occurring structure-specific nucleases remaining after digestion of the internal RNA segment, those include, but are not limited to Pyrococcus woesii FEN-1 DNA segments dissociate, thus allowing another DNA-RNA endonuclease, thermostable Methoanoeoccus jannaschii DNA oligomer to associate with the target polynucleotide. FEN-1 endonucleases, yeast Rad2, and yeast Rad1/Rad10 Repeated hybridization, RNA cleavage, and dissociation of complex (Kaiser et al., U.S. Pat. No. 6,090,606, Cleavage the flanking DNA segments amplifies the level of detectable Reagents: Kaiser, et al. U.S. Pat. No. 5,843,669, Cleavage of dissociated DNA segments. The reaction temperature, in one nucleic acid using thermostable Methoanococcus jannasehii embodiment, is held constant during the amplification pro FEN-1 endonucleases). Other structure-specific enzymes cess, thus obviating any need for thermal cycling (Duck, P. suitable for the cleaving reaction are those derived from (1990), Probe amplifier system based on chimeric cycling modifications of known nucleases and polymerases (Dahl oligonucleotides, Biotechniques 9: 142-48; Modrusan, Z. berget al., U.S. Pat. No. 5,795,763, Synthesis Deficient Ther (1998) Spermine-mediated improvement of cycling probe mostable DNA Polymerase; Dahlberg et al., U.S. Pat. No. reaction, Mol. Cell Probes 12: 107-16). In this aspect of the 6,614.402, 5' Nucleases Derived from Thermostable DNA present invention, the sequence-specific DNA-RNA-DNA Polymerase). Modified polymerase that lack polymerase nucleobase polymer used comprise at least one mobility activity but still retain 5'-nuclease activity, are also used as modifying polymer and at least one reporter molecule cleaving reagents. attached to either or both of the flanking DNA segments, 0209 Another embodiment is directed toward the use of thereby providing a labeled digestion product having a dis the mobility-modifying polymers of the present invention in tinctive ratio of charge to translational frictional drag. “invader assays, which are SNP-identifying procedures 0207. In another aspect, detection of selected nucleotide based upon flap endonuclease cleavage of structures formed sequences within one or more target nucleic acids based on by two overlapping nucleobase polymers that hybridize to a cleavage of a mobility-modified nucleobase polymer relies target nucleic acid (see e.g. Cooksey et al., 2000, Antimicro upon cleavage Substrates formed by invasive hybridization, as bial Agents and Chemotherapy 44: 1296-1301). Such cleav described in Brow et al. U.S. Pat. No. 5,846,717. In this age reactions release products corresponding to the 5'-termi embodiment, the 5'-portion of a mobility-modified sequence nal nucleobase(s) of the “downstream nucleobase polymer. specific nucleobase polymer, which comprises a reporter Where those cleavage products are labeled and can be sepa molecule and which is hybridized to a target nucleic acid, is rated from the uncleaved nucleobase polymer, an invader displaced by a second nucleobase polymer that hybridizes to assay can be used to discriminate single base differences in, the same region and thereby exposing that displaced sequence for example, genomic sequences or PCR-amplified genomic to cleavage with a cleaving reagent. In practicing the embodi Sequences. ment, the target nucleic acid is contacted with a mobility 0210 Attachment of the mobility-modifying polymers of modified sequence-specific nucleobase polymer and with a the present invention to the labeled 5'-terminus of the down second nucleobase polymer. The mobility-modified stream nucleobase polymer used in an invader assay provides sequence-specific nucleobase polymer has a 5'-segment delectably-labeled cleavage products with distinctive charge complementary to a second region of the selected nucleotide to translational frictional drag ratios. Accordingly, a plurality sequence contained within a target nucleic acid and a 3'-por of SNP's are analyzed simultaneously using a plurality of tion complementary to a third region of the selected nucle sequence-specific downstream nucleobase polymers, otide sequence contained within a target nucleic acid, wherein the sequence-specific downstream nucleobase poly wherein the second region is downstream from the third mers comprise a mobility-modifying polymer of the present region. The second nucleobase polymer has a 5'-segment invention attached to the labeled 5'-terminus, such that the complementary to a first region of the selected nucleotide labeled product generated by flap endonuclease cleavage at sequence contained within a target nucleic acid and a 3'-seg each SNP has a distinctive charge to translational frictional ment complementary to the second region of the selected drag ratio. nucleotide sequence contained within a target nucleic acid, 0211. In a further aspect of the invader assay, for example, wherein the first region is downstream from the second the downstream nucleobase polymer, which carries a label region. Under selected conditions, hybrids form in which the and a first mobility-modifying polymer of the present inven mobility-modified sequenced specific nucleobase polymer tion attached to the 5'-terminus, further comprises a second and the second nucleobase polymer hybridize to the target mobility-modifying polymer attached to the 3'-terminus. The US 2011/0160082 A1 Jun. 30, 2011 28 presence of the second mobility-modifying polymer nucleobase polymer, or the mobility-modifying polymer of increases the sensitivity of the invader assay by enhancing the the present invention may be incorporated within the first difference between the electrophoretic mobility of the flap region of the downstream nucleobase polymer, providing a endonuclease generated product, comprising the 5'-terminus, molecule according to Structural formula (IV). Therefore, in label, and first mobility-modifying polymer, and the electro each of these embodiments, the presence of the second high phoretic mobility of the uncleaved downstream nucleobase molecular weight mobility-modifying polymer attached to polymer. Accordingly, the second mobility-modifying poly the second region of the downstream nucleobase polymer mer has a molecular weight of at least 2000. In other embodi increases the sensitivity of the invader assay by enhancing the ments, the second mobility-modifying polymer has a molecu difference between the electrophoretic mobility of the flap lar weight of at least 5,000, at least 10,000, at least 20,000, endonuclease generated product comprising a label and a and at least 100,000. In one embodiment, the second mobil mobility-modifying polymer of the present invention, the first ity-modifying polymer is a mobility-modifying polymer of region of the downstream oligonucleobase polymer, and the the present invention, while in other embodiments, the second electrophoretic mobility of the uncleaved downstream mobility-modifying polymer is a mobility-modifying poly nucleobase polymer. mer of the art, which is, in one illustrative, non-limiting 0213. In a still further embodiment of an invader assay, the example, an uncharged mono methyl polyethyleneglycol downstream nucleobase polymer carries a label and a first polymer. Moreover, the second mobility-modifying polymer mobility-modifying polymer, which is in one non-limiting may comprise a mixture of species of different molecular embodiment, a standard PEO mobility-modifying polymer of weight, provided that those species do not interfere Substan the art, that is attached to the first region of the downstream tially with detection of the signal product, i.e., the flap endo nucleobase polymer, and a second, high molecular weight nuclease generated product, comprising the 5'-terminus, mobility-modifying polymer attached to the second region of label, and first mobility-modifying polymer (see Example 5, the downstream nucleobase polymer. As above, the presence below). of the second mobility-modifying polymer increases the sen 0212 More generally, in other embodiments of the present sitivity of the invader assay by enhancing the difference invention, invader assays are performed in which the down between the electrophoretic mobility of the flap endonuclease stream oligonucleobase polymer comprises a label and a generated product, i.e., the first region of the downstream mobility-modifying polymer of the present invention nucleobase polymer, which comprises a label and a first attached to a first region of the downstream oligonucleobase mobility-modifying polymer, and the electrophoretic mobil polymer, and a second, high-molecular weight mobility ity of the uncleaved downstream nucleobase polymer. modifying polymer attached to a second region of the down Accordingly, the second mobility-modifying polymer has a stream oligonucleobase polymer, wherein first and second molecular weight of at least 2000. In other embodiments, the regions are separated by the flap endonuclease cleavage site. second mobility-modifying polymer has a molecular weight One aspect of this embodiment is described above and in of at least 5,000, at least 10,000, at least 20,000, and at least Example 5, wherein the label and mobility-modifying poly 100,000. In one embodiment, the second mobility-modifying mer of the present invention are attached to the 5'-end of the polymer is a mobility-modifying polymer of the present sequence-specific oligonucleobase polymer and a second, invention, while in other embodiments, the second mobility high molecular weight mobility-modifying polymer is modifying polymer is a mobility-modifying polymer of the attached to the 3'-end of the sequence-specific oligonucleo art, which is, in one illustrative, non-limiting example, an base polymer. In other embodiments, for example, a second, uncharged mono methyl polyethyleneglycol polymer. high molecular weight mobility-modifying polymer is 0214. In another aspect of the present invention, the mobil attached, via a linker arm nucleotide residue, to the sequence ity-modified sequence-specific nucleobase polymer serves as specific nucleobase polymer, rather than at the 5'-end or a cleavage Substrate in detection reactions involving multiple 3'-end of the sequence-specific nucleobase polymer. Accord sequential cleavage reactions, as described in Hall, J. G. et al., ingly, the second, high molecular weight mobility-modifying U.S. Pat. No. 5,994,069. In this embodiment, a first cleavage polymer, is attached at any nucleobase residue within the structure is thrilled as set forth above, except that in the second region of the downstream nucleobase polymer, or to present embodiment, the first nucleobase polymer is option the 5'-end or 3'-end, whichever is included within the second ally a mobility-modified sequence-specific nucleobase poly region of the downstream oligonucleobase polymer. Simi mer. The reaction mixture further includes a second target larly, in some embodiments, the label, which is a fluorescent nucleic acid and a third nucleobase polymer, which is a dye in certain non-limiting examples, is also attached via a mobility-modified sequence-specific nucleobase polymer, linker arm nucleotide residue at any nucleobase residue and further comprises at least one attached reporter molecule. within the first region of the downstream nucleobase polymer. The second target polynucleotide has a first, a second and a Synthesis of such linker arm nucleotides and the coupling of third region, wherein the first region is downstream of the inter alia, a fluorescent dye or an uncharged mono methyl second region, and the second region is downstream of the polyethyleneglycol polymer to the linker, are within the scope third region. The third nucleobase polymer has a 5 portion of the art (see e.g., Section 4.5 above). Moreover, e.g., linker fully complementary to the second region of the second target arm nucleoside phosphoramidite monomers, as well as linker polynucleotide and a 3' portion fully complementary to the arm nucleoside phosphoramidite monomers comprising fluo third region of the second target polynucleotide. Treatment of rescent moieties, are commercially available (Glen Research, the first cleavage structure results in release of a fourth Inc., Sterling, Va.). In these embodiments, the mobility-modi nucleobase polymer, which has a 5" portion complementary to fying polymer of the present invention is attached to the first the first region of the second target polynucleotide and a 3' region of the downstream nucleobase polymer, where the portion fully complementary to the second region of the sec point of attachment may be at the 5'-end or the 3'-end, which ond target polynucleotide. This released fourth nucleobase ever is encompassed within the first region of the downstream polymer forms a cleavage structure with the second target US 2011/0160082 A1 Jun. 30, 2011 29 polynucleotide and the third nucleobase polymer under con tion to target nucleic acids of different length alters their ratio ditions where the 3' portion of the third nucleobase polymer of charge to translational frictional drag of the target nucleic and the 5' portion of the fourth nucleobase polymer remains acids in a manner and to a degree Sufficient to effect their annealed to the second target polynucleotide. Cleavage of the electrophoretic separation in non-sieving media. Further third nucleobase polymer with a cleavage reagent generates a more, and in contrast to electrophoretic separations in sieving fifth and sixth nucleobase polymer, either or both of which media, longer nucleic acids to which a mobility-modified comprise a reporter molecule and a mobility-modifying poly sequence-specific nucleobase polymer of the present inven mer, thereby providing a digestion product having a distinc tion has been attached will migrate more rapidly than a tive ratio of charge to translational frictional drag. The fifth shorter nucleic acid to which the same mobility-modified nucleobase polymer is released upon cleavage, while the sequence-specific nucleobase polymer has been attached. sixth nucleobase polymer remains hybridized to the second Applicants believe, although without wishing to be held to target polynucleotide until dissociated by denaturation. Sub that belief, that such separations are based upon the propor sequent separation and detection of the fifth or sixth nucleo tionately smaller effect of attachment of a mobility-modify base polymer provides information about the presence of the ing, sequence-specific nucleobase polymer of defined mass first and second selected nucleotide sequence within the tar and size to a longer chain nucleic acid molecule than to a get nucleic acid. shorter chain nucleic acid molecule. Consequently, the ratio 0215. In a further aspect of the present invention relating of charge to frictional translational drag will be greater for the to a nucleotide sequence detection method involving multiple longer chain, providing the longer chain nucleic acid with a sequential cleavage reactions, a first cleavage structure is greater Velocity in an electric field. formed by first and second nucleobase polymerand a selected 0218. Attachment of a mobility-modified sequence-spe nucleotide sequence within a target nucleic acid, as set forth cific nucleobase polymers selected from the group consisting above. This aspect of the method further comprises a mobil of Structural formulae (ID and (III) to a population of nucleic ity-modified sequence-specific second target nucleobase acids of different length can be accomplished using a variety polymer, which has a first, a second, and a third region, of approaches, including but not limited to enzymatic ligation wherein the first region is downstream of the second region, or direct, synthetic incorporation of the mobility-modifying and wherein the third region upstream of the second region, is sequence-specific nucleobase polymers of the present inven fully self complementary and also complementary to the sec tion into the population of nucleic acids of different lengths ond region, Such that it forms a hairpin structure under that are to be separated. selected conditions. Cleavage of the first cleavage structure 0219. In one aspect of this method, a mobility-modifying with a cleaving reagent generates a fourth nucleobase poly sequence-specific nucleobase polymer is enzymatically mer, which has a 5'-portion complementary to the first region ligated to a population of nucleic acids of different length but and a 3'-portion fully complementary to the second region of having a common nucleotide sequence at the 5'-end, as is seen the nucleobase polymer. Hybridization of the released fourth within the products of a chain termination nucleic acid nucleobase polymer to the first and second regions of the sequencing reaction or, effectively, in chemical cleavage mobility-modified sequence-specific nucleobase polymer sequencing reactions which are transparent to all sequences forms a second cleavage structure with a displaced third other than those comprising the labeled 5'-end of the nucleic region that is complementary to the second region. Cleavage acid substrate. In this embodiment a synthetic template oli of this second cleavage structure generates a fifth and sixth gonucleotide, having two distinct sequence regions would be nucleobase polymers, either of which comprises a mobility used as a template to align the hybridized 3'-end of a mobility modifying polymer and a label, thereby providing a digestion modifying sequence-specific nucleobase polymer so that it product having a distinctive ratio of charge to translational would directly abut the hybridized 5'-end, which is generally frictional drag, and whose separation and detection provides phosphorylated, that is common to the population of nucleic information about the presence of the first target nucleic acid acids to be separated, and permit the two molecules to be and the second nucleobase polymer. covalently joined. Therefore the 5'-region of the synthetic 0216 Methods for labeling and detecting the cleaved template oligonucleotide would consist of a nucleotide nucleobase polymers, as set forth infra., are equally appli sequence complementary to the common 5'-end sequence of cable to the labeling and detection of products of the cleavage the molecules to be separated, while the 3'-region of the reactions. Moreover, labeling of released cleavage products is synthetic template would consist of sequences complemen also accomplished by extension of the product by template tary to the 3'-end of the mobility-modifying sequence-spe independent polymerases, including but not limited to termi cific nucleobase polymer to be joined. In another embodiment nal transferase and polyA polymerase as described in U.S. of this approach, the common 5'-end of the population of Pat. No. 6,090,606, which is hereby specifically incorporated nucleic acids to be separated corresponds to that generated by by reference. a sequence-specific restriction endonuclease. Therefore the 0217. In yet another aspect, the mobility-modified synthetic template nucleic acid consists of at least eight sequence-specific nucleobase polymers of the present inven nucleobases, of which at least three would be complementary tion are employed within a general method to effect the elec to a common 5'-sequence of the population of molecules to be trophoretic separation of target nucleic acids of different sizes separated. The design of Such template nucleic acids, as well in non-sieving media. Normally, nucleic acids of different as the conditions under which the enzymatic joining of the length, i.e. consisting of different numbers of nucleobase hybridized target nucleic acid and the mobility-modified residues, nevertheless display an essentially invariant ratio of sequence-specific nucleobase polymer would be carried out, charge to translational frictional drag. Accordingly, such mol are well known to those of ordinary skill in the art. Accord ecules cannot be separated electrophoretically in non-sieving ingly, this embodiment of the invention is applicable to any media. However, attachment of a mobility-modified population of molecules of different sizes, provided each has sequence-specific nucleobase polymer of the present inven a common 5'-end sequence of at least three nucleotides, in US 2011/0160082 A1 Jun. 30, 2011 30 certain embodiments, at least four nucleotides, and in further kits may also comprise a second nucleobase polymer, typi embodiments, at least eight nucleotides. Similar procedures, cally an oligonucleotide, which is optionally mobility-modi wherein the sequence common to a population of molecules fied, where the intended assay requires a second oligonucle of different sizes occurs at the 3'-end, and consequently, the otide; for example, kits for oligonucleotide ligation assays mobility-modifying sequence-specific nucleic to be attached and PCR analysis. Similarly, kits designed for ligase chain has a phosphorylated 5'-end with the mobility-modifying reaction amplification will further comprise at least two addi polymer attached to the 3'-end, are also included within the tional nucleobase polymers, which together are complemen Scope of the present invention. tary to a diagnostic ligase reaction product. The kits further 0220. In a further embodiment, a mobility-modifying sequence-specific nucleobase polymer is synthesized so as to may also comprise treating reagents such as restriction be complementary to a nucleotide sequence within, for enzymes, DNA polymerases, RNases, mismatch binding pro example, a sequencing vector, that is upstream of, i.e. toward teins, ligases, and exonucleases. Primer extension kits appro the 5'-end of the binding site of a sequencing printer used in priate for sequencing or oligonucleotide extension assays for Sanger, enzymatic chain termination sequencing reaction. In detecting single nucleotide polymorphisms, may further this embodiment, the mobility-modified sequence-specific comprise nucleoside triphosphates and/or chain terminating nucleobase polymer is enzymatically ligated to the sequenc nucleotides. The kit may also comprise reaction buffers for ing primer either before or after extension of the sequencing carrying out hybridizations and enzymatic treatments. primer during a chain termination sequencing reaction. In this 0225. The invention further comprises kits comprising embodiment, the mobility-modified sequence-specific one or more of the mobility-modifying phosphoramidite nucleic acid is synthesized so that, once hybridized to the reagents of present invention. One or more of the mobility template polynucleotide, its 3'-end would either directly abut modifying phosphoramidite reagents, in Such kits, may fur the 5'-end of the hybridized sequencing primer, or that 3'-end ther comprise one or more protecting groups, reporter mol would hybridize to sequences upstream of the 5'-end of the ecules, or ligands. Such kits may also comprise one or more sequencing primer. In the latter instance, the resulting gap is Solvents, reagents, or Solid Surface-bound nucleobase mate filled with a nucleic, acid polymerase and the extended mol rials for use in the synthesis of mobility-modified sequence ecule is then enzymatically ligated to the sequencing primer. specific nucleobase polymers. 0221) Another embodiment of the invention is related to the separation and detection of mobility-modified sequence specific nucleobase polymers and polynucleotides. Separa 6. EXAMPLES tion of oligonucleotides is effected by electrophoresis, chro matography, or mass spectroscopy. In methods employing 0226. The following examples serve to illustrate certain electrophoresis, the format may be thin flat chambers. In preferred embodiments and aspects of the present invention another embodiment, the separation is carried out by electro and are not to be construed as limiting the scope thereof. phoresis in capillary tubes. The advantage of capillary elec trophoresis is efficient heat dissipation, which increases reso Example 1 lution and permits rapid separation under high electrical fields. Moreover, the small diameters of the capillary tubes allow separation of numerous samples in arrays of capillaries. Synthesis of DMT-Protected Poly(ethylene oxide) 0222 Sieving or nonsieving media are applicable to sepa Alkyl Phosphoramidite ration of mobility-modified nucleobase polymers including but not limited to the reaction products generated in the detec 0227 Bis(diisopropylamino) chlorophosphine was syn tion methods disclosed herein. Sieving media include thesized by reacting phosphorous trichloride and diisopropy covalently crosslinked matrices, such as polyacrylamide lamine in toluene. Fifty ml of the resulting bis(diisopropy crosslinked with bis-acrylamide (see e.g. Cohen, A. S. et al. lamino)phosphine was placed in a 250 ml flaskand 3.01 ml of (1988) Rapid separation and purification of oligonucleotides ethanol added slowly over a two minute period and the reac by high performance capillary gel electrophoresis, Proc. Natl tion allowed to proceed for several days. After filtering the Acad. Sci. USA 85:9660; Swerdlow, H. et al., (1990), Capil mixture and removing the solvent, the structure of the bis lary gel electrophoresis for rapid, high resolution DNA (diisopropylamino)ethylphosphite ester reagent was ana sequencing, Nuc. Acids Res. 18 (6): 1415-1419) or linear lyzed by P and "H NMR (PNMR in CD.CN 125.9 ppm). polymers, for example hydroxypropylmethylcellulose, 0228 Mono dimethoxytrityl (DMT) protected pentaeth methyl cellulose, or hydroxylethylcellulose (Zhu et al. ylene oxide (3.0 gm, 5.5 mmole) tetrazole diisopropylamine (1992), J. Chromatogr. 489: 3.11-319; Nathakarnkitkool, S., salt (0.095 gm, 0.055 mmole) were dissolved in 10 ml of et al. (1992), Electrophoresis 13:18–31). methylene chloride and reacted with bis(diisopropylamino) 0223) In one embodiment, the electrophoretic medium is a non-sieving medium. Although polynucleotides are not ethylphosphite ester (1.75 gm; 7.2 mmole) for 15 hours. The readily separable in a non-sieving medium, mobility-modi phosphoramidite product; DMT-pentaethyleneoxide ethyl fied nucleobase polymers and polynucleotides have distinc N,N-diisopropylphosphoramidite, was washed two times tive ratios of charge to translational frictional drag that permit with saturated NaHCO solution, followed by a third wash separation in a non-sieving media, even when the nucleobase with saturated NaCl solution and dried over NaSO. The polymer and polynucleotides are of the same length. solution was basified by addition of triethylamine (TEA) (100 ul), and the solvent was removed. Recovered crude (4.2 gm) was purified using silica gel chromatography, yielding 2.8 gm 5.7 Kits product ('P NMR in CD.CN 145.3 ppm). To synthesize 0224 Kits of the invention comprise one or more mobil mobility-modifying phosphoramidite reagents lacking the ity-modified sequence-specific nucleobase polymers. The ethyl ester, DMT protected tetraethylene oxide was reacted US 2011/0160082 A1 Jun. 30, 2011 with B-cyanoethyl chloro N,N-diisopropylaminophosphite understandard conditions (see e.g. Levenson. C., et al., U.S. TABLE 1-continued Pat. No. 4,914,210). Oligonucleotide: Mobility Mobility change relative to Example 2 3'TMRA labeled 12 mer (base size) unmodified oligonucleotide 5' (PEE). 60.0 35.3 Synthesis of Mobility-Modified Sequence-Specific 5' (PEE). 116.0 91.3 Nucleobase Polymers TMRA; carboxytetramethylrhodamine PEO; phosphate diester linked pentaethylene oxide 0229 Sequence-specific nucleobase polymers labeled at PEE: ethyl phosphate triester linked pentaethylene oxide the 3'-nucleoside with tetramethyl rhodamine were synthe sized on a Applied Biosystems 394 synthesizer using stan dard phosphoramidite chemistry. To synthesize mobility Example 4 modifying nucleobase polymers, the phosphoramidite Analysis of Synthetic Modified Oligonucleotide reagents of Example 1 were reacted with the 5’ OH end of the Products of Invader Assay immobilized nucleobase polymer. Subsequent oxidation with 0232. The SNP-identifying procedure generally referred iodine and deprotection with base converts B-cyanoeth to as an invader assay is based upon flap endonuclease cleav ylphosphite linkages to phosphate diester linkages while age of structures formed by two overlapping oligonucleotides ethyl phosphite linkages are converted to ethyl phosphate that hybridize to a target nucleic acid (see e.g. Cooksey et al., triester linkages. After cleavage from the Solid Support and 2000, Antimicrobial Agents and Chemotherapy 44: 1296 deprotection with base at 55° C. for 4 hrs, the nucleobase 1301). Such cleavage reactions release products correspond polymers were purified by high performance liquid chroma ing to the 5'-terminal nucleotide or 5'-terminal oligonucle tography (HPLC). Treatment with 100 ul of 80% acetic acid otide of the downstream oligonucleotide. Where those for 15 min removed the final DMT protecting group. The cleavage products are labeled, and can be separated from the mobility-modified nucleobase polymers were purified on a uncleaved oligonucleotide, the invader assay can be used to PD 10 column. discriminate single base differences in, for example, genomic or PCR-amplified genomic sequences. Example 3 0233. In order to demonstrate the utility of the mobility modifying polymer segments of the present invention for use, Separation Characteristics of Mobility-Modified e.g., in invader assays, the compounds of Table 2, which Sequence-Specific Nucleobase Polymers represent exemplary reaction products that could be gener ated within an invader assay, were synthesized using methods 0230. A series of twelve-residue nucleobase polymers disclosed supra. Each of the compounds of Table 2 was (“12 mers') were synthesized to which were attached differ labeled at the 5'-end with a fluorescent dye as indicator. The ent mobility-modifying monomeric units of pentaethylene relative mobility of each of these compounds was determined oxide via either charged phosphate diester (PEO) or by capillary electrophoresis using an Applied Biosystems uncharged ethyl phosphate triester (PEE) linkages, which were synthesized as set forth in Example 2. The relative instrument, model number 310, and data analysis was per electrophoretic mobility profile for each compound was formed using GeneScan Software, version 2.1 fic4. evaluated by separation by capillary electrophoresis in a non sieving medium (Applied Biosystems 310 Genetic Ana TABLE 2 lyzer). Fluorescent internal size standards provided the refer Oligonucleotide: Mobility Mobility change relative to ence markers for peak retention analysis using GeneScan R. 5' FAM labeled (base size) unmodified oligonucleotide software G 39.6 0231. An unmodified 12-mer nucleobase polymer 5'' (PEO)-(PEE) 75.5 35.9 migrates with an apparent base size of 24.7. Attachment of 5'' (PEE) 95.1 55.5 5' (PEO)-(PEE). 65.5 25.9 three units of pentaethylene oxide covalently linked to the 5'' (PEE). 250 210.4 nucleobase polymer moiety through negatively charged 5'' (PEE), 160 120.4 phosphate diester linkages, retards the mobility of the 12 mer 5'' (PEO)-(PEE), 104 64.4 by 5.3 bases, while modification with only a single mono 5'' (PEE). 1300 1260 meric unit of pentaethylene oxide attached through an FAM: carboxyfluorescein uncharged ethyl phosphate triester linkage retards the mobil PEO; phosphate diester linked pentaethylene oxide ity by 8.3 bases. Furthermore, the attachment of additional PEE: ethyl phosphate triester linked pentaethylene oxide pentaethylene oxide units linked through uncharged ethyl phosphate triester linkages produces non-linear decreases in 0234. The data in Table 2 demonstrate the unexpectedly mobility of the oligonucleotide. large effect on mobility provided by the mobility-modifying polymer segments of the present invention, especially incom parison to the phosphate diester linked pentaethylene oxide TABLE 1. monomers of the art. The data of Table 2 further demonstrate Oligonucleotide: Mobility Mobility change relative to the extent of electrophoretic separation that can be obtained 3'TMRA labeled 12 mer (base size) unmodified oligonucleotide using the mobility-modifying phosphoramidite functionaliz 5’ OH 24.7 ing reagents of the present invention. Moreover, the data of 5'' (PEO) 3O.O 5.3 Table 2 also demonstrate that compounds of intermediate 5'' (PEE) 33.0 8.3 mobility are obtained by combining the mobility-modifying phosphoramidite functionalizing reagents of the present US 2011/0160082 A1 Jun. 30, 2011 32 invention with, as a non-limiting example, phosphate diester a total reaction volume of 10 ul further comprising 10 mM linked pentaethylene oxide monomers of the art. MOPS, pH 8.0, 7.5 mM MgCl, 0.05% Tween 20, and 0.05% Nonidet P40. The invader assay is incubated for 15 hours at Example 5 66°C. The reaction is terminated and a 1 ul aliquot thereof is electrophoresed on an Applied Biosystems instrument, model Analysis of Cleaved Modified Olignucleotide Prod number 310, with data analysis performed using GeneScan ucts of Invader Assay software, version 2.1 fea. Analysis of the results demonstrates 0235 An invader assay probe is synthesized that com the presence of the cleavage product 5'-FAM-(PEE)-G, prises a fluorescent dye (FAM) coupled to a first mobility which is well separated from the uncleaved probe, 5'-FAM modifying polymer, a dimer of a mobility-modifying phos (PEE)-GGGACGGGGTTCAGC-3'-PEG 5000. The phoramidite functionalizing reagent of the present invention uncleaved probe was detected as a plurality of closely-spaced ((PEE)), linked to the following oligonucleotide: 5'-GG peaks arising from the plurality of molecular weight species GACGGGGTTCAGC-3'-NH, using standard DNA synthe included within the commercially-available mono methyl sis methods and PEE phosphoramidite reagents. After cleav polyethylene glycol polymer product attached to the 3'-end of age from the support and deprotection with base at 55° C. for the labeled probe, 5'-FAM-(PEE)-GGGACGGGGT 4 hours, the oligonucleotide is purified by HPLC. The oligo TCAGC-3" as the second mobility-modifying polymer. nucleotide: 5'-FAM-(PEE)-GGGACGGGGTTCAGC-3'- 0237 All publications and patents referred to herein are NH, is dissolved in water, coupled with a second mobility hereby incorporated, by reference in their entirety. As recog modifying polymer, mono-methyl polyethylene glycol 5000 nized by those skilled in the art of molecular biology, the use propionic acid N-Succinimidyl ester (Fluka) in the presence of mobility-modified sequence-specific nucleobase polymers of NaHCO for two hours, and purified by HPLC, yielding the are adaptable to a variety of methods. Various modifications derivatived, mobility-modified product 5'-FAM-(PEE)-GG and variations of the above described method and composi GACGGGGTTCAGC-3'-PEG SOOO. tion will be apparent to those skilled in the art without depart 0236. The invader assay is performed using, as template, a ing from the spirit and scope of the invention. As a specific 527 bp PCR product generated by amplification of a segment example, although various embodiments of the invention may of human genomic DNA corresponding to the TNF-C. gene, be descriptively exemplified with DNA or RNA oligonucle using the following PCR primers: 5'-GAGTCTCCGGGT otides, skilled artisans will recognize that the described CAGAATGA (forward) and 5'-TCTCGGTTTCTTCTC embodiments may also work with other nucleobase poly CATCG (reverse). In the first step of the invader assay, mers, including analogs and derivatives of RNA and DNA approximately 0.2 pmole of the PCR product is denatured at oligonucleotides. Although specific preferred embodiments 95° C. for 5 min. in the presence of 0.5 pmole of invading of the claims are described, the invention as claimed should probe (5'-GAGGCAATAGTTTTTGAGGGGCATGT). In not be limited to the specific embodiments. Various modifi the second step, 50 ng of Cleavase VII is added along with 10 cation of the described modes which are obvious to those pmole of 5'-mobility-modified oligonucleobase probe, skilled in the art are intended to be within the scope of the 5'-FAM-(PEE)-GGGACGGGGTTCAGC-3'-PEG 5000, in following claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 12

<21 Os SEQ ID NO 1 &211s LENGTH: 8 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA <4 OOs SEQUENCE: 1 atgcatgc

<21 Os SEQ ID NO 2 &211s LENGTH: 8 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA

<4 OOs SEQUENCE: 2 atgcatgc

<21 Os SEQ ID NO 3 &211s LENGTH: 9 US 2011/0160082 A1 Jun. 30, 2011 33

- Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA <4 OOs, SEQUENCE: 3 cgitacgitat

<210s, SEQ ID NO 4 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA <4 OOs, SEQUENCE: 4 gggacggggt to agc 15

<210s, SEQ ID NO 5 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA <4 OOs, SEQUENCE: 5 gggacggggt to agc 15

<210s, SEQ ID NO 6 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA <4 OOs, SEQUENCE: 6 gggacggggt to agc 15

<210s, SEQ ID NO 7 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA <4 OO > SEQUENCE: 7 gagt ct cogg gtcagaatga

<210s, SEQ ID NO 8 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA <4 OOs, SEQUENCE: 8 t citcggitttc ttcticcatcg

<210s, SEQ ID NO 9 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA US 2011/0160082 A1 Jun. 30, 2011 34

- Continued

<4 OOs, SEQUENCE: 9 gaggcaatag tittittgaggg gCatgt 26

<210s, SEQ ID NO 10 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA <4 OOs, SEQUENCE: 10 gggacggggt to agc 15

<210s, SEQ ID NO 11 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA <4 OOs, SEQUENCE: 11 gggacggggt to agc 15

<210s, SEQ ID NO 12 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic DNA <4 OOs, SEQUENCE: 12 gggacggggt to agc 15

What is claimed is: R is selected from the group consisting of hydrogen, pro 1. A mobility-modified sequence-specific nucleobase tecting group, reporter molecule, and ligand; polymer comprising a mobility-modifying polymer linked to a sequence-specific nucleobase polymer, according to Struc tural formula (II) or (III): O

(II) R3 is R x-cha-ol-ch-0--0 s O OR

R-x-Ech-oh-ch-0--0-oricoOR2 each R" is independently selected, from the group consist (III) ing of hydrogen and R: O each X is independently selected from the group consisting of O, S, NH and NH CO); R3 X-FCH)-O-(CH,-O--O OLIGO each a is independently an integer from 1 to 6: OR O d each b is independently an integer from 0 to 40; each d is independently an integer from 1 to 200; and OLIGO is a sequence-specific nucleobase polymer, or a salt thereof, wherein: R is selected from the group consisting of alkyl compris with the proviso that at least one R' or at least one R is other ing at least two carbon atoms, aryl, (R),Si– where than hydrogen. each R is independently selected, from the group con 2. The mobility-modified sequence-specific nucleobase sisting of linear and branched chain alkyl and aryl, base polymer of claim 1 in which each X is O. stable protecting groups, and R X-(CH2), Ola 3. The mobility-modified sequence-specific nucleobase (CH2) ; polymer of claim 1 in which each a is 2. each R' is independently selected from the group consist 4. The mobility-modified sequence-specific nucleobase ing of hydrogen and R: polymer of claim 3 in which each b is 4. US 2011/0160082 A1 Jun. 30, 2011

5. The mobility-modified sequence-specific nucleobase 25. The mobility-modified sequence-specific nucleobase polymer of claim 1 in which OLIGO is a DNA, RNA, DNA polymer of claim 22, wherein the polyethlyene oxide polymer analog, or RNA analog oligonucleotide. has a molecular weight of at least 2000 daltons. 6. The mobility-modified sequence-specific nucleobase 26. The mobility-modified sequence-specific nucleobase polymer of claim 1 in which OLIGO is an analog of a DNA or polymer of claim 22, wherein the polyethlyene oxide polymer RNA oligonucleotide. has a molecular weight of at least 5000 daltons. 27. A composition comprising a plurality of mobility 7. The mobility-modified sequence-specific nucleobase modified sequence-specific nucleobase polymers, wherein polymer of claim 1 in which OLIGO comprises at least one each said nucleobase polymer is a compound according to non-negatively charged internucleotide linkage. claim 1, and wherein each said nucleobase polymer has a 8. The mobility-modified sequence-specific nucleobase distinctive ratio of charge to translational frictional drag. polymer of claim 7, wherein said internucleotide linkage is a 28. The composition of claim 27, wherein said each mobil mono alkyl phosphate triester. ity-modified sequence-specific nucleobase polymer of said 9. The mobility-modified sequence-specific nucleobase plurality comprises an OLIGO, and wherein each OLIGO has polymer of claim 1 in which R is a reporter molecule. the same number of nucleobase units. 10. The mobility-modified sequence-specific nucleobase 29. A method for detecting a plurality of selected nucle polymer of claim 9 in which the reporter molecule is a fluo otide sequences within one or more target nucleic acids, com rophore, a chemiluminescent moiety, or a ligand. prising: 11. The mobility-modified sequence-specific nucleobase contacting at least one or more target nucleic acids with a polymer of claim 1 in which OLIGO includes a detectable plurality of mobility-modified sequence-specific label. nucleobase polymers under conditions that distinguish 12. The mobility-modified sequence-specific nucleobase those nucleobase polymers that hybridize to the target polymer of claim 9 in which the detectable label is a fluoro nucleic acid, wherein each said nucleobase polymer is a phore. compound according to claim 1, and wherein each said 13. The mobility-modified sequence-specific nucleobase nucleobase polymer has a distinctive ratio of charge to polymer of claim 1 in which OLIGO comprises a polyethly translational frictional drag, and ene oxide polymer. detecting those nucleobase polymer that have hybridized to 14. The mobility-modified sequence-specific nucleobase the target nucleic acid. polymer of claim 13, wherein the polyethlyene oxide polymer 30. The method of claim 29, in which the OLIGO portions is a mono methyl polyethlyene oxide polymer. of the nucleobase polymers are composed of the same num 15. The mobility-modified sequence-specific nucleobase ber of nucleobase units. polymer of claim 13, wherein the polyethlyene oxide polymer 31. The method of claim 29, wherein the one or more target has a molecular weight of at least 2000 daltons. nucleic acids are immobilized on a Solid Support. 16. The mobility-modified sequence-specific nucleobase 32. The method of claim 29, wherein each nucleobase polymer of claim 13, wherein the polyethlyene oxide polymer polymer includes a detectable label. has a molecular weight of at least 5000 daltons. 33. The method of claim32, wherein the detectable label is 17. The mobility-modified sequence-specific nucleobase a radioisotope, a chemiluminescent moiety, a fluorophore, or polymer of claim 1, wherein the mobility-modifying polymer a ligand. is attached to the 5'-end of the sequence-specific nucleobase 34. The method of claim 29, wherein said detecting com polymer. prises the steps of 18. The mobility-modified sequence-specific nucleobase recovering those nucleobase polymers that are hybridized polymer of claim 17, further comprising a polyethlyene oxide to the target nucleic acid; and polymer attached to the 3'-end of the sequence-specific separating the recovered nucleobase polymers by electro nucleobase polymer. phoresis. 35. The method of claim34, wherein said electrophoresis is 19. The mobility-modified sequence-specific nucleobase carried out by capillary electrophoresis in a non-sieving polymer of claim 18, wherein the polyethlyene oxide polymer medium. is a mono methyl polyethlyene oxide polymer. 36. A method for detecting a plurality of selected nucle 20. The mobility-modified sequence-specific nucleobase otide sequences within one or more target nucleic acids, com polymer of claim 18, wherein the polyethlyene oxide polymer prising: has a molecular weight of at least 2000 daltons. contacting the target nucleic acids with a first plurality of 21. The mobility-modified sequence-specific nucleobase mobility-modified sequence-specific nucleobase poly polymer of claim 18, wherein the polyethlyene oxide polymer mer probes and a second plurality of sequence-specific has a molecular weight of at least 5000 daltons. nucleobase polymer probes under conditions that distin 22. The mobility-modified sequence-specific nucleobase guish between those probes that hybridize to the target polymer of claim 1, wherein the mobility-modifying polymer nucleic, wherein each mobility-modified sequence-spe is attached to the 3'-end of the sequence-specific nucleobase cific nucleobase polymer is a compound according to polymer. claim 1 and has a distinctive ratio of charge to transla 23. The mobility-modified sequence-specific nucleobase tional frictional drag, polymer of claim 22, further comprising a polyethlyene oxide covalently joining first and second probes that adjacently polymer attached to the 5'-end of the sequence-specific hybridize to the same target nucleic acid molecules to nucleobase polymer. formaligation product, wherein each said ligation prod 24. The mobility-modified sequence-specific nucleobase uct has a distinctive ratio of charge to translational fric polymer of claim 22, wherein the polyethlyene oxide polymer tional drag, and is a mono methyl polyethlyene oxide polymer. detecting said ligation products. US 2011/0160082 A1 Jun. 30, 2011 36

37. The method of claim 36, wherein each ligation product wherein each said polymerase chain reaction product comprises the same number of nucleobases. has a distinctive ratio of charge to translational frictional 38. The method of claim 36, wherein the one or more target drag, and nucleic acids are immobilized, on a solid Support. e) detecting said polymerase chain reaction products. 39. The method of claim 36, wherein at least one of said 49. The method of claim 48, wherein each said polymerase first probe and said second probe includes a detectable label. chain reaction product comprises the same number of nucleo 40. The method of claim39, wherein the detectable label is bases. a radioisotope, a chemiluminescent moiety, a bioluminescent 50. The method of claim 48, wherein the one or more target moiety, a fluorophore, or a ligand. nucleic acids are immobilized on a Solid Support. 51. The method of claim 48, wherein at least one of said 41. The method of claim 40, wherein said detecting com first nucleobase polymer primer, said second nucleobase prises the steps of polymer primer, or a substrate deoxyribonucleoside triphos recovering the ligation products; and phate comprises a detectable label. separating the recovered ligation products by electrophore 52. The method of claim 48, wherein said detecting com S1S. prises fractionation of said plurality of polymerase chain 42. The method of claim 41, wherein said electrophoresis is reaction products by capillary electrophoresis in a non-siev carried out by capillary electrophoresis in a non-sieving ing medium. medium. 53. A mobility-modifying phosphoramidite reagent having 43. The method of claim 40, wherein the covalent joining is the structure: accomplished by a ligase. 44. The method of claim 43, wherein the ligase is a ther mostable ligase. (I) 45. The method of claim 44, wherein said contacting, R6 hybridizing, joining, and releasing steps are repeated a plu rality of times. -R 46. A method of separating a plurality of target nucleic acid R-x-Echa-ol-cha-o-K molecules, comprising: O-R2 attaching a mobility-modified sequence-specific nucleo base polymer according to claim 1 to each target nucleic wherein: acid of the plurality, thereby forming a plurality of R is selected from the group consisting of alkyl compris mobility-modified target nucleic acids, wherein each ing at least two carbon atoms, aryl, (R)-Si- where target nucleic acid having the same number of nucle each R is independently selected from the group con otide residues has a distinctive ratio of charge to trans sisting of linear and branched chain alkyl and aryl, base lational frictional drag, and stable protecting groups, and R X-(CH2), Ola - fractionating the plurality of mobility-modified target (CH2) ; nucleic acids. R is selected from the group consisting of hydrogen, pro 47. The method of claim 46, wherein said plurality of target tecting group, reporter molecule, and ligand; nucleic acids is generated by a sequencing method selected Rand Rare each independently selected from the group from the group consisting of chain termination sequencing consisting of C-C alkyl, C-Co cycloalkyl, Co-Co and chemical cleavage sequencing methods. aryl, and Co-C27 arylalkyl, 48. A method for detecting a plurality of selected nucle X is selected from the group consisting of O, S, NH, otide sequences within one or more target nucleic acids, com NH CO); prising: each a is independently an integer from 1 to 6; and b is an integer from 0 to 40. a) contacting the target nucleic acids with a plurality of 54. A kit comprising at least one mobility-modified nucleobase polymer primers whereby a first nucleobase sequence specific nucleobase polymer, wherein the mobility polymer primer and a second nucleobase polymer modified sequence specific nucleobase polymer comprises a primer each hybridize to complementary strands and at mobility-modifying polymer linked to a sequence-specific opposite ends of each of a plurality of selected nucle otide sequences, wherein at least one of each said first nucleobase polymer, according to structural formula (II) or nucleobase polymer primer and said second nucleobase (III): polymer primer is a mobility-modified sequence-spe cific nucleobase polymer according to claim 3: (II) b) extending each said first nucleobase polymer primer and O each said second nucleobase polymer primer with a DNA polymerizing activity in the presence deoxyribo R-x-cyl-ol-cha-0--0-olco nucleoside triphosphate Substrates; OR2 c) denaturing the plurality of base-paired structures formed (III) by base pairing interactions between each extended first O nucleobase polymer primer and the target nucleic acid and each extended second nucleobase polymer primer R3 X-ECH)-O-(CH-O--O OLIGO and the target nucleic acid; OR O d d) repeating steps (a) through (c) a plurality of times to form a plurality of polymerase chain reaction products, US 2011/0160082 A1 Jun. 30, 2011 37

or a salt thereof, wherein: R is selected from the group consisting of alkyl compris R is selected from the group consisting of alkyl compris ing at least two carbon atoms, aryl, (R)-Si- where ing at least two carbon atoms, aryl, (R),Si– where each R is independently selected from the group con each R is independently selected from the group con sisting of linear and branched chain alkyl and aryl, base sisting of linear and branched chain alkyl and aryl, base stable protecting groups, and R X-(CH2), Ola stable protecting groups, and R X-(CH2), Ola (CH2)—, and b is an integer from 0 to 40. (CH2) ; 56. A mobility-modified sequence-specific nucleobase R is selected from the group consisting of hydrogen, pro polymer comprising a mobility-modifying polymer linked to tecting group, reporter molecule, and ligand; the 3'-end of a first sequence-specific nucleobase polymerand to the 5'-end of a second sequence-specific nucleobase poly mer according to Structural formula (IV): O

R3 is R X-(CH-OH,-(CH-O--O (IV) OR4 d

each R" is independently selected from the group consist OLIGO X-FCH)-OH-CH)-O--O OLIGO ing of hydrogen and R: OR d each R is independently selected from the group consist ing of hydrogen and R: or a salt thereof, wherein: each X is independently selected from the group consisting each R'' is independently selected, from the group consist of O, S, NH and NH CO); ing of hydrogen, alkyl comprising at least two carbon each a is independently an integer from 1 to 6: atoms, aryl, (R) Si where each R is independently each b is independently an integer from 0 to 40; Selected from the group consisting of linear and each d is independently an integer from 1 to 200; and branched chain alkyl and aryl, base-stable protecting OLIGO is a sequence-specific nucleobase polymer, groups, R X-(CH2), O-(CH2). , protecting with the proviso that if at least one R' or at least one R is not group, reporter molecule, and ligand, with the proviso hydrogen. that at least one R' is not hydrogen; each X is independently selected from the group consisting 55. A kit comprising at least one mobility-modifying phos of O, S, NH and NH CO); phoramidite reagent, wherein said reagent has a structure each a is independently an integer from 1 to 6: according to: each b is independently an integer from 0 to 40; d is an integer from 1 to 200; OLIGO" is a first sequence-specific nucleobase polymer; (I) and R6 OLIGO is a second sequence-specific nucleobase poly C. -R 57. The mobility-modified sequence-specific nucleobase polymer of claim 56 in which at least one of OLIGO" and R-x-Echa-ol-ch-o-KO-R2 OLIGO comprises a polyethlyene oxide polymer. 58. The mobility-modified sequence-specific nucleobase polymer of claim 57, wherein the polyethlyene oxide polymer wherein: is a mono methyl polyethlyene oxide polymer. R is selected from the group consisting of hydrogen, pro 59. The mobility-modified sequence-specific nucleobase tecting group, reporter molecule, and ligand; polymer of claim 57, wherein the polyethlyene oxide polymer RandR are each independently selected from the group has a molecular weight of at least 2000 daltons. consisting of C1-C alkyl, Cs-Co cycloalkyl, Co-Co 60. The mobility-modified sequence-specific nucleobase aryl, and Co-C27 arylalkyl, polymer of claim 57, wherein the polyethlyene oxide polymer X is selected from the group consisting of O, S, NH, has a molecular weight of at least 5000 daltons. NH CO); a is an integer from 1 to 6: c c c c c