|HAO WATATU WITHUS009815916B2 TO TAMIL MONTE (12 ) United States Patent (10 ) Patent No. : US 9 ,815 ,916 B2 Brown et al. (45 ) Date of Patent: Nov. 14 , 2017

(54 ) POLYMERS AND DNA COPOLYMER 5 , 599 ,675 A 2 / 1997 Brenner COATINGS 5 .641 . 658 A 6 /1997 Adams et al. 5 ,658 , 734 A 8 / 1997 Brock et al . 5 , 750 , 341 A 5 / 1998 Macevicz (71 ) Applicant: Illumina Cambridge Limited , Nr 5 , 837 , 858 A 11/ 1998 Brennan Saffron Walden , Essex (GB ) 5 , 874 ,219 A 2 / 1999 Rava et al . 5 ,919 ,523 A 7 / 1999 Sundberg et al. (72 ) Inventors : Andrew A . Brown, Nr Saffron Walden 6 , 136 , 269 A 10 /2000 Winkler et al . (GB ) ; Wayne N . George , Nr Saffron 6 , 210 , 891 B1 4 / 2001 Nyren et al. Walden (GB ) ; Alexandre Richez , Nr 6 , 214 , 587 B1 4 /2001 Dattagupta et al. Saffron Walden (GB ); Anne -Cecile 6 , 258, 568 B1 7 / 2001 Nyren Dingwall , Nr Saffron Walden (GB ) ; 6 , 274, 320 B1 8/ 2001 Rothberg et al . 6 ,287 , 768 B1 9 /2001 Chenchik et al . Xavier von Hatten , Nr Saffron Walden 6 ,287 , 776 B1 9 / 2001 Hefti (GB ) 6 , 288 , 220 B1 9 /2001 Kambara et al. 6 ,291 , 193 B1 9 /2001 Khodadoust ( 73 ) Assignee : ILLUMINA CAMBRIDGE 6 , 297 , 006 B1 10 /2001 Drmanac et al . LIMITED , Nr Saffron Walden , Essex 6 , 346, 413 B1 2 / 2002 Fodor et al . (GB ) 6 ,355 , 431 B1 3 /2002 Chee et al. 6 , 416, 949 B1 7 /2002 Dower et al. ( * ) Notice : Subject to any disclaimer, the term of this 6 ,482 , 591 B2 11 /2002 Lockhart et al. patent is extended or adjusted under 35 6 , 514 , 751 B2 2 / 2003 Johann et al. 6 ,524 ,793 B12 /2003 Chandler et al. U .S .C . 154 (b ) by 119 days . 6 , 610 , 482 B1 8 / 2003 Fodor et al. 6, 890 , 741 B2 5 / 2005 Fan et al. (21 ) Appl. No. : 14 / 927, 252 6 , 913 , 884 B2 7 / 2005 Stuelpnagel et al. 7 , 057 , 026 B2 6 / 2006 Barnes et al . (22 ) Filed : Oct . 29 , 2015 7 , 115 ,400 B1 10 / 2006 Adessi et al . 7 , 211, 414 B2 5 / 2007 Hardin et al . (65 ) Prior Publication Data 7 ,244 ,559 B2 7 / 2007 Rothberg et al. 7 , 315, 019 B2 1 / 2008 Turner et al. US 2016 /0122816 A1 May 5 , 2016 7 , 329 , 492 B2 2 / 2008 Hardin et al . 7, 405 ,281 B2 7 / 2008 Xu et al. Related U . S . Application Data NNN7 ,582 , 420 B2 9 / 2009 Oliphant et al . 7 ,595 ,883 B1 9 /2009 El Gamal et al . (60 ) Provisional application No . 62/ 073 ,764 , filed on Oct. 7 ,622 , 294 B2 11/ 2009 Walt et al . 31, 2014 8 , 778 , 848 B2 7 / 2014 Lin et al. (51 ) Int. Ci. ( Continued ) C08F 20/ 60 ( 2006 .01 ) FOREIGN PATENT DOCUMENTS C120 1/ 68 ( 2006 .01 ) C08F 220 / 56 ( 2006 . 01 ) EP 0 742 287 A2 11 / 1996 C08F 8 /32 (2006 .01 ) EP 0 799 897 Al 10 / 1997 C08F 29300 ( 2006 .01 ) WO WO 89 / 10977 11/ 1989 C40B 50 / 18 ( 2006 .01 ) WO WO 91/ 066785 / 1991 (52 ) U . S . CI. ( Continued ) CPC ...... C08F 20 /60 (2013 .01 ) ; C08F 8 /32 ( 2013. 01 ) ; C08F 220/ 56 ( 2013 . 01 ) ; C08F OTHER PUBLICATIONS 293 /005 (2013 . 01 ); C12Q 1 /6874 ( 2013 .01 ) ; B01J 2219 / 00637 ( 2013 . 01 ); B01J 2219 / 00722 Sumerlin , et al. , “ Highly Efficient “ Click ” Functionalization of (2013 .01 ) ; C08F 2438 /03 (2013 .01 ) ; C40B Poly ( 3 - azidopropyl methacrylate ) Prepared by ATRP ” , Macromol 50 / 18 ( 2013 .01 ) ecules, 2005 , vol. 38 , pp . 7540 -7545 . ( 58 ) Field of Classification Search ( Continued ) CPC ...... C08F 8/ 32 ; C08F 293 /005 ; C08F 220 / 56 ; C08F 2438 /03 ; C12Q 1 /6874 ; C40B 50 / 18 ; B01J 2219/ 00637 ; B01J Primary Examiner — Robert Jones , Jr . 2219 / 00722 See application file for complete search history . (57 ) ABSTRACT (56 ) References Cited Some embodiments described herein relate to new polymer U . S . PATENT DOCUMENTS coatings for surface functionalization and new processes for grafting pre - grafted DNA -copolymers to surface ( s ) of sub 5 , 130 , 238 A 7 / 1992 Malek et al. strates for use in DNA sequencing and other diagnostic 5 , 429 ,807 A 7 / 1995 Matson et al . 5 , 436 , 327 A 7 / 1995 Southern et al. applications. 5 , 455 , 166 A 10 / 1995 Walker 5 , 561, 071 A 10 / 1996 Hollenberg et al. 5 ,583 ,211 A 12 / 1996 Coassin et al . 18 Claims, 11 Drawing Sheets US 9, 815 ,916 B2 Page 2 ( 56 ) References Cited Bains et al. , A Novel Method for Nucleic Acid Sequence Determi nation , Journal of Theoretical Biology, 1988 , 135 ( 3 ) : 303 - 7 . U . S . PATENT DOCUMENTS Barker et al. , Tetrazine -Norbornene Click Reactions to Functional ize Degradable Polymers Derived From Lactide, Macromolecular 8 ,778 , 849 B2 7 /2014 Bowen et al. 2002 /0055100 Al 5 / 2002 Kawashima et al . Rapid Communications, 2011 , 32 ( 17 ): 1362 - 1366 . 2002 /0102578 A1 8 /2002 Dickinson et al. Bentley et al ., Accurate Whole Human Genome Sequencing Using 2004 / 0002090 A1 1/ 2004 Mayer et al . Reversible Terminator Chemistry , Nature, 2008 , 456 : 53 -59 . 2004 /0096853 Al 5 / 2004 Mayer Chen et al. , Clicking 1 , 2 , 4 , 5 -tetrazine and Cyclooctynes with Tun 2005 / 0053980 A 3 /2005 Gunderson et al. able Reaction Rates, Chem . Commun ., 2012 , 48 : 1736 - 1738 . 2005 /0064460 A1 3 /2005 Holliger et al. Cheng et al ., A Facile Method for the Preparation of Thermally 2005/ 0130173 Al 6 / 2005 Leamon et al. Remendable Cross -Linked , Journal of Polymer 2005 /0181440 Al 8 / 2005 Chee et al . 2005 /0191698 A1 9 / 2005 Chee et al . Science , Part A : Polymer Chemistry, 2013 , 51: 1205 - 1214 . 2007 /0099208 Al 5 / 2007 Drmanac et al. Dean et al. , Comprehensive Human Genome Amplification Using 2007 /0128624 Al 6 / 2007 Gormley et al. Multipole Displacement Amplification , Proc . Natl . Acad . Sci ., 2002 , 2008/ 0009420 A1 1 / 2008 Schroth et al . 99 : 5261- 66 . 2008 /0108082 A1 5 / 2008 Rank et al. Dressman et al ., Transforming Single DNA Molecules into Fluo 2009 /0026082 Al 1 / 2009 Rothberg et al . rescent Magnetic Particles for Detection and Enumeration of 2009 /0127589 Al 5 / 2009 Rothberg et al . 2009 /0186349 Al 7 / 2009 Gunderson et al . Genetic Variations, Proc . Natl . Acad . Sci. , 2003 , 100 : 8817 -8822 . 2010 /0111768 A15 / 2010 Banerjee et al. Drmanac et al. , Accurate Sequencing by Hybridization for DNA 2010 /0137143 A1 6 / 2010 Rothberg et al. Diagnostics and IndividualGenomics , Nature Biotechnology , 1998, 2010 /0282617 A111/ 2010 Rothberg et al. 16 : 54 - 58 . 2011/ 0059865 Al 3 / 2011 Smith et al . Fodor et al. , Light - Directed , Spatially Addressable Parallel Chemi 2011 /0172118 A1 7 / 2011 Kain et al. cal Synthesis , Science , 1991, 251( 4995 ) : 767- 773 . 2012 / 0270305 Al 10 /2012 Reed et al. Korlach et al ., Selective Aluminum Passivation for Targeted Immo 2014 / 0243224 AL 8 / 2014 Barnard et al. bilization of Single DNA Polymerase Molecules in Zero -Mode Waveguide Nanostructures , Proc . Natl. Acad . Sci. , 2008 , 105 , FOREIGN PATENT DOCUMENTS 1176 - 1181 . Lage et al. , Whole Genome Analysis of Genetic Alterations in Small WO WO 93 / 17126 AL 9 / 1993 DNA Samples Using Hyperbranched Strand Displacement Ampli WO WO 95 / 11995 AL 5 / 1995 fication and Array - CGH , Genome Research , 2003 , 13 :294 -307 . WO WO 95 / 35505 A1 12 / 1995 Levene et al ., Zero -Mode Waveguides for Single -Molecule Analysis WO WO 00 /63437 10 / 2000 WO WO 2004 /018497 3 /2004 at High Concentrations, Science , 2003 , 299 : 682 -686 . WO WO 2005 /010145 2 / 2005 Lizardi et al. , Mutation Detection and Single -Molecule Counting WO WO 2007 / 123744 11 / 2007 Using Isothermal Rolling -Circle Amplification , Nat. Genet ., 1998 , WO WO 2012 /058096 5 / 2012 19 : 225 -232 . Wo WO 2013 / 184796 12 / 2013 Lundquist et al ., Parallel Confocal Detection of Single Molecules in Real Time, Opt. Lett . , 2008 , 33 , 1026 - 1028 . Qiu et al ., Reverse Self - Assemblies Based on Amphiphilic OTHER PUBLICATIONS Polyphosphazenes for Encapsulation of Water - Soluble Molecules, Nanotechnology , 2007 , 18 , 475602 . Chen , “ Surface Modification with Polymers Using Living Radical Ronaghi et al. , A Sequencing Method Based on Real- Time Polymerisation and Click Chemistry ” , A Thesis Submitted for the Pyrophosphate , Science , 1998 , 281( 5375 ), 363 . Degree of PhD at the University of Warwick , 2007 , Retrieved from Ronaghi , et al. , Real - Time DNA Sequencing Using Detection of the Internet on Apr . 11 , 2016 at URL : http : // wrap .warwick .ac . uk / Pyrophosphate Release , Analytical Biochemistry , 1996 , 242 ( 1 ) , 4462/ 1 /WRAP _ THESIS _ Chen _ 2007 . pdf . 84 - 9 . International Search Report and Written Opinion of the Interna Ronaghi , Pyrosequencing Sheds Light on DNA Sequencing , tional Searching Authority issued in PCT/ EP2015 /074759 dated Genome Res ., 2001, 11 (1 ), 3 - 11. Apr. 20 , 2016 Shendure et al ., Accurate Multiplex Polony Sequencing of an Walker et al . , ( 1995 ) A chemiluminescent DNA probe test based on Evolved Bacterial Genome, Science , 2005, 309 :1728 - 1732 . strand displacement amplification . In Danny L . Wiedbrauk and Walker et al. , Strand Displacement Amplification - An Isothermal , Daniel H . Farkas (Eds . ), Molecular Methods for Virus Detection In Vitro DNA Amplification Technique, Nucl . Acids Res. , 1992 , (pp . 329 -349 ). San Diego: Academic Press , Inc . 20 : 1691 -96 . atent Nov . 14 , 2017 Sheet 1 of 11 US 9 ,815 ,916 B2

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A- STERIIDROM A A A A A US 9 ,815 ,916 B2 POLYMERS AND DNA COPOLYMER Some embodiments described herein are related to a COATINGS polymer for surface functionalization , comprising a recur ring unit of Formula ( I ) and a recurring unit of Formula ( II ) : INCORPORATION BY REFERENCE TO PRIORITY APPLICATIONS 5 The present application claims the benefit of priority to U .S . Provisional PatentApplication No . 62 /073 , 764 , filed on -E3 Oct. 31, 2014 , which is hereby expressly incorporatedod byby 10 reference in its entirety . N — R3a , REFERENCE TO SEQUENCE LISTING Rla The present application includes a sequence listing in R2a Electronic format. The Sequence Listing is provided as a file ( II) entitled ILLINC .267A . txt, created Jan . 7 , 2016 which is NH2 approximately 5 KB in size . The information in the elec Z-Y tronic format of the sequence listing is incorporated herein by reference in its entirety . 20 N - R36 FIELD In general, the present application relates to the fields of R 1b chemistry, biology and material science . More specifically, 25 R26 the present application relates to novel polymer coatings and grafted DNA - copolymers to support substrate surface func wherein each Ria , R20 , R1b and R26 is independently tionalization and downstream applications , such as DNA selected from hydrogen , optionally substituted alkyl or sequencing and other diagnostic applications. Methods for optionally substituted phenyl; each R ” and R ” is indepen preparing such functionalized surface and the use thereof are 30 dently selected from hydrogen , optionally substituted alkyl, optionally substituted phenyl, or optionally substituted C7- 14 also disclosed . aralkyl; and each L and L is independently selected from BACKGROUND an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker . In some embodiments , the Polymer or hydrogel- coated substrates are used in many 35 polymer may further comprise one or more recurring units technological applications. For example , implantable medi selected from the group consisting of polyacrylamides, cal devices can be coated with biologically inert polymers . polyacrylates, polyurethanes , polysiloxanes , Silicones, poly In another example , polymer or hydrogel coated substrates acroleins , polyphosphazenes , polyisocyanates , poly -ols , and are used for the preparation and / or analysis of biological polysaccharides , or combinations thereof. In some such molecules . Molecular analyses , such as certain nucleic acid 40 embodiments , the polymer may further comprise one or sequencing methods, rely on the attachment of nucleic acid more recurring units of polyacrylamide of Formula ( IIIa ) or strands to a polymer or hydrogel- coated surface of a sub - (111b ) or both : strate . The sequences of the attached nucleic acid strands can then be determined by a number of different methods that are well known in the art . 45 ( IIIa ) In certain Sequencing - by - Synthesis (“ SBS ” ) processes , one ormore surfaces of a flow cell are coated with a polymer or a hydrogel to which primers (single stranded DNA or ssDNA ) are then grafted . However , there is an inherent cost associated with performing the coating, grafting and quality 50 control steps . R4aLe ( IIIb ) SUMMARY The present application discloses polymer coatings that 55 are useful for SBS applications and processes of incorpo R 46 rating the primer polymer coupling steps into the initial ROBROD R76 RS polymer synthesis . This may eliminate some or all of the X grafting process undertaken to manufacture a sequencing flow cell or other substrate used for SBS . These processes 60 wherein each R40 , R4h and RS is selected from hydrogen may maximize primer accessibility to the downstream bio - or C1- 3 alkyl; and each RS4, R6a , Rob and R75 is indepen chemistry, minimize side reactions and yield a more efficient dently selected from hydrogen , optionally substituted C1- 6 surface chemistry. The coatings and processes disclosed alkyl or optionally substituted phenyl. herein are useful for other analytical apparatus and processes Some embodiments described herein are related to a including, but not limited to , those used for synthesis or 65 substrate having a first surface comprising a polymer with a detection of nucleic acids and other biologically active recurring unit of Formula ( I ) and a recurring unit of Formula molecules. ( II ) as described herein . In some embodiments , the polymer US 9 ,815 ,916 B2 may further comprise one or more recurring units of various different polymer backbones as described above , for example , one or more recurring units of polyacrylamide of Formula ( IIIa ) or ( IIIb ) or both . mi In some embodiments , when the polymer is covalently 5 Z=> attached to the first surface of the substrate , at least one w covalent bond is formed between the amino group of the LZ- winy - recurring unit of Formula ( II ) and the first surface of the - substrate . Therefore, as described herein , a substrate having 10 z a first surface comprising a polymer with a recurring unit of N - R3a or N - R3a Formula ( I ) and a recurring unit of Formula ( II ) covalently bonded thereto , should be understood to also include the polymer with a modified recurring unit of the structure Rla Rla 15 R20 R20 showing the covalent bonding position with functionalized oligonucleotide , wherein - - - - - is a single or double bond . porno Some embodiments described herein are related to a 24 20 polymer for surface functionalization , comprising a recur N — R35 ring unit of Formula ( IV ) :

(IV ) R 16 25 R 25 ???|PIN showing the covalent bonding position with the substrate surface . 30 N — R3 Some embodiments described herein are related to a grafted polymer comprising functionalized oligonucleotides covalently bonded to a polymer with a recurring unit of Formula ( I) and a recurring unit of Formula ( II ) as described RIC herein . In some embodiments , the polymer may further 35 Rc comprise one or more recurring units of various different polymer backbones as described above , for example , one or wherein each Rle and R2C is independently selected from more recurring units of polyacrylamide of Formula ( IIIa ) or hydrogen , optionally substituted alkyl or optionally substi tuted phenyl; R3c is selected from hydrogen , optionally ( IIIb ) or both . 40 substituted alkyl, optionally substituted phenyl, or option In some embodiments, when functionalized oligonucle - * ally substituted C7. 14 aralkyl; Ar is selected from an option otides are covalently bonded to the polymer, at least two ally substituted C6- 10 aryl or an optionally substituted 5 or 6 covalent bonds are formed as the result of a reaction between membered heteroaryl; R4 is optionally substituted tetrazine ; the azido group of the recurring unit of Formula (I ) and a and L is selected from a single bond , an optionally substi functionalized oligonucleotide . Therefore , as described 45 tuted alkylene linker or an optionally substituted heteroalky herein , a grafted polymer comprising functionalized oligo lene linker . In some embodiments , the polymer may further nucleotides covalently bonded to a polymer of a recurring comprise one or more recurring units selected from the unit of Formula ( 1 ) and a recurring unit of Formula ( II ) , group consisting of polyacrylamides , polyacrylates, poly should be understood to also include the polymer with a urethanes , polysiloxanes, silicones , polyacroleins, poly modified recurring unit of the structure 50 phosphazenes , polyisocyanates , poly -ols , and polysaccha rides, or combinations thereof. In some such embodiments , the polymer may further comprise one or more recurring units of polyacrylamide of Formula ( IIIa ) or ( IIIb ) or both , mm with the structure shown above . SS Some embodiments described herein are related to a substrate having a first surface comprising a polymer with a ZZ recurring unit of Formula ( IV ) as described herein . In some embodiments , the polymer may further comprise one or more recurring units of various different polymer backbones 60 as described above , for example , one or more recurring units N — R3a , of polyacrylamide of Formula (IIIa ) or ( IIIb ) or both . In some embodiments , when the polymer is covalently attached to the first surface of the substrate , at least two Rla covalentbonds are formed as the result of a reaction between R20 65 the tetrazine group of the recurring unit of Formula ( IV ) and the first surface ofthe substrate . In some other embodiments, at least two covalent bonds are formed between the tetrazine US 9 ,815 ,916 B2 group of the recurring unit of Formula (IV ). Therefore, as wherein the moiety Ar - RAB is selected from described herein , a substrate having a first surface compris ing a polymer with a recurring unit of Formula ( IV ) as described herein , should be understood to also include the ? polymer with a modified recurring unit of the structure 5 mi NH NH or . N , er m m 10 Ar Ar showing the covalent bonding position with the oligonucle ?????N — R3c otide ; and wherein - - - - - is a single or double bond . RAB may be optionally substituted . Some embodiments described herein are related to a polymer for surface functionalization , comprising a recur RIC ring unit of Formula (V ): R 2c

20 wherein the moiety Ar - R44 is selected from HO . 1 - RB

R3d NH 25 Z- wy ? NH or mi Rld mi Ar mi AL R2d 30 wherein each Rid and R20 is independently selected from showing the covalent bonding position with the substratea hydrogen , optionally substituted alkyl or optionally substi surface; and wherein - - - - - is a single or double bond . RAM tuted phenyl; each R3d is selected from hydrogen , optionally may be optionally substituted . 35 substituted alkyl , optionally substituted phenyl, or option Some embodiments described herein are related to a ally substituted C7- 4 aralkyl; RB is selected from azido , grafted polymer comprising functionalized oligonucleotides optionally substituted amino , Boc- protected amino , covalently bonded to a polymer with a recurring unit of hydroxy, thiol, alkynyl , alkenyl, halo , epoxy, tetrazinyl or Formula ( IV ) as described herein . In some embodiments , the aldehyde; each L4 and L is independently selected from an polymer may further comprise one or more recurring units 40 optionally substituted alkylene linker or an optionally sub of various different polymer backbones as described above, stituted heteroalkylene linker. In some embodiments , the for example , one or more recurring units of polyacrylamide polymer may further comprise a recurring unit of Formula of Formula ( IIIa ) or ( IIIb ) or both . (VIa ) or (VIb ) , or both : In some embodiments , when functionalized oligonucle otides are covalently bonded to the polymer , at least two ( VIa ) covalent bonds are formed as the result of a reaction between the tetrazine group of the recurring unit of Formula ( IV ) and a functionalized oligonucleotide . Therefore , as described herein , a grafted polymer comprising functionalized oligo nucleotides covalently bonded to a polymer of a recurring unit of Formula ( IV ) , should be understood to also include the polymer with a modified recurring unit of the structure Rle 55 R2e ( VID ) OH

OH

N - R3C,

RIC RIf R20 R ? US 9 ,815 ,916 B2 wherein each Rle , Rze , R1 and Raf is independently showing the covalent bonding position with the substrate selected from hydrogen , optionally substituted alkyl or surface . optionally substituted phenyl. In some embodiments , the In some other embodiments , when the polymer is cova polymer may further comprise one or more recurring units lently attached to the first surface of the substrate , at least selected from the group consisting of polyacrylamides, 5 one covalent bond is formed between the amino group of the polyacrylates , polyurethanes , polysiloxanes , silicones , poly - recurring unit of Formula ( V ) and the first surface of the acroleins, polyphosphazenes , polyisocyanates, poly -ols , and substrate . Therefore , as described herein , a substrate having polysaccharides , or combinations thereof. a first surface comprising a polymer with a recurring unit of Some embodiments described herein are related to a Formula ( V ) covalently bonded thereto , should be under substrate having a first surface comprising a polymer with a stood to also include the polymer with a modified recurring recurring unit of Formula ( V ) as described herein . In some unit of the structure embodiments , the polymer may further comprise a recurring unit of Formula (Vla ) or (Vb ) , or both . In some embodi ments , the polymer may further comprise one or more recurring units of various different polymer backbones as described above . 15 HOA L – NH . In some embodiments , when the polymer is covalently 1. 4 wenn attached to the first surface of the substrate , at least two R3d covalent bonds are formed as the result of a reaction between the azido group of the recurring unit of Formula ( V ) and the first surface of the substrate . Therefore , as described herein , 20 a substrate having a first surface comprising a polymer with a recurring unit of Formula ( V ) covalently bonded thereto , should be understood to also include the polymer with a Rld modified recurring unit of the structure R2d 25 showing the covalent bonding position with the substrate surface . Some embodiments described herein are related to a min grafted polymer comprising functionalized oligonucleotides min 30 covalently bonded to a polymer with a recurring unit of HO L - N Formula ( V ) as described herein . In some embodiments , the polymer may further comprise a recurring unit of Formula NEN (Vla ) or ( VIb ), or both . In some embodiments , the polymer R3d may further comprise one or more recurring units of various different polymer backbones as described above . In some embodiments , when functionalized oligonucle otides are covalently bonded to the polymer , at least one covalent bond is formed as the result of a reaction between Rld the epoxy group of the recurring unit of Formula (Vla ) and R2d a functionalized oligonucleotide . Therefore , as described 40 herein , a grafted polymer comprising functionalized oligo nucleotides covalently bonded to a polymer of a recurring mm unit of Formula ( V ) and a recurring unit of Formula ( VIa ) , should be understood to also include the polymer with a HO . 1 - N modified recurring unit of the structure Z N = N or 45 - 15 R3d min 50 Ria OH R2d

55 HO . L - N mam Rle ' N R2e ?A R3d 60 showing the covalent bonding position with the oligonucle otide . Some embodiments described herein are related processes for immobilizing a grafted polymer to a first surface of a Rld substrate , comprising : R2d 65 providing a substrate having a first surface comprising a first plurality of functional groups covalently attached thereto ; US 9 ,815 ,916 B2 10 providing a grafted polymer comprising functionalized wherein n is an integer in the range of 1 - 20 , 000 , and m is oligonucleotides covalently bonded to a polymer, an integer in the range of 1 - 100 , 000 . In some such embodi wherein the polymer comprises a second plurality of ments , the unsaturated moieties of the functionalized silane functional groups ; and reacting the first plurality functional groups of the first 5 comprise norbornene . surface with the second plurality of functional groups 5 Some embodiments described herein are related processes of the polymer such that the polymer is covalently or methods for immobilizing a polymer described herein to bonded to the first surface of the substrate . a first surface of a substrate , comprising: providing a sub In some embodiments of the methods described herein , strate having a first surface comprising a first plurality of when the surface is treated with functionalized silane com functional groups covalently attached thereto ; providing a prising unsaturated moieties selected from cycloalkenes , polymer described herein ; and reacting the first plurality cycloalkynes, heterocycloalkenes, heterocycloalkynes ; and functional groups of the first surface with the polymer such the functionalized oligonucleotides comprise bicyclo [6 . 1. 0 ] that the polymer is covalently bonded to the first surface of non - 4 - yne ; then the polymer is not a poly ( N - ( 5 - azidoacet the substrate . amidylpentyl) acrylamide -co - acrylamide ) ( PAZAM ) of the 15 following structure : BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A to 1D show the Typhoon florescence image of the polymers coated flow cell with norbornene silanemono N3 layer surface and the related bar chart of median Typhoon 20 intensity of the polymers of Example 1 ( Table 2 ) . FIGS. 2A to 2D show the Typhoon florescence image of NH the polymers coated flow cell with norbornene silane mono layer surface and the related bar chart of median Typhoon intensity of the polymers of Example 1 ( Table 3 ) . 25 FIG . 3A is a line and bar chart that illustrates the TET QC intensity data ( Table 4 ) after coating a norbornene surface with different polymers as listed in Example 1 ( Table 2 ) and surface loss percentage as measured after a thermal Stress Test. NH OYNH2 30 FIG . 3B is a line and bar chart that illustrates the TET QC intensity data ( Table 5 ) after coating an epoxy surface with different polymers as listed in Example 1 ( Table 3 ) and surface loss percentage as measured after a thermal Stress A Test . N3; 35 FIGS . 4A to 4D show the Typhoon florescence image of ?? the polymers coated flow cell with norbornene silane mono layer surface and the related bar chart of median Typhoon intensity of the polymers of Example 1 ( Table 6 ) . FIG . 4E is a line and bar chart that illustrates the TET OC 40 intensity data ( Table 7 ) after coating a norbornene surface with different polymers as listed Example 1 ( Table 6 ) and surface loss percentage as measured after a thermal Stress ON NHA O , NH ON NHA Test . FIG . 5 shows a series of NMR images of a reaction 45 between norbornene and a bipyridyl tetrazine at different time points. FIG . 6 shows a line graph of a UV - Vis absorption pattern of a reaction between bipyridyl tetrazine and bicyclo [ 6 . 1 . 01 non - 4 - yn - 9 - yl methanol. 50 FIG . 7 shows the coupling reaction between a grafted dendrimer and a functionalized dendrimer with surface y Nz; attachment groups. NH DETAILED DESCRIPTION 55 The present application relates to nucleic acid -copoly mers ( for example , DNA - copolymers and processes for grafting such nucleic acid - copolymers to the surface of a substrate . Some embodiments of polymers used for pre 60 conjugation with single stranded DNA ( ssDNA ” ) primers include acrylamide/ azido -acrylamide / aminoethyl- acrylam ide ternary copolymers , tetrazine modified polyacrylamide, and the reaction products of poly ( glycidyl methacrylate ) with amino - PEG - azide or amino - PEG -Boc - amide. The 65 nucleic acid -copolymer can then be covalently attached to a surface of a substrate , in some instances, a silane function alized surface of a substrate , such as a surface of a flow cell US 9 ,815 ,916 B2 12 or a surface of a molecular array. The present disclosure also CRP Controlled radical relates to methods of preparing such nucleic acid -copolymer CVD Chemical vapor deposition coated surfaces and methods of using substrates comprising ° C . Temperature in degrees Centigrade such nucleic acid - copolymer coated surfaces in sequencing DATP Deoxyadenosine triphosphate by -synthesis reactions . 5 dCTP Deoxycytidine triphosphate Some embodiments relate to flow cells for performing dGTP Deoxyguanosine triphosphate sequencing -by -synthesis reactions that include functional dTTP Deoxythymidine triphosphate ized oligonucleotides pre - conjugated to a polymer described DCA Dichloroacetic acid herein through one or more functional moieties , such as DCE 1 , 2 -Dichloroethane bicyclo [ 6 . 1 . 0 ]non - 4 -yne , alkyne, amido or azido derivatized 10 DCM Methylene chloride linkage . In some embodiments , the primers are a P5 or P7 DIEA Diisopropylethylamine primer . The P5 and P7 primers are used on the surface of DIPEA Diisopropylethylamine commercial flow cells sold by Illumina Inc . for sequencing DMA Dimethylacetamide on the HiSeq® , MiSeq® , NextSeq® and Genome Ana DME Dimethoxyethane lyzer® platforms. 15 DMF N .NP - Dimethylformamide Further process and cost savings may be achieved by DMSO Dimethylsulfoxide incorporating quality control ( QC ) markers within the poly DPPA Diphenylphosphoryl azide mer along with the primer attachment . Analytical tests may Et Ethyl be used to determine the quality and consistency of the EtOAc or EA Ethyl acetate DNA - copolymer with or without QC markers , and used 20 g Gramme( s ) again to determine effectiveness of deposition when working h or hr Hour( s ) with an open wafer format. These additional quality control iPr Isopropyl checkpoints should reduce flow cell batch to batch variation KPi 10 mM potassium phosphate buffer at pH 7 .0 to yield more consistent products, and also help narrow KPS Potassium persulfate down where process deviation has occurred when failures 25 IPA Isopropyl Alcohol during manufacturing appear. IPHA .HCI N - Isopropylhydroxylamine hydrochloride m or min Minute ( s ) Definitions MeOH Methanol MeCN Acetonitrile Unless defined otherwise , all technical and scientific 30 mL Milliliter ( s ) terms used herein have the same meaning as is commonly NaNz Sodium Azide understood by one of ordinary skill in the art. The use of the NHS N -hydroxysuccinimide term “ including ” as well as other forms, such as “ include” , NMP Nitroxide- mediated radical polymerisation " includes, ” and “ included , ” is not limiting . The use of the PAZAM poly ( N - ( 5 - azidoacetamidylpentyl) acrylamide term “ having” as well as other forms, such as " have ” , " has , " 35 co - acrylamide) of any acrylamide to Azapa ratio and “ had ,” is not limiting . As used in this specification , PEG Polyethylene glycol whether in a transitional phrase or in the body of the claim , PG Protecting group the terms " comprise ( s ) ” and “ comprising ” are to be inter PGMA Poly ( glycidyl methacrylate ) preted as having an open -ended meaning That is , the above Ph Phenyl terms are to be interpreted synonymously with the phrases 40 ppt Precipitate “ having at least” or “ including at least .” For example , when RAFT Reversible addition - fragmentation chain transfer used in the context of a process , the term “ comprising " polymerisation means that the process includes at least the recited steps, but rt Room temperature may include additional steps . When used in the context of a SFA Silane Free Acrylamide as defined in U .S . Pat. Pub . compound , composition , or device , the term “ comprising” 45 No . 2011 /0059865 meansm that the compound , composition , or device includes Sulfo -HSAB or SHSAB N -Hydroxysulfosuccinimidyl - 4 at least the recited features or components , but may also azidobenoate include additional features or components . TEA Triethylamine As used herein , common organic abbreviations are Tert, t tertiary defined as follows : 50 THF Tetrahydrofuran Ac Acetyl TEMED Tetramethylethylenediamine Ac20 Acetic anhydride YES Yield Engineering Systems APTS aminopropyl silane uL Microliter (s ) APTES ( 3 - aminopropyl) triethoxysilane As used herein , the term “ array ” refers to a population of APTMS ( 3 - aminopropyl) trimethoxysilane 55 different probe molecules that are attached to one or more aq . Aqueous substrates such that the different probe molecules can be ATRP Atom -transfer radical polymerization differentiated from each other according to relative location . Azapa N -( 5 - azidoacetamidylpentyl) acrylamide An array can include different probe molecules that are each BCN Bicyclo [ 6 . 1 . O ]non - 4 -yne located at a different addressable location on a substrate . Bn Benzyl 60 Alternatively or additionally , an array can include separate Brapa or BRAPA N - (5 -bromoacetamidylpentyl ) acrylam substrates each bearing a different probe molecule , wherein ide the different probemolecules can be identified according to Bz Benzoyl the locations of the substrates on a surface to which the BOC or Boc tert - Butoxycarbonyl substrates are attached or according to the locations of the Bu n - Butyl 65 substrates in a liquid . Exemplary arrays in which separate cat . Catalytic substrates are located on a surface include, without limita CMP Chemical mechanical polishing tion , those including beads in wells as described , for US 9 ,815 ,916 B2 13 14 example, in U . S . Pat. No . 6 , 355 ,431 B1, US 2002 /0102578 the present definition also covers the occurrence of the term and PCT Publication No. WO 00 /63437 . Exemplary formats " alkenyl” where no numerical range is designated . The that can be used in the invention to distinguish beads in a alkenyl group may also be a medium size alkenyl having 2 liquid array, for example , using a microfluidic device , such to 9 carbon atoms. The alkenyl group could also be a lower as a fluorescent activated cell sorter (FACS ) , are described , 5 alkenyl having 2 to 4 carbon atoms. The alkenyl group may for example , in U . S . Pat . No. 6 ,524 ,793 . Further examples of be designated as “ C2- 4 alkenyl” or similar designations. By arrays that can be used in the invention include , without way of example only , “C2 - 4 alkenyl” indicates that there are limitation , those described in U . S . Pat . Nos. 5 ,429 , 807 ; two to four carbon atoms in the alkenyl chain , i. e . , the 5 ,436 , 327 ; 5 , 561, 071; 5 , 583 ,211 ; 5 , 658, 734 ; 5 ,837 , 858 ; alkenyl chain is selected from the group consisting of 5, 874 ,219 ; 5 ,919 ,523 ; 6, 136 , 269 ; 6 ,287 , 768 ; 6, 287 , 776 ; 10 ethenyl, propen - 1 - yl, propen - 2 - yl, propen - 3 - yl , buten - 1 - yl, 6 ,288 ,220 ; 6 , 297 ,006 ; 6 ,291 , 193 ; 6 ,346 ,413 ; 6 ,416 , 949 ; buten -2 -yl , buten -3 - yl, buten -4 -yl , 1 -methyl - propen - 1- yl, 6 ,482 ,591 ; 6 ,514 ,751 and 6 ,610 ,482 ; and WO 93 / 17126 ; 2 -methyl - propen - 1 - yl, 1 - ethyl- ethen - 1 - yl, 2 -methyl - propen WO 95 / 11995 ; WO 95 / 35505 ; EP 742 287 ; and EP 799 897 . 3 -yl , buta - 1 ,3 -dienyl , buta - 1 ,2 ,- dienyl, and buta - 1 ,2 -dien - 4 As used herein , the term " covalently attached ” or onding“ cova 15 yl. Typical alkenyl groups include, but are in no way limited that is characterized by the sharing of pairs of electrons to , ethenyl, propenyl , butenyl, pentenyl, and hexenyl, and between atoms. For example , a covalently attached polymer the like . The alkenyl group can be optionally substituted . coating refers to a polymer coating that forms chemical As used herein , “ alkynyl” refers to a straight or branched bonds with a functionalized surface of a substrate , as com hydrocarbon chain containing one or more triple bonds. The pared to attachment to the surface via other means , for 20 alkynyl group may have 2 to 20 carbon atoms, although the example, adhesion or electrostatic interaction . It will be present definition also covers the occurrence of the term appreciated that polymers that are attached covalently to a “ alkynyl ” where no numerical range is designated . The surface can also be bonded via means in addition to covalent alkynyl group may also be a medium size alkynyl having 2 attachment. to 9 carbon atoms. The alkynyl group could also be a lower As used herein , “ C , to Ch ” or “ Col” in which “ a ” and “ b ” 25 alkynyl having 2 to 4 carbon atoms. The alkynyl group may are integers refer to the number of carbon atoms in the be designated as “ C2. 4 alkynyl” or similar designations . By specified group . That is , the group can contain from “ a ” to way of example only , “ C2- 4 alkynyl” indicates that there are “ b ” , inclusive, carbon atoms. Thus , for example , a " C , to C4 two to four carbon atoms in the alkynyl chain , i .e ., the alkyl” or “ C , . , alkyl” group refers to all alkyl groups having alkynyl chain is selected from the group consisting of from 1 to 4 carbons, that is , CH3 – CH2CH2- , 30 ethynyl , propyn - 1 - yl, propyn - 2 - yl , butyn - 1 - yl, butyn - 3 - yl, CH2CH ,CH , – ( CHZ) , CH? , CH2CH2CH ,CH , — , butyn - 4 -yl , and 2 -butynyl . Typical alkynyl groups include , CH2CH2CH (CH3 ) and (CH3 ) 3C — . but are in no way limited to , ethynyl, propynyl, butynyl, The term “ halogen ” or “ halo , " as used herein , means any pentynyl , and hexynyl, and the like . The alkynyl group can one of the radio -stable atoms of column 7 of the Periodic be optionally substituted . Table of the Elements , e . g . , fluorine , chlorine, bromine , or 35 As used herein , " heteroalkyl” refers to a straight or iodine , with fluorine and chlorine being preferred . branched hydrocarbon chain containing one or more het As used herein , “ alkyl” refers to a straight or branched eroatoms, that is , an element other than carbon , including hydrocarbon chain that is fully saturated ( i. e ., contains no but not limited to , , oxygen and sulfur, in the chain double or triple bonds ) . The alkyl group may have 1 to 20 backbone. The heteroalkyl group may have 1 to 20 carbon carbon atoms (whenever it appears herein , a numerical range 40 atoms, although the present definition also covers the occur such as “ 1 to 20 ” refers to each integer in the given range ; rence of the term " heteroalkyl” where no numerical range is e . g ., “ 1 to 20 carbon atoms” means that the alkyl group may designated . The heteroalkyl group may also be a medium consist of 1 carbon atom , 2 carbon atoms, 3 carbon atoms, size heteroalkyl having 1 to 9 carbon atoms. The heteroalkyl etc ., up to and including 20 carbon atoms, although the group could also be a lower heteroalkyl having 1 to 4 carbon present definition also covers the occurrence of the term 45 atoms. The heteroalkyl group may be designated as “ C1- 4 " alkyl” where no numerical range is designated ). The alkyl heteroalkyl” or similar designations. The heteroalkyl group group may also be a medium size alkyl having 1 to 9 carbon may contain one or more heteroatoms. By way of example atoms. The alkyl group could also be a lower alkyl having only , “ C1- 4 heteroalkyl” indicates that there are one to four 1 to 4 carbon atoms. The alkyl group may be designated as carbon atoms in the heteroalkyl chain and additionally one “ C1- 4 alkyl” or similar designations . By way of example 50 or more heteroatoms in the backbone of the chain . only, “ C1- 4 alkyl” indicates that there are one to four carbon As used herein , " alkylene ” means a branched , or straight atoms in the alkyl chain , i .e ., the alkyl chain is selected from chain fully saturated di -radical chemical group containing the group consisting of methyl, ethyl, propyl, iso -propyl , only carbon and hydrogen that is attached to the rest of the n -butyl , iso -butyl , sec - butyl, and t - butyl. Typical alkyl molecule via two points of attachment (i . e ., an alkanediyl) . groups include , but are in no way limited to , methyl, ethyl, 55 The alkylene group may have 1 to 20 ,000 carbon atoms, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, although the present definition also covers the occurrence of hexyl, and the like . The alkyl group can be optionally the term alkylene where no numerical range is designated . substituted . The alkylene group may also be a medium size alkylene As used herein , “ alkoxy ” refers to the formula OR having 1 to 9 carbon atoms . The alkylene group could also wherein R is an alkyl as is defined above , such as “ C1. , 60 be a lower alkylene having 1 to 4 carbon atoms. The alkoxy " , including but not limited to methoxy, ethoxy , alkylene group may be designated as " C , 4 alkylene " or n - propoxy , 1 -methylethoxy (isopropoxy ) , n -butoxy , iso -bu - similar designations. By way of example only , “ C1- 4 alky toxy , sec - butoxy, and tert- butoxy, and the like . The alkoxy l ene” indicates that there are one to four carbon atoms in the group can be optionally substituted . alkylene chain , i. e ., the alkylene chain is selected from the As used herein , “ alkenyl” refers to a straight or branched 65 group consisting of methylene , ethylene, ethan - 1, 1 - diyl, hydrocarbon chain containing one or more double bonds. propylene , propan - 1 , 1 - diyl, propan - 2 , 2 - diyl, 1 -methyl - eth The alkenyl group may have 2 to 20 carbon atoms, although ylene, butylene , butan -1 , 1 -diyl , butan -2 , 2 -diyl , 2 -methyl US 9 ,815 ,916 B2 15 16 propan - 1 , 1 - diyl, 1 -methyl - propylene, 2 -methyl - propylene , An " aralkyl ” or “ arylalkyl” is an aryl group connected , as 1 , 1 -dimethyl - ethylene , 1, 2 - dimethyl- ethylene, and 1 - ethyl- a substituent, via an alkylene group , such as “ C7- 14 aralkyl” ethylene . and the like , including but not limited to benzyl, 2 -pheny As used herein , the term " heteroalkylene” refers to an lethyl, 3 - phenylpropyl, and naphthylalkyl . In some cases, alkylene chain in which one or more skeletal atoms of the 5 the alkylene group is a lower alkylene group ( i . e ., a C1 - 4 alkylene are selected from an atom other than carbon , e . g ., alkylene group ) . oxygen , nitrogen , sulfur, or combinations As used herein , " heteroaryl” refers to an aromatic ring or thereof. The heteroalkylene chain can have a length of 2 to ring system ( i .e ., two or more fused rings that share two 20 ,000 . Exemplary heteroalkylenes include , but are not adjacent atoms) that contain ( s ) one or more heteroatoms, limited to , — OCH , — , - OCH (CH3 ) - , - OC (CH3 ) 2 — 10 that is , an element other than carbon , including but not - OCH CH2 – CH ( CH3) O - , - CH2OCH — , limited to , nitrogen , oxygen and sulfur , in the ring backbone . - CHOCH2CH2- , SCH , — , - SCH (CH3 ) , SC When the heteroaryl is a ring system , every ring in the (CH3 ) 2 - , - SCH CH2 – CH SCH2CH2- , system is aromatic . The heteroaryl group may have 5 - 18 ring – NHCH , , NHCH ( CH?) , _ NHC( CH3) , , members (i . e ., the number of atoms making up the ring — NHCH2CH2 – CH NHCH2 – , CH NHCH2CH2 — , 15 backbone , including carbon atoms and heteroatoms) , and the like . although the present definition also covers the occurrence of As used herein , " alkenylene " means a straight or branched the term "heteroaryl ” where no numerical range is desig chain di- radical chemical group containing only carbon and nated . In some embodiments , the heteroaryl group has 5 to hydrogen and containing at least one carbon - carbon double 10 ring members or 5 to 7 ring members . The heteroaryl bond that is attached to the rest of the molecule via two 20 group may be designated as “ 5 - 7 membered heteroaryl, " points of attachment. The alkenylene group may have 2 to “ 5 - 10 membered heteroaryl, ” or similar designations . 20 ,000 carbon atoms, although the present definition also Examples of heteroaryl rings include , but are not limited to , covers the occurrence of the term alkenylene where no furyl , thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, numerical range is designated . The alkenylene group may imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, also be a medium size alkenylene having 2 to 9 carbon 25 thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, atoms. The alkenylene group could also be a lower alk - triazinyl, quinolinyl, isoquinlinyl, benzimidazolyl, benzox enylene having 2 to 4 carbon atoms. The alkenylene group azolyl, benzothiazolyl, indolyl, isoindolyl, and benzothie may be designated as “ C2- 4 alkenylene” or similar designa - nyl. The heteroaryl group can be optionally substituted . tions . By way of example only, “ C24 alkenylene ” indicates As used herein , " heteroarylene ” refers to an aromatic ring that there are two to four carbon atoms in the alkenylene 30 or ring system containing one or more heteroatoms in the chain , i. e . , the alkenylene chain is selected from the group ring backbone that is attached to the rest of the molecule via consisting of ethenylene, ethen - 1 , 1 - diyl, propenylene , pro - two points of attachment. pen - 1 , 1 -diyl , prop - 2 - en - 1 , 1 -diyl , 1 -methyl - ethenylene, but- A " heteroaralky ” or “ heteroarylalkyl” is heteroaryl group 1 - enylene, but- 2 - enylene , but- 1 , 3 -dienylene , buten - 1 , 1 -diyl , connected , as a substituent, via an alkylene group . Examples but- 1 , 3 - dien - 1 , 1 - diyl, but- 2 - en - 1 , 1 -diyl , but- 3 - en - 1 , 1 - diyl, 35 include but are not limited to 2 - thienylmethyl , 3 - thienylm 1 -methyl - prop - 2 - en - 1 , 1 - diyl, 2 -methyl - prop - 2 - en - 1 , 1 - diyl, ethyl, furylmethyl, thienylethyl , pyrrolylalkyl , pyridylalkyl, 1 - ethyl- ethenylene , 1 , 2 -dimethyl - ethenylene , 1 -methyl - pro isoxazollylalkyl, and imidazolylalkyl. In some cases, the penylene, 2 -methyl - propenylene , 3 -methyl -propenylene , alkylene group is a lower alkylene group ( i. e ., a C1- 4 2 -methyl - propen - 1 , 1 -diyl , and 2 , 2 -dimethyl - ethen - 1 , 1 - diyl. alkylene group ) . As used herein , “ alkynylene” means a straight or 40 As used herein , “ carbocyclyl” means a non -aromatic branched chain di- radical chemical group containing only cyclic ring or ring system containing only carbon atoms in carbon and hydrogen and containing at least one carbon - the ring system backbone . When the carbocyclyl is a ring carbon triple bond that is attached to the rest of the molecule system , two or more rings may be joined together in a fused , via two points of attachment . bridged or spiro - connected fashion . Carbocyclyls may have The term " aromatic ” refers to a ring or ring system having 45 any degree of saturation provided that at least one ring in a a conjugated pi electron system and includes both carbocy - ring system is not aromatic . Thus , carbocyclyls include clic aromatic ( e . g ., phenyl) and heterocyclic aromatic groups cycloalkyls , cycloalkenyls , and cycloalkynyls . The carbo ( e . g . , ) . The term includes monocyclic or fused -ring cyclyl group may have 3 to 20 carbon atoms, although the polycyclic ( i . e ., rings which share adjacent pairs of atoms) present definition also covers the occurrence of the term groups provided that the entire ring system is aromatic . 50 " carbocyclyl” where no numerical range is designated . The As used herein , " aryl” refers to an aromatic ring or ring carbocyclyl group may also be a medium size carbocyclyl system ( i. e . , two or more fused rings that share two adjacent having 3 , 4 , 5 , 6 , 7 , 8 , 9 or 10 carbon atoms. The carbocyclyl carbon atoms) containing only carbon in the ring backbone . group could also be a carbocyclyl having 3 to 6 carbon When the aryl is a ring system , every ring in the system is atoms. The carbocyclyl group may be designated as “ C3- 6 aromatic . The aryl group may have 6 to 18 carbon atoms, 55 carbocyclyl” or similar designations . Examples of carbocy although the presentdefinition also covers the occurrence of clyl rings include , but are not limited to , cyclopropyl, the term “ aryl ” where no numerical range is designated . In cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2 , 3 -di some embodiments , the aryl group has 6 to 10 carbon atoms. hydro - indene , bicycle / 2 . 2 . 2 ]octanyl , adamantyl, and spiro The aryl group may be designated as “ C6 -10 aryl, ” “ Coor C1 [4 .4 ]nonanyl . aryl, ” or similar designations. Examples of aryl groups 60 As used herein , " cycloalkyl” means a fully saturated include , but are not limited to , phenyl , naphthyl, azulenyl, carbocyclyl ring or ring system . Examples include cyclo and anthracenyl . The aryl group can be optionally substi - propyl , cyclobutyl, cyclopentyl , and cyclohexyl . tuted . As used herein , “ cycloalkylene ” means a fully saturated As used herein , " arylene” refers to an aromatic ring or carbocyclyl ring or ring system that is attached to the rest of ring system containing only carbon and hydrogen that is 65 the molecule via two points of attachment. attached to the rest of the molecule via two points of As used herein , " cycloalkenyl” or “ cycloalkene ” means a attachment. carbocyclyl ring or ring system having at least one double US 9 ,815 ,916 B2 18 bond , wherein no ring in the ring system is aromatic . An membered heterocyclyl, as defined herein . Non - limiting example is cyclohexenyl or cyclohexene. Another example examples include formyl , acetyl, propanoyl, benzoyl, and is norbornene or norbornenyl. acryl. As used herein , “ heterocycloalkeny?” or “ heterocycloalk - An “ O -carboxy ” group refers to a " _ OCCOR” group ene ” means a carbocyclyl ring or ring system with at least 5 in which R is selected from hydrogen , C . , alkyl, C2. 6 one heteroatom in ring backbone, having at least one double alkenyl, C2- 6 alkynyl, C3- 7 carbocyclyl, C6- 10 aryl, 5 - 10 bond , wherein no ring in the ring system is aromatic . In membered heteroaryl, and 5 - 10 membered heterocyclyl, as some embodiments , heterocycloalkenyl or heterocycloalk - defined herein . ene ring or ring system is 3 -membered , 4 -membered , A “ C - carboxy” group refers to a “ _ CEO )OR ” group in 5 -membered , 6 -membered , 7 -membered , 8 -membered , 9 10 which R is selected from hydrogen , C1- 6 alkyl, C2- 6 alkenyl, membered , or 10 membered . C2- 6 alkynyl, C3- 7 carbocyclyl, C6- 10 aryl, 5 - 10 membered As used herein , " cycloalkynyl" or " cycloalkyne ” means a heteroaryl , and 5 - 10 membered heterocyclyl, as defined carbocyclyl ring or ring system having at least one triple herein . A non - limiting example includes carboxyl ( i . e . , bond , wherein no ring in the ring system is aromatic . An C ( O )OH ) . example is cyclooctyne. Another example is bicyclononyne. 15 An " acetal ” group refers to RC ( H ) (OR ' ) 2 , in which R and As used herein , " heterocycloalkynyl” or “ heterocy R ' are independently selected from hydrogen , C1- 6 alkyl, cloalkyne” means a carbocyclyl ring or ring system with at C2- 6 alkenyl, C2- 6 alkynyl, C3- 7 carbocyclyl, C6- 10 aryl , 5 - 10 least one heteroatom in ring backbone , having at least one membered heteroaryl , and 5 - 10 membered heterocyclyl, as triple bond , wherein no ring in the ring system is aromatic . defined herein . In some embodiments , heterocycloalkynyl or heterocy - 20 A " cyano ” group refers to a “ — CN ” group . cloalkyne ring or ring system is 3 -membered , 4 -membered , A " sulfinyl” group refers to an “ _ SEOR” group in 5 -membered , 6 -membered , 7 -membered , 8 -membered , 9 which R is selected from hydrogen , C1- 6 alkyl, C2- 6 alkenyl , membered , or 10 membered . C2- 6 alkynyl, C3- 7 carbocyclyl, C6- 10 aryl, 5 - 10 membered As used herein , “ heterocyclyl” means a non - aromatic heteroaryl, and 5 - 10 membered heterocyclyl, as defined cyclic ring or ring system containing at least one heteroatom 25 herein . in the ring backbone . Heterocyclyls may be joined together A “ sulfonyl” group refers to an “ _ SO , R ” group in which in a fused , bridged or spiro -connected fashion . Heterocy R is selected from hydrogen , C1- 6 alkyl, C2- 6 alkenyl, C2- 6 clyls may have any degree of saturation provided that at least alkynyl, Cz. , carbocyclyl, C6- 10 aryl, 5 - 10 membered het one ring in the ring system is not aromatic . The heter - eroaryl, and 5 - 10 membered heterocyclyl, as defined herein . oatom (s ) may be present in either a non -aromatic or aro - 30 An “ S - sulfonamido ” group refers to a “ — SO ,NR , R , " matic ring in the ring system . The heterocyclyl group may group in which RA and RB are each independently selected have 3 to 20 ring members ( i. e ., the number of atomsmaking from hydrogen , C1- 6 alkyl, C2- 6 alkenyl, C2- 6 alkynyl, C3- 7 up the ring backbone, including carbon atoms and heteroa - carbocyclyl, C6- 10 aryl , 5 - 10 membered heteroaryl, and 5 - 10 toms) , although the present definition also covers the occur- membered heterocyclyl, as defined herein . rence of the term “ heterocycly1” where no numerical range 35 An “ N -sulfonamido ” group refers to a “ _ N (RA )SO , R0 " is designated . The heterocyclyl group may also be amedium group in which RA and Rg are each independently selected size heterocyclyl having 3 to 10 ring members . The hetero from hydrogen , C1- 6 alkyl, C2- 6 alkenyl, C2- 6 alkynyl, C3- 7 cyclyl group could also be a heterocyclyl having 3 to 6 ring carbocyclyl, Co. 10 aryl, 5 - 10 membered heteroaryl, and 5 - 10 members . The heterocyclyl group may be designated as “ 3 - 6 membered heterocyclyl, as defined herein . membered heterocyclyl” or similar designations. In pre - 40 An “ O -carbamyl ” group refers to a “ _ OCCO) NR RB ” ferred six membered monocyclic heterocyclyls , the heteroa - group in which RA and Rg are each independently selected tom ( s ) are selected from one up to three of O , N or S , and from hydrogen , C1- 6 alkyl, C2- 6 alkenyl, C2- 6 alkynyl, C3- 7 in preferred five membered monocyclic heterocyclyls , the carbocyclyl, C6- 10 aryl, 5 - 10 membered heteroaryl, and 5 - 10 heteroatom ( s ) are selected from one or two heteroatoms membered heterocyclyl, as defined herein . selected from O , N , or S . Examples of heterocyclyl rings 45 An “ N - carbamyl” group refers to an “ _ N ( R ) OCEO ) include , but are not limited to , azepinyl, acridinyl , carba - RB" group in which R and Rg are each independently zolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, selected from hydrogen , C1- 6 alkyl, C2- 6 alkenyl, C2- 6 alky morpholinyl, oxiranyl , oxepanyl , thiepanyl, piperidinyl, pip - nyl, C3- 7 carbocyclyl, C6- 10 aryl, 5 - 10 membered heteroaryl , erazinyl, dioxopiperazinyl, pyrrolidinyl , pyrrolidonyl, pyr- and 5 - 10 membered heterocyclyl, as defined herein . rolidionyl, 4 -piperidonyl , pyrazolinyl, pyrazolidinyl, 1, 3 - 50 A “ C - amido " group refers to a “ _ CEO ) NRARB” group dioxinyl , 1, 3 - dioxanyl , 1 ,4 - dioxinyl, 1 , 4 - dioxanyl, 1 , 3 - in which RA and Rg are each independently selected from oxathianyl, 1 , 4 - oxathiinyl , 1 , 4 -oxathianyl , 2H - 1 , 2 - oxazinyl, hydrogen , C1- 6 alkyl, C2- 6 alkenyl, C2- 6 alkynyl , C3- 7 carbo trioxanyl, hexahydro - 1 , 3 , 5 - triazinyl, 1 , 3 - dioxolyl, 1 , 3 -di cyclyl, C6 - 10 aryl , 5 - 10 membered heteroaryl , and 5 - 10 oxolanyl, 1 , 3 - dithiolyl, 1 , 3 - dithiolanyl, isoxazolinyl, isox - membered heterocyclyl , as defined herein . azolidinyl, oxazolinyl, oxazolidinyl , oxazolidinonyl, thiaz - 55 An “ N -amido ” group refers to a “ _ N ( R ) C ( O ) RE" olinyl, thiazolidinyl, 1 , 3 -oxathiolanyl , indolinyl, group in which RA and Rg are each independently selected isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahy from hydrogen , C1- 6 alkyl, C2- 6 alkenyl, C2- 6 alkynyl, C3- 7 drothiophenyl, tetrahydrothiopyranyl, tetrahydro - 1 , 4 - thiazi carbocyclyl , C6- 10 aryl , 5 - 10 membered heteroaryl , and 5 - 10 nyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazo membered heterocyclyl, as defined herein . lidinyl, and tetrahydroquinoline . 60 An " amino ” group refers to a “ — NR RB” group in which As used herein , " heterocyclylene ” means a non -aromatic R , and Rg are each independently selected from hydrogen , cyclic ring or ring system containing at least one heteroatom C1- 6 alkyl, C2- 6 alkenyl, C2- 6 alkynyl, C3- 7 carbocyclyl, C6- 10 that is attached to the rest of the molecule via two points of aryl, 5 - 10 membered heteroaryl, and 5 - 10 membered het attachment. erocyclyl , as defined herein . A non - limiting example As used herein , " acyl” refers to CEO ) R , wherein R is 65 includes free amino (i . e ., - NH2) . hydrogen , C1- 6 alkyl , C2- 6 alkenyl, C2- 6 alkynyl , C3- 7 carbo - The term “ hydrazine ” or “ hydrazinyl” as used herein cyclyl, C6- 10 aryl , 5 - 10 membered heteroaryl, and 5 - 10 refers to a — NHNH2 group . US 9 ,815 ,916 B2 19 20 The term “ hydrazone” or “ hydrazonyl” as used herein bered heterocyclyl- C , -Co - alkyl ( optionally substituted with refers to a halo , C , -C . alkyl, C ,- C . alkoxy , C . - C . haloalkyl, and C , -C6 haloalkoxy) , aryl (optionally substituted with halo , C1 -C6 alkyl, C -C6 alkoxy, C1- C6 haloalkyl , and C1- C6 _ NH2 5 haloalkoxy ), aryl( C ,- C )alkyl ( optionally substituted with halo , C1- Coalkyl , C1- Coalkoxy , C1 - C haloalkyl, and C1 -C6 haloalkoxy ), 5 - 10 membered heteroaryl ( optionally substi a Ry tuted with halo , C , -C . alkyl, C , -C6 alkoxy, C . -C . haloalkyl , and C -Co haloalkoxy ), 5 - 10 membered heteroaryl( C , - C . ) group . alkyl (optionally substituted with halo , C , -C . alkyl, C , -C6 The term “ formyl” as used herein refers to a C (OH alkoxy , C .- C . haloalkyl , and C . - C . haloalkoxy ), halo , group . cyano, hydroxy , C - Co alkoxy, C1- C6 alkoxy ( C - C6 ) alkyl The term “ hydroxy ” as used herein refers to a _ OH ( i. e ., ether ), aryloxy , sulfhydryl (mercapto ) , halo ( C1 - C ) group . 15 alkyl ( e . g ., CF3) , halo ( C . - C . ) alkoxy ( e . g . , OCF3) , The term “ azido ” as used herein refers to a N , group . C -C6 alkylthio , arylthio , amino , amino ( C . - C .) alkyl, nitro , The term “ thiol” as used herein refers to a SH group . O -carbamyl , N - carbamyl, O - thiocarbamyl, N - thiocarbamyl, C - amido , N - amido , S - sulfonamido , N -sulfonamido , C - car The term " glycidyl ether ” as used herein refers to boxy , O -carboxy , acyl, cyanato , isocyanato , thiocyanato , 20 isothiocyanato , sulfinyl, sulfonyl, and oxo ( O ). Wherever a group is described as “ optionally substituted ” that group can be substituted with the above substituents . It is to be understood that certain radical naming conven A tions can include either a mono - radical or a di - radical, mu 25 depending on the context. For example , where a substituent requires two points of attachment to the rest of the molecule , The term " epoxy ” as used herein refers to it is understood that the substituent is a di- radical. For example , a substituent identified as alkyl that requires two points of attachment includes di- radicals such as — CH , — , 30 - CH2CH2 - , - CH2CH (CH3 ) CH2 - , and the like . Other or radical naming conventions clearly indicate that the radical mm is a di- radical such as " alkylene ” or “ alkenylene .” ma Wherever a substituent is depicted as a di- radical ( i . e . , has man two points of attachment to the rest of the molecule ) , it is to 35 be understood that the substituent can be attached in any or directional configuration unless otherwise indicated . Thus , for example , a substituent depicted as - AE - or www min 40 mu The term “ ester” as used herein refers to R - C ( O ) O man R ', wherein R and R ' can be independently alkyl, alkenyl, 45 includes the substituent being oriented such that the A is alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, het attached at the leftmost attachment point of the molecule as eroaryl, heteroalicyclyl, aralkyl, (heteroalicyclyl )alkyl , or w ell as the case in which A is attached at the rightmost optionally substituted variants thereof. attachment point of the molecule . The term “ carboxylic acid ” or “ carboxyl ” as used herein Where the compounds disclosed herein have at least one refers to C ( O )OH . 50 stereocenter, they may exist as individual enantiomers and As used herein , the term “ tetrazine ” or “ tetrazinyl” refers diastereomers or as mixtures of such isomers , including to six -membered heteroaryl group comprising four nitrogen racemates . Separation of the individual isomers or selective atoms. Tetrazine can be optionally substituted . synthesis of the individual isomers is accomplished by As used herein , a substituted group is derived from the application of various methods which are well known to unsubstituted parent group in which there has been an 55 practitioners in the art. Unless otherwise indicated , all such exchange of one or more hydrogen atoms for another atom isomers and mixtures thereof are included in the scope of the or group . Unless otherwise indicated , when a group is compounds disclosed herein . Furthermore , compounds dis deemed to be “ substituted ,” it is meant that the group is closed herein may exist in one or more crystalline or substituted with one or more substituents independently amorphous forms. Unless otherwise indicated , all such selected from C , - C alkyl, C , - C . alkenyl, C , - C . alkynyl, 60 forms are included in the scope of the compounds disclosed C , - Cg heteroalkyl , Cz- C , carbocyclyl (optionally substituted herein including any polymorphic forms. In addition , some with halo , C . - C . alkyl, C . -Co alkoxy, C . - C . haloalkyl, and of the compounds disclosed herein may form solvates with C -Co haloalkoxy ) , Cz - Cz- carbocyclyl- C / - Co - alkyl (option water ( i . e . , hydrates ) or common organic solvents . Unless ally substituted with halo , C . - C . alkyl, C . - C . alkoxy , C . - C . otherwise indicated , such solvates are included in the scope haloalkyl, and C , -Co haloalkoxy) , 5 - 10 membered hetero - 65 of the compounds disclosed herein . cyclyl (optionally substituted with halo , C1 -C6 alkyl, C1 - C . As used herein , a “ nucleotide ” includes a nitrogen con alkoxy , C , -Chaloalkyl , and C , - C haloalkoxy ) , 5 - 10 mem - taining heterocyclic base , a sugar, and one or more phos US 9 ,815 ,916 B2 21. phate groups . They can be monomeric units (whether pre - polymers , brush polymers , dendronized polymers , ladders , cursors or linked monomers ) of a nucleic acid sequence . In and dendrimers . The polymers described herein can also be RNA , the sugar is a ribose, and in DNA a deoxyribose , i. e . in the form of polymer nanoparticles . Other examples of a sugar lacking a hydroxyl group that is present at the 2 polymer architectures include , but not limited to ring block position in ribose . The nitrogen containing heterocyclic base 5 polymers , coil - cycle - coil polymers , etc . can be purine or pyrimidine base . Purine bases include As used herein , the prefixes " photo " or " photo - ” mean adenine ( A ) and guanine (G ) , and modified derivatives or relating to light or electromagnetic radiation . The term can analogs thereof. Pyrimidine bases include cytosine ( C ) , encompass all or part of the electromagnetic spectrum thymine ( T ) , and uracil ( U ) , and modified derivatives or including, but not limited to , one or more of the ranges analogs thereof. The C - 1 atom of deoxyribose is bonded to 10 commonly known as the radio , microwave, infrared , visible , N - 1 of a pyrimidine or N - 9 of a purine . , X - ray or gamma ray parts of the spectrum . The As used herein , a “ nucleoside ” is structurally similar to a part of the spectrum can be one that is blocked by a metal nucleotide , but lacks any phosphate moieties at the 5 region of a surface such as those metals set forth herein . position . The term “ nucleoside ” is used herein in its ordinary Alternatively or additionally , the part of the spectrum can be sense as understood by those skilled in the art . Examples 15 one that passes through an interstitial region of a surface include , but are not limited to , a ribonucleoside comprising such as a region made of glass , plastic, silica , or other a ribose moiety and a deoxyribonucleoside comprising a material set forth herein . In particular embodiments , radia deoxyribose moiety . A modified pentose moiety is a pentose tion can be used that is capable of passing through a metal. moiety in which an oxygen atom has been replaced with a Alternatively or additionally, radiation can be used that is carbon and /or a carbon has been replaced with a sulfur or an 20 masked by glass , plastic , silica , or other material set forth oxygen atom . A “ nucleoside” is a monomer that can have a herein . substituted base and / or sugar moiety . Additionally , a nucleo As used herein , the term “ YES method ” refers to the side can be incorporated into larger DNA and/ or RNA chemical vapor deposition tool provided by Yield Engineer polymers and oligomers . ing Systems (" YES " ) with chemical vapor deposition pro As used herein , the term “ polynucleotide” refers to 25 cess developed by Illumina , Inc. It includes three different nucleic acids in general, including DNA ( e . g . genomic DNA vapor deposition systems. The automated YES - VertaCoat cDNA ) , RNA ( e . g . mRNA ), synthetic oligonucleotides and silane vapor system is designed for volume production with synthetic nucleic acid analogs. Polynucleotidesmay include a flexible wafer handling module that can accommodate 200 natural or non - natural bases , or combinations thereof and or 300 mm wafers . The manual load YES - 1224P Silane natural or non -natural backbone linkages , e . g . phosphoro - 30 Vapor System is designed for versatile volume production thioates , PNA or 2 - O -methyl - RNA , or combinations with its configurable large capacity chambers. Yes - Labkote thereof. is a low - cost , tabletop version that is ideal for feasibility As used herein , the term “ primer ” is defined as a nucleic studies and for R & D . acid having a single strand with a free 3 ' OH group . A primer As used herein , the term “ percent surface remaining ” can can also have a modification at the 5 ' terminus to allow a 35 refer to the intensity measured using a TET QC to stain the coupling reaction or to couple the primer to another moiety . P5 / P7 surface primers. The P5 and P7 primers are used on The primer length can be any number of bases long and can the surface of commercial flow cells sold by Illumina Inc. include a variety of non - natural nucleotides . As used herein , for sequencing on the HiSeq? , MiSeq? , Genome Ana “ BCN primer ” or “ BCN modified primer ” refers to a primer lyzer® and NextSeq platforms. The primer sequences are comprising covalently attached bicyclo [6 . 1. 0 ]non - 4 -yne at 40 described in U . S . Pat. Pub . No. 2011/ 0059865 A1, which is the 5 ' terminus. incorporated herein by reference . The P5 and P7 primer As used herein , the term “ silane” refers to an organic or sequences comprise the following : inorganic compound containing one or more silicon atoms. Non - limiting example of an inorganic silane compound is SiH4, or halogenated SiH , where hydrogen is replaced by 45 Paired end set : one or more halogen atoms. Non - limiting example of an P5 : paired end 5 ' - 3 ' organic silane compound is X - RC — Si( ORP ) 3, wherein X AATGATACGGCGACCACCGAGAUCTACAC is a non - hydrolyzable organic group , such as amino , vinyl, P7 : paired end 5 ' - 3 ' epoxy , methacrylate, sulfur, alkyl, alkenyl , alkynyl; RC is a CAAGCAGAAGACGGCATACGAG * AT spacer, for example ( CH ), - , wherein n is 0 to 1000 ; R? 50 Single read set : is selected from hydrogen , optionally substituted alkyl, P5 : single read : 5 ' 3 ' optionally substituted alkenyl, optionally substituted alky AATGATACGGCGACCACCGA nyl, optionally substituted carbocyclyl, optionally substi tuted aryl , optionally substituted 5 - 10 membered heteroaryl, P7 : single read 5 ' 3 '. and optionally substituted 5 - 10 membered heterocyclyl, as 55 CAAGCAGAAGACGGCATACGA defined herein . As used herein , the term “ silane ” can com In some embodiments , the P5 and P7 primers may com prise mixtures of different silane compounds. prise a linker or spacer at the 5 ' end . Such linker or spacer As used herein , the term “ polymer” refers to a molecule may be included in order to permit cleavage, or to confer composed of many repeated subunits or recurring units . some other desirable property , for example to enable cova Non - limiting examples of polymer structures include linear, 60 lent attachment to a polymer or a solid support, or to act as branched , or hyper- branched polymers . Non - limiting spacers to position the site of cleavage an optimal distance examples of linear polymers comprising block copolymers from the solid support . In certain cases , 10 spacer nucleo or random copolymers . Non - limiting examples of branched tides may be positioned between the point of attachment of polymers include star polymers , star - shaped or star -block the P5 or P7 primers to a polymer or a solid support . In some polymers comprising both hydrophobic and hydrophilic 65 embodiments polyT spacers are used , although other nucleo segments, H - shaped polymers comprising both hydrophobic tides and combinations thereof can also be used . In one and hydrophilic segments , dumbbell shaped polymers , comb embodiment, the spacer is a 10T spacer. TET is a dye labeled US 9 ,815 ,916 B2 23 24 oligonucleotide having complimentary sequence to the optionally substituted ethylene. In some further embodi P5 / P7 primers . TET can be hybridized to the P5 /P7 primers ments, L2 is optionally substituted propylene . In some on a surface ; the excess TET can be washed away, and the embodiments , the recurring unit of Formula ( I) is also attached dye concentration can be measured by fluorescence represented by Formula ( la ) or ( Ib ) and Formula ( II ) is also detection using a scanning instrument such as a Typhoon 5 represented by Formula ( Ila ) : Scanner (General Electric ). Polymers and DNA - Copolymers Polymers and Nucleic Acid -Copolymers with Recurring ( Ia ) Units of Formulae ( I ) and ( II ) Some embodiments described herein are related to a 10 polymer for surface functionalization , comprising a recur ring unit of Formula ( I ) and a recurring unit of Formula ( II ) as described above . O NHNH 15

Rla ( Ib ) R32 20 Y N3, NH

Rla R2a 25 NH2 ( II)

NH LR35 ONH

Rla R 1b ( IIa ) R20 NH2 wherein each Rla, R20 , R16 and R25 is independently selected from hydrogen , optionally substituted alkyl or optionally substituted phenyl; each R3a and R35 is indepen NH dently selected from hydrogen , optionally substituted alkyl, 40 optionally substituted phenyl, or optionally substituted C7- 14 aralkyl, and each L1 and L2 is independently selected from an optionally substituted alkylene linker or an optionally Rla substituted heteroalkylene linker. In some embodiments , R "“ is hydrogen . In some embodi- 45 wherein each Ria and Rlb is selected from hydrogen or ments , R20 is hydrogen . In some embodiments , R3a is hydro methyl . In someembodiments , the polymer comprises recur gen . In some embodiments, Rla is selected from hydrogen or optionally substituted alkyl , preferably C1- 6 alkyl and each ring units of Formula ( la ) and (Ila ) . In some other embodi of R2a and R3a is hydrogen . In some embodiments , each of ments , the polymer comprises recurring units of Formula Rla , R24 and R3a is hydrogen . In some embodiments , Rib is 50 ( lb ) and ( IIa ). In some embodiments of the recurring unit of hydrogen . In some embodiments , R2 is hydrogen . In some Formula ( II ) or ( IIa ), the amino functional group is in the embodiments , R36 is hydrogen . In some embodiments , R16 form of an inorganic salt, for example , hydrochloride salt. In is selected from hydrogen or optionally substituted alkyl, some embodiments , the recurring units of Formulae ( I ) and preferably C alkyl and each of R25 and R36 is hydrogen . ( II ) are about 1 : 1 in molar ratio . In some such embodiments , In some embodiments , each of R1b, R2b and R3b is hydrogen . 55 the recurring units of Formulae ( la ) and ( IIa ) are about 1 : 1 In some embodiments , L ' is an optionally substituted alky - in molar ratio . In some such embodiments , the recurring lene. In some such embodiments, L ' is optionally substituted units of Formulae ( lb ) and ( IIa ) are about 1 : 1 in molar ratio . methylene . In some other embodiments , L ' is optionally In some embodiments , the polymer may further comprise substituted ethylene . In some further embodiments , Ll is so one or more recurring units selected from the group con optionallyis an optionally substituted substituted propylene heteroalkylene . In some embodiments linker . In some, L ' 60 sisting of polyacrylamides , polyacrylates , polyurethanes , such embodiments , L ' is – (CH2 ) m -NH — (CH2 ) n - option - polysiloxanes , silicones , polyacroleins, polyphosphazenes , ally substituted with one or more oxo groups, and wherein polyisocyanates, poly -ols , and polysaccharides, or combi each m and n is an integer independently selected from 1 to 10 . In some embodiments , L² is an optionally substituted 65* s nations thereof . In some such embodiments , the polymer alkylene . In some such embodiments , L ’ is optionally sub may further comprising one or more recurring units of stituted methylene . In some other embodiments , L² is polyacrylamide of Formula ( IIIa ) or (IIIb ) or both : US 9 ,815 ,916 B2 26 mole percent of Formulae ( lb ) and ( IIa ) is about 5 % each . In (IIIa ) one embodiment, the polymer comprises recurring units of Formulae ( Illal) , ( lb ) and ( Ila ) in the mole percent of about 5 90 % to about 5 % to about 5 % . In another embodiment, the Roa polymer comprises recurring units of Formulae (IIlal ) , ( Ib ) and ( Ila ) in themolar percent ratio of about 85 % to about 5 % R4a to about 10 % . In yet another embodiment , the polymer ( llib ) 1n comprises recurring units of Formulae (IIIa2 ) , ( Ib ) and (IIa ) in themolar percent ratio of about 90 % to about 5 % to about NN 5 % . In some further embodiments , the polymer may further R45 - Sb R6b R76 comprise about 0 .5 mol % to about 2 mol % of a recurring 15 unit of Formula ( IIIb1 ) . Some embodiments described herein are related to a wherein each R40 , R45 and RS6 is selected from hydrogen grafted polymer comprising functionalized oligonucleotides or C - 3 alkyl; and each RS , RO , R and R ' ' is indepen - covalently bonded to a polymer with a recurring unit of dently selected from hydrogen , optionally substituted C1- 6 20 Formula (1 ) and a recurring unit of Formula ( II ) as described alkyl or optionally substituted phenyl. In sometuted embodi 1 6 20 Formula (I ) and a recurring unit of Formula ( II) as described ments , each R40, R46 and R5b is selected from hydrogen or herein . In some embodiments , the polymer may further methyl. In some embodiments , Rob and R75 are both hydro - comprise one or more recurring units of various different gen . In some embodiments , at least one of Rsa or Røa is polymer backbones as described above, for example , one or hydrogen . In some such embodiments, both R $ a and Rºa are 25 more recurring units of polyacrylamide of Formula ( IIIa ) or hydrogen . In some other embodiments , at least one of R5a or Rais methyl. In some such embodiments , both R5a and R6a (IIIb ) or both . In some embodiments, the covalent bonding are methyl. In some such embodiments , the recurring units between the functionalized oligonucleotide and the polymer of Formula ( IIIa ) is also represented by ( IIIal ) , ( IIIa2 ) or comprises the structure moiety ( IIIa3 ) : 30

( IIIal) / * 05 NH min üle

( IIIa2 ) min min or Ê min w NH2 ( IIIa3 ) KAmi or combinations thereof, wherein * indicates the point of connection with the functionalized oligonucleotide . In some In some such embodiments , the recurring unit of Formula 50 such embodiments , the covalent bonding between the func ( IIIb ) is also represented by (IIIb1 ) : tionalized oligonucleotide and the polymer comprises struc ture moiety (IIIb1 ) N NH mu

60 In some specific embodiments , the polymer comprises mi recurring units of Formulae ( lb ) , ( IIa ) and ( IIIa ). In some further embodiments , the polymer comprises recurring units of Formulae ( lb ), ( IIa ), ( IIIa ) and ( IIIb ). In some such 65 In some other such embodiments , the covalent bonding embodiments , the mole percent of Formula ( IIIa ) is from between the functionalized oligonucleotide and the polymer about 85 % to about 90 % . In some such embodiments , the comprises structure moiety US 9, 815 ,916 B2 28 and L is selected from a single bond , an optionally substi tuted alkylene linker or an optionally substituted heteroalky min lene linker . w In some embodiments , Rlc is hydrogen . In some embodi 5 ments , R2° is hydrogen . In some embodiments , R3° is hydro gen . In some embodiments , R ' is selected from hydrogen or optionally substituted alkyl, preferably C . , alkyl and each R2C and R30 is hydrogen . In some embodiments , each Ric, R2C and R3c is hydrogen . In some embodiments , Ar is an optionally substituted phenyl. In some embodiments , L ' is a In some such embodiments , the covalent bonding between single bond . In some embodiments , the recurring unit of the functionalized oligonucleotide and the polymer com Formula ( IV ) is also represented by Formula (IVa ): prises structure moiety ( IVA ) NN mm mm =

?

In some embodiments* , the grafted* polymer is prepared by reacting one or more functional moieties of the functional - 25 NH ized oligonucleotide with the polymer, said one or more functional moieties comprise or are selected from alkynes , cycloalkenes, cycloalkynes , heterocycloalkenes , heterocy cloalkynes , or optionally substituted variants or combina RIC tions thereof. In some such embodiments , said one or more 30 wherein Rlc is selected from hydrogen or methyl. functional moieties comprise alkyne or are selected from In some embodiments , the polymer may further comprise alkyne. In some other embodiments , said one or more one or more recurring units selected from the group con functional moieties comprise or are selected from nor sisting of polyacrylamides, polyacrylates , polyurethanes , bornene , cyclooctyne, or bicyclononyne , or optionally sub 5 polysiloxanes, silicones, polyacroleins, polyphosphazenes, stituted variants or combinations thereof. In one embodi- 35 polyisocyanates , poly -ols , and polysaccharides, or combi ment, the bicyclononyne is bicyclo [6 . 1. 0 ]non - 4 - yne. In nations thereof. In some such embodiments , the polymer some such embodiments , the grafted polymer is prepared by may further comprising one or more recurring units of reacting the azido groups of the polymer with said one or polyacrylamide of Formula ( IIIa ) or (IIIb ) with the structure more functional moieties of the functionalized oligonucle to shown above. otides, for example , bicyclo [ 6 . 1 . 0 ]non - 4 - yne . Some embodiments described herein are related to a Polymers and Nucleic Acid - Copolymers with Recurring grafted polymer comprising functionalized oligonucleotides Units of Formula ( IV ) covalently bonded to a polymer with a recurring unit of Some embodiments described herein are related to a Formula (IV ) as described herein . In some embodiments , the polymer for surface functionalization , comprising a recur 10 polymer may further comprise one or more recurring units ring unit of Formula (IV ) as described above : of various different polymer backbones as described above , for example , one or more recurring units of polyacrylamide of Formula (IIIa ) or (IIIb ) or both . In some embodiments, the (IV ) covalent bonding between the functionalized oligonucle t otide and the polymer comprises the structure moiety

N - R3C 55 N , or Rlc mi R20 mm * 60 wherein each Rlc and R2C is independently selected from NH . hydrogen , optionally substituted alkyl or optionally substi tuted phenyl; R3c is selected from hydrogen , optionally substituted alkyl, optionally substituted phenyl, or option ally substituted C7- 14 aralkyl; Ar is selected from an option - 65 ma ally substituted C6 - 10 aryl or an optionally substituted 5 or 6 membered heteroaryl; R4 is optionally substituted tetrazine ; US 9 ,815 ,916 B2 29 30 or combinations thereof, and wherein * indicates the point of wherein each Rid and R20 is independently selected from connection of the polymer with the functionalized oligo - hydrogen , optionally substituted alkyl or optionally substi nucleotide . In some such embodiments , the covalent bond - tuted phenyl ; each R3d is selected from hydrogen , optionally ing between the functionalized oligonucleotide and the 5 substituted alkyl, optionally substituted phenyl, or option polymer comprises structure moiety ally substituted C7- 4 aralkyl; R? is selected from azido , optionally substituted amino , Boc -protected amino , hydroxy, thiol , alkynyl, alkenyl , halo , epoxy, tetrazine or 10 aldehyde; each L4 and L is independently selected from an mus SN optionally substituted alkylene linker or an optionally sub stituted heteroalkylene linker. win In some embodiments , Rid is alkyl group , preferably C1- 6 15 alkyl. In some such embodiments , Ria is methyl. In some other embodiments , Rid is hydrogen . In some embodiments , In some other such embodiments , the covalent bonding eR4is hydrogen . In some embodiments ,R4 is hydrogen . In between the functionalized oligonucleotide and the polymer some embodiments , each R20 and R3d is hydrogen . In some comprises structure moiety embodiments , RB is selected from azido , amino or Boc 20 protected amino , or combinations thereof. In some embodi ments , L4 is an optionally substituted alkylene linker. In some such embodiments , L4 is a methylene linker. In some embodiments , L is an optionally substituted heteroalkylene mu NH linker. In some such embodiments , L ' is nu 25

In some embodiments , the grafted polymer is prepared by 30 min reacting one or more functional moieties of the functional ized oligonucleotide with the polymer , said one or more and wherein n is an integer of 1 to 50 . In some embodiments , functional moieties comprise , or are selected from , alkynes , the recurring unit of Formula ( V ) is also represented by cycloalkenes , cycloalkynes , heterocycloalkenes, heterocy - 35 Formula ( Va ) or (Vb ): cloalkynes, or optionally substituted variants or combina tions thereof. In some such embodiments , the one or more ( Va ) functional moieties may include or be selected from nor OH bornene, cyclooctyne, or bicyclononyne, or optionally sub - 40 stituted variants or combinations thereof. In one embodi owns ment, the bicyclononyne is bicyclo [6 .1 . 0 ]non -4 -yne . In some such embodiments , the grafted polymer is prepared by reacting the tetrazine groups of the polymer with said one or 45 more functional moieties of the functionalized oligonucle Rld otides, for example , bicyclo [6 . 1 . 0 ]non - 4 - yne . ( Vb ) Polymers and Nucleic Acid -Copolymers with Recurring OH Units of Formula ( V ) Some embodiments described herein are related to a 50 NHBoc , polymer for surface functionalization , comprising a recur or NHB?C, ring unit of Formula ( V ) as described above : (V ) SS HO . L - RB N Rld R3d wherein each Rid is independently selected from hydro 60 gen or methyl. In some such embodiments , n is an integer of 1 to 20 . In some such embodiments , n is an integer of 1 to 10 . In some such embodiments , n is an integer of 1 to 5 . In one embodiment, n is 3 . In some embodiments , the polymer Rld R20 ss comprises both Formulae ( Va ) and ( Vb ). In some embodiments , the polymer may further comprise a recurring unit of Formula ( Vla ) or (VID ), or both : US 9, 815 ,916 B2 32 (Vla ) min -HN OH mi

10 In some embodiments , the grafted polymer is prepared by Rle Re reacting one or more functional moieties of the functional (VID ) ized oligonucleotide with the polymer , said one or more OH functional moieties comprise or are selected from optionally substituted amino , hydroxy , thiol, carboxyl, acid anhydride , LOH or combinations thereof. In one embodiment, the function alized oligonucleotide comprises one or more optionally substituted amino groups . In some such embodiments , the amino group is unsubstituted . In some such embodiments , the grafted polymer is prepared by reacting the glycidyl ether or epoxy groups of the polymer with said one or more 20 RIS amino groups of the functionalized oligonucleotides . In R ? some such embodiments , the epoxy groups of the polymer are derived from recurring unit of Formula (Vla ) . Substrates selectedwherein from each hydrogen R " , R , , optionallyR " and R4substituted is independently alkyl or 25 su Some embodiments described herein are related to a optionally substituted phenyl . In some such embodiments , 25 substrate having a first surface comprising a polymer with a Rle is alkyl , preferably C1- 6 alkyl, for example , methyl. In recurring unit of Formula ( I ) and a recurring unit of Formula some other embodiments , Rle is hydrogen . In some embodi ( II ) covalently bonded thereto as described herein . In some ments , R2e is hydrogen . In some such embodiments , Rl is embodiments , the polymer may further comprise one or alkyl, preferably C . alkyl, for example , methyl. In some more recurring units selected from the group consisting of other embodiments , R '' is hydrogen . In some embodiments , 30 polyacrylamides, polyacrylates , polyurethanes , polysilox R2f is hydrogen . anes, silicones , polyacroleins , polyphosphazenes , polyiso In some embodiments , the polymer may further comprise cyanates , poly -ols , and polysaccharides , or combinations one or more recurring units selected from the group con - thereof. In some such embodiments , the polymer may fur sisting of polyacrylamides , polyacrylates , polyurethanes , ther comprising one or more recurring units of polyacryl polysiloxanes, silicones , polyacroleins, polyphosphazenes , 35 amide of Formula ( IIIa ) or ( IIIb ) with the structure shown polyisocyanates, poly -ols , and polysaccharides, or combi- above. In some embodiments , the covalent bonds between nations thereof. In some such embodiments , the polymer the polymer and the substrate are formed by amine epoxy may further comprising one or more recurring units of ring opening reaction . In some embodiments , the covalent polyacrylamide of Formula ( IIIa ) or (IIIb ) with the structure bonding between the polymer and the first surface of the shown above . substrate comprises the structure moiety Some embodiments described herein are related to a 40 grafted polymer comprising functionalized oligonucleotides covalently bonded to a polymer with a recurring unit of Formula ( V ) as described herein . In some embodiments , the mu polymer may further comprise a recurring unit of Formula HN HN OH or (Vla ) or (VIb ) , or both . In some embodiments , the covalent 45 bonding between the functionalized oligonucleotide and the mi polymer comprises the structure moiety mu minn

m 50 wat HN HN wa - HN OH , or mi mu mi w HN OH ,

mm 60 min mi HN or combinations thereof, wherein * indicates the polymer' s mim point of connection with the functionalized oligonucleotide . mu In some such embodiments , the covalent bonding between 65 * the functionalized oligonucleotide and the polymer com prises structure moiety US 9 ,815 ,916 B2 33 34 or combinations thereof, wherein the substituted amino is - continued derived from the recurring unit of Formula ( II ) and * indicates the polymer ' s point of connection with the first surface of the substrate . In some such embodiment, the mi covalent bonding between the polymer and the first surface 5 comprises structure moiety

wenn HN OH 10 or min mm 15 mah In some embodiments , the substrate is prepared by reacting the polymer with a first plurality of functional groups mo covalently attached thereto the first surface, wherein the first plurality of functional groups comprise or are selected from 20 vinyl, acryloyl , alkenyl, cycloalkenyl , heterocycloalkenyl, or combinations thereof, wherein * indicates the polymer ' s alkynyl, cycloalkynyl, heterocycloalkynyl, nitrene, alde - point of connection with the functionalized oligonucleotide. hyde , hydrazinyl, glycidyl ether, epoxy, carbene , isocyanate In some such embodiments , the functionalized oligonucle or maleimide , or optionally substituted variants or combi otides are covalently bonded to the polymer by reacting one nations thereof. In some such embodiments , the first plural or more functional moieties of the functionalized oligo ity of functional groups comprises or are selected from nucleotides with the azido groups of the polymer , said one epoxy groups. In one embodiment, said epoxy group has the or more functional moieties comprise or are selected from structure 30 alkynes , cycloalkenes, cycloalkynes, heterocycloalkenes , heterocycloalkynes , or optionally substituted variants or combinations thereof. In some such embodiments , said one or more functional moieties comprise alkyne. In some other nin 33 embodiments , said one or more functional moieties com prise or are selected from norbornene , cyclooctyne, or 006- bicyclononyne, or optionally substituted variants or combi

m nations thereof. In one embodiment, the bicyclononyne is 40 bicyclo [6 .1 . O ]non -4 -yne . In some embodiments, the func tionalized oligonucleotides are covalently bonded to the In some embodiments , the substrate is prepared by reacting polymer by reacting the azido groups of the polymer with the amino groups of the polymer with the epoxy groupsroups ofof one or more alkyne moieties of the functionalized oligo the first surface. In some embodiments , the surface is 45 nucleotides . In some other embodiments , the functionalized pretreated with a functionalized silane comprising said first oligonucleotides are covalently bonded to the polymer by plurality of the functional groups described above , and the polymer is covalently bonded to the first surface through reacting the azido groups of the polymer with one or more reaction with the first plurality of the functional groups of bicyclo [ 6 .1 .0 ]non -4 -yne moieties of the functionalized oli the functionalized silane . 50 gonucleotides . In some embodiments, the substrate further comprises Some embodiments described herein are related to a functionalized oligonucleotides covalently bonded to the substrate having a first surface comprising a polymer with a polymer. In some embodiment, the covalent bond between recurring unit of Formula (IV ) covalently bonded thereto as the oligonucleotide and the polymer is formed by azide click described herein . In some embodiments , the polymer may cycloaddition reaction . In some embodiments , the covalent 55 further comprise one or more recurring units selected from bonding between the functionalized oligonucleotide and the the group consisting of polyacrylamides, polyacrylates, polymer comprises the structure moiety polyurethanes, polysiloxanes, silicones , polyacroleins, poly phosphazenes, polyisocyanates, poly -ols , and polysaccha rides , or combinations thereof. In some such embodiments , / * 60 the polymer may further comprising one or more recurring units of polyacrylamide of Formula ( IIIa ) or ( IIIb ) with the min www mm structure shown above . In some embodiments , the covalent bonds between the polymer and the substrate are formed by tetrazine Diels - Alder reactions , which results in the elimi ??? 65 nation of nitrogen gas . In some embodiments, wherein the or covalent bonding between the polymer and the first surface mu mi of the substrate comprises the structure moiety US 9, 815 ,916 B2 35 36 groups of the polymer with the norbornene groups of the first surface . In some embodiments , the surface is pretreated with a functionalized silane comprising said first plurality of the mi functional groups described above, and the polymer is 7-? 5 covalently bonded to the first surface through reaction with or the first plurality of the functional groups of the function alized silane . mu wie In some embodiments , the substrate further comprises functionalized oligonucleotides covalently bonded to the 10 nopolymer . In some embodiments , the covalent bonding between the oligonucleotide and the polymer is formed by a tetrazine Diels - Alder reaction , which results in the elimina Wim tion of nitrogen gas . In some embodiments , the covalent bonding between the functionalized oligonucleotide and the 15 polymer comprises the structure moiety Or

20 NH , wa V , or mm 25 mn * or combinations thereof, wherein * indicates the polymer ' s point of connection with the first surface . In some such embodiments , the covalent bonding between the polymer 30 and the first surface comprises structure moiety mah

35 Z

Z- w ma NH , 40 w In some other such embodiments , the covalent bonding between the polymer and the first surface comprises struc ture moiety or combinations thereof, and wherein * indicates the poly 45 mer ' s point of connection with the functionalized oligo nucleotide . In some such embodiments , the covalent bond ing between the functionalized oligonucleotide and the polymer comprises structure moiety NH . 50

Z

Z- In some embodiments , the substrate is prepared by reacting 55 the polymer with a first plurality of functional groups covalently attached thereto the first surface , wherein the first plurality of functional groups comprise or are selected from vinyl, acryloyl, alkenyl, cycloalkenyl, heterocycloalkenyl, In some such embodiments, the functionalized oligonucle alkynyl, cycloalkynyl, heterocycloalkynyl, nitrene, alde - 60 otides are covalently bonded to the polymer by reacting one hyde, hydrazinyl, glycidyl ether , epoxy, carbene , isocyanate or more functional moieties of the functionalized oligo or maleimide , or optionally substituted variants or combi- nucleotides with the tetrazine groups of the polymer , said nations thereof. In some such embodiments , the first plural one or more functional moieties comprise or are selected ity of functional groups comprises, or are selected from , from alkynes, cycloalkenes , cycloalkynes , heterocycloalk optionally substituted cycloalkenyl groups . In one embodi- 65 enes, heterocycloalkynes, or optionally substituted variants ment, said cycloalkenyl is norbornene . In some embodi- or combinations thereof. In some such embodiments, said ments , the substrate is prepared by reacting the tetrazine one or more functional moieties comprise or are selected US 9 ,815 ,916 B2 37 38 from norbornene, cyclooctyne, or bicyclononyne, or option ally substituted variants or combinations thereof . In one embodiment, the bicyclononyne is bicyclo [ 6 . 1. O ] non - 4 -yne . minma In some such embodiments , the functionalized oligonucle - 5 otides are covalently bonded to the polymer by reacting the tetrazine groups of the polymer with one or more bicyclo [ 6 . 1 . 0 ]non - 4 - yne moieties of the functionalized oligonucle otides. 10 In some other embodiments , the covalent bonding between Some embodiments described herein are related to a the polymer and the first surface comprises the structure substrate having a first surface comprising a polymer with a moiety recurring unit of Formula (V ) covalently bonded thereto as described herein . In some embodiments , the polymer may 15 further comprise one or more recurring units selected from HN OH , the group consisting of polyacrylamides , polyacrylates , or polyurethanes , polysiloxanes , silicones, polyacroleins, poly butyin phosphazenes , polyisocyanates , poly -ols , and polysaccha- 20 mi m rides, or combinations thereof. In some embodiments , the polymer may further comprise a recurring unit of Formula HN ?? (Vla ) or (VIb ) , or both . In some embodiments , the covalent bonds between the polymer and the substrate are formed by 25 amine epoxy ring opening reaction . In some other embodi im ments , the covalent bonds between the polymer and the substrate are formed by azide click cycloaddition reaction . or In some embodiments , the covalent bonding between the 30 polymer and the first surface of the substrate comprises the structure moiety HN OH 35 min mi mim min www 40 or combinations thereof, wherein * indicates the point of connection of polymer with the first surface . In some such or embodiments , the covalent bonding between the polymer and the first surface comprises structure moiety

mu 45 HN OH min winan 50 movie In some other such embodiments , the covalent bonding between the polymer and the first surface comprises struc maw mi 55 ture moiety

HN N mm 60 minn min or combinations thereof, wherein * indicates the point of connection of polymer with the first surface . In some such 65 embodiments , the covalent bonding between the polymer In some embodiments , the substrate is prepared by reacting and the first surface comprises structure moiety the polymer with a first plurality of functional groups US 9 ,815 ,916 B2 39 e covalently attached thereto the first surface, wherein the first plurality of functional groups comprise or are selected from vinyl, acryloyl, alkenyl, cycloalkenyl, heterocycloalkenyl, HN ??OH alkynyl , cycloalkynyl , heterocycloalkynyl , nitrene , alde hyde , hydrazinyl, glycidyl ether, epoxy , carbene , isocyanate min or maleimide , or optionally substituted variants or combi ni nations thereof. In some such embodiments , the first plural ity of functional groups comprises or are selected from In some such embodiments , the functionalized oligonucle optionally substituted cycloalkenyl groups. In one embodi- 10 otides are covalently bonded to the polymer by reacting one ment, said cycloalkenyl is norbornene . In some embodi or more functional moieties of the functionalized oligo ments , the substrate is prepared by reacting the azido groups nucleotides with the epoxy groups of the polymer, said one of the polymer with the norbornene groups of the first or more functional moieties comprise or are selected from surface. In some other embodiments , the first plurality of 15 optionally substituted amino , hydroxy, thiol, carboxyl, acid functional groups comprises or are selected from glycidyl a nhydride , or combinations thereof. In some such embodi ether or epoxy groups . In some embodiments , the substrate ments , said one or more functional moieties comprise or are is prepared by deprotecting the Boc -protected amino of the selected from optionally substituted amino groups . In some such embodiments , the functionalized oligonucleotides are polymer and then reacting the amino groups of the polymer 20 covalently bonded to the polymer by reacting the epoxy with the glycidyl ether or epoxy groups of the first surface . groups of the polymer with the amino groups of the func In some embodiments , the surface is pretreated with a tionalized oligonucleotides . functionalized silane comprising said first plurality of the In embodiments described herein , the substrate material functional groups described above , and the polymer is may comprise glass, silica, plastic , quartz , metal, metal covalently bonded to the first surface through reaction with 25 oxide , or combinations thereof. In some embodiments , the the first plurality of the functional groups of the function substrate comprises glass . In some embodiments , the first surface of the substrate comprises both polymer coated alized silane . regions and inert regions. In some embodiments, the substrate further comprises Substrate Surface Preparations functionalized oligonucleotides covalently bonded to the 30 Some embodiments described herein are related to pro polymer . In some embodiments , the covalent bonding cesses or methods for immobilizing a grafted polymer to a between the oligonucleotide and the polymer is formed by first surface of a substrate by providing a substrate having a amine epoxy ring opening reaction . In some embodiments, first surface having a first plurality of functional groups the covalent bonding between the functionalized oligonucle covalently attached thereto ; providing a grafted polymer otide and the polymer comprises the structure moiety 35 having functionalized oligonucleotides covalently bonded to a polymer , wherein the polymer comprises a second plural ity of functional groups ; and reacting the first plurality many functional groups of the first surface with the second plu HN * OH rality of functional groups of the polymer such that the ?HN 40 polymer is covalently bonded to the first surface of the or substrate . In some embodiments of the methods described herein , mi mm the first surface of the substrate is pretreated with a func tionalized silane , wherein said functionalized silane com 45 prises the first plurality of the functional groups . In some HN OH embodiments , the functionalized silane is deposited onto the ? surface by Chemical Vapor Deposition (CVD ) method . In min some such embodiments, functionalized silane can be we applied onto the first surface by CVD method using Yield 50 Engineering Systems ( YES ) oven . In some embodiments of the methods described herein , Or the grafted polymer is formed by reacting a third plurality of functional groups of the polymer with one or more func tional moieties of the functionalized oligonucleotides . In 55 some other embodiments , the grafted polymer is formed by reacting said one or more functional moieties of function HN OH alized oligonucleotides with monomers comprising a third min plurality of functional groups; polymerizing the reacted m ! monomers to form the polymer such that the functionalized wa 60 oligonucleotides are covalently bonded to the polymer. In some embodiments of the methods described herein , the second plurality of functional groups of the polymer are or combinations thereof, wherein * indicates the point of the same as the third plurality of functional groups of the connection of polymer with the functionalized oligonucle - polymer . For example , the second plurality and the third otide . In some embodiments , the covalent bonding between 65 plurality of functional groups of the polymer can both be the functionalized oligonucleotide and the polymer com - tetrazines . In some other embodiments, the functional prises structure moiety groups in the second plurality of functional groups of the US 9 ,815 ,916 B2 42 polymer are different from the functional groups in the third cycloalkynyl, nitrene, aldehyde , hydrazinyl, ormaleimide or plurality of functional groups of the polymer . optionally substituted variants or combinations thereof. In The polymer backbone used in the methods described some further embodiments , said one or more functional herein can be linear, branched , hyper - branched or dendritic . moieties of the functionalized oligonucleotides comprise or The final polymer structure can be in different architectures, 5 are selected from alkynyl, cycloalkenyl, cycloalkynyl, including, for example , random copolymer , block copoly amino , azido , hydroxy, thiol, carboxyl, acid anhydride , or mer , comb - shaped polymer or star- shaped polymer archi optionally substituted variants or combinations thereof. In tectures . Different classes of polymer backbones can be used some such embodiments , said one or more functionalmoi in the methods described herein , including but not limited to eties comprise cycloalkynyl, for example , bicyclo [ 6 . 1 . 0 ] polyacrylamides , polyacrylates , polyurethanes , polysilox - 10 non -4 -yne (BCN ). In some other embodiments , said one or anes , silicones , polyacroleins, polyphosphazenes, polyiso more functional moieties comprise alkynyl. In still some cyanates , poly -ols , polysaccharides , etc . In some embodi other embodiments, said one or more functional moieties ments, the polymer comprises polyacrylamide backbone. In comprise azido . In still some other embodiments, said one or some other embodiments , the polymer comprises polyacry - more functional moieties comprise optionally substituted late backbone . In still some other embodimentsliments , the polymer 1515 amino . comprises polyurethane backbone . In still some other In some embodiments of the methods described herein , embodiments , the polymer comprises polyphosphazenes the grafted polymer comprises functionalized oligonucle backbone. In still some other embodiments , the polymer otides covalently bonded to a polymer with a recurring unit comprises a dendrimer backbone . of Formula (I ) and a recurring unit of Formula ( II ) as In some embodiments of the methods described herein , 20 described herein . In some such embodiments , the first plu the first plurality of functional groups of the first surface comprise or are selected from vinyl, acryloyl, alkenyl, rality of functional groups of the first surface comprise cycloalkenyl, heterocycloalkenyl, alkynyl, cycloalkynyl, epoxy groups . In one embodiment , said epoxy group is heterocycloalkynyl, nitrene, aldehyde , hydrazinyl, glycidyl ether , epoxy , carbene , isocyanate or maleimide , or option - 25 ally substituted variants or combinations thereof. In some such embodiments , the first plurality of functional groups ma comprises or is selected from cycloalkenyl, glycidyl ether , epoxy , or optionally substituted variants or combinations thereof. In some further embodiments , the first plurality of 30 functional groups comprises or is selected from norbornene . In some other embodiments, the first plurality of functional groups comprises an epoxy . In still some other embodi ments , the first plurality of functional groups comprises glycidyl ether. 35 In some such embodiments , the grafted polymer is cova In some embodiments of themethod described herein , the lently bonded to the first surface by reacting the amino functional groups of the polymer may comprise or are groups of the polymer with the epoxy groups of the first selected from amino , tetrazinyl, azido, carboxyl, hydroxy, surface . thiol, aldehyde , halo , alkenyl, alkynyl, epoxy , glycidyl ether , In some embodiments of the methods described herein , etc . In some embodiments of the methods described herein , 40 the grafted polymer comprises functionalized oligonucle the second plurality of functional groups of the polymer otides covalently bonded to a polymer with a recurring unit comprise or are selected from amino , tetrazinyl, azido , of Formula (IV ) as described herein . In some such embodi carboxyl, hydroxy, thiol, aldehyde , or optionally substituted ments , the first plurality of functional groups of the first variants or combinations thereof. In some such embodi surface comprises optionally substituted cycloalkenyl ments , the second plurality of functional groups comprise or 45 groups, for example , optionally substituted norbornene . In are selected from amino or protected amino , for example , some such embodiments , the grafted polymer is covalently Boc - protected amino . In some other embodiments, the sec - bonded to the first surface by reacting the tetrazine groups of ond plurality of functional groups comprises optionally the polymer with the norbornene groups of the first surface . substituted tetrazinyl. In still some other embodiments, the In some embodiments of the methods described herein , second plurality of functional groups comprises azido . 50 the grafted polymer comprises functionalized oligonucle In some embodiments of the methods described herein , otides covalently bonded to a polymer with a recurring unit the third plurality of functional groups of the polymer of Formula ( V ) as described herein . In some embodiments , comprises or is selected from azido , tetrazinyl, glycidyl, the polymer may further comprise a recurring unit of For epoxy, alkynyl, or optionally substituted variants or combi- mula (Vla ) or (Vb ) , or both , as described herein . In some nations thereof. In some such embodiments , the third plu - 55 such embodiments , the first plurality of functional groups rality of functional groups comprises azido . In some other comprises optionally substituted cycloalkenyl groups , for embodiments , the third plurality of functional groups com - example , optionally substituted norbornene. In some such prises optionally substituted tetrazinyl. In yet some other embodiments , the grafted polymer is covalently bonded to embodiments , the third plurality of functional groups com - the first surface by reacting the azido groups of the polymer prises alkynyl. In yet some other embodiments , the third 60 with the norbornene groups of the first surface . In some plurality of functional groups comprises optionally substi - other embodiments , the first plurality of functional groups tuted glycidyl ether. comprises glycidyl ether or epoxy groups . In some other In some embodiments of the methods described herein , embodiments , the grafted polymer is covalently bonded to said one or more functional moieties of the functionalized the first surface by deprotecting the Boc - protected amino oligonucleotides comprise or are selected from amino , 65 groups of the polymer ; and reacting the amino groups of the azido , carboxyl, acid anhydride, tetrazine , epoxy , glycidyl polymer with the glycidyl ether or epoxy groups of the first ether, vinyl, acryloyl , alkenyl, cycloalkenyl, alkynyl, surface . US 9 ,815 , 916 B2 43 44 Some embodiments described herein are related to pro - include plastic materials such as polyethylene, polystyrene , cesses or methods for immobilizing a polymer described poly ( vinyl chloride ) , polypropylene, nylons, polyesters , herein to a first surface of a substrate , comprising: providing polycarbonates and poly (methyl methacrylate ). Preferred a substrate having a first surface comprising a first plurality plastics material is poly (methyl methacrylate ), polystyrene of functional groups covalently attached thereto ; providing 5 and cyclic olefin polymer substrates . In some embodiments , a polymer with recurring units of Formulae ( I) and ( II ) , the substrate is a silica -based material or plastic material. In Formula (IV ) , or Formula ( V ) as described herein ; and particular embodiments, the substrate has at least one sur reacting the first plurality functional groups of the first face comprising glass. surface with the polymer such that the polymer is covalently In some embodiments , the substrates can be , or can bonded to the first surface of the substrate . In some such 10 contain , a metal. In some such embodiments , the metal is embodiments , the processes or methods further comprises gold . In some embodiments , the substrate has at least one providing functionalized oligonucleotides comprising one or surface comprising a metal oxide . In one embodiment, the more functionalized moieties ; and reacting said one or more surface comprises a tantalum oxide or tin oxide . functionalized moieties with the polymer such that the Acrylamide , enone , or acrylate may also be utilized as a functionalized oligonucleotides are covalently bonded to the 15 substrate material. Other substrate materials can include , but polymer. In some such embodiments , the first plurality of are not limited to gallium aresnide, indium phosphide , functional groups of the first surface comprises or are aluminum , ceramics, polyimide, quartz , resins, polymers selected from vinyl, acryloyl, alkenyl, cycloalkenyl, hetero - and copolymers . The foregoing lists are intended to be cycloalkenyl, alkynyl, cycloalkynyl , heterocycloalkynyl, illustrative of, but not limiting to the present application . nitrene , aldehyde , hydrazinyl, glycidyl ether, epoxy, car - 20 In some embodiments , the substrate and /or the substrate bene , isocyanate or maleimide , or optionally substituted surface can be quartz. In some other embodiments , the variants or combinations thereof. In some such embodi - substrate and /or the substrate surface can be semiconductor, ments , said one or more functionalized moieties comprise or i .e . GaAs or ITO . are selected from amino, azido , carboxyl, acid anhydride , Substrates can comprise a single material or a plurality of tetrazine , epoxy , glycidyl ether , vinyl, acryloyl, alkenyl, 25 different materials . Substrates can be composites or lami cycloalkenyl, alkynyl, cycloalkynyl, nitrene , aldehyde , nates . Substrate can be flat , round , textured and patterned . hydrazinyl , or maleimide or optionally substituted variants Patterns can be formed , for example , by metal pads that form or combinations thereof. features on non -metallic surfaces, for example , as described In any embodiments of the methods described herein , the in U . S . Pat. No. 8 ,778 , 849, which is incorporated herein by polymer or grafted polymer with recurring units of Formulae 30 reference . Another useful patterned surface is one having ( I ) and ( II) , Formula ( IV ) , or Formula ( V ) may further well features formed on a surface , for example , as described comprise one or more recurring units selected from the in U . S . Pat . App . Pub . No. 2014 /0243224 A1, U . S . Pat. App . group consisting of polyacrylamides , polyacrylates , poly - Pub . No. 2011 /0172118 A1 or U . S . Pat. No . 7, 622 ,294 , each urethanes, polysiloxanes , silicones, polyacroleins , poly of which is incorporated herein by reference in its entirety . phosphazenes , polyisocyanates , poly - ols , and polysaccha - 35 For embodiments that use a patterned substrate , a gel can be rides , or combinations thereof. In some such embodiments , selectively attached to the pattern features ( e . g . gel can be the polymer may further comprise one or more recurring attached to metal pads or gel can be attached to the interior units of polyacrylamide of Formula ( IIIa ) or ( IIIb ) with the of wells) or alternatively the gel can be uniformly attached structure shown above . across both the pattern features and the interstitial regions. In any embodiments of the methods described herein , the 40 Advantages in using plastics- based substrates in the method further comprises a washing step to remove excess preparation and use of molecular arrays include cost : the unbounded functionalized oligonucleotides . In some preparation of appropriate plastics -based substrates by , for embodiments , the method further comprises a drying step . example injection -molding , is generally cheaper than the In any of the embodiments described herein , the substrate preparation , e . g . by etching and bonding , of silica -based can comprise a material selected from glass, silica , quartz , 45 substrates . Another advantage is the nearly limitless variety plastic , metal, metal oxide , patterned or not or combinations of plastics allowing fine - tuning of the optical properties of thereof. In one embodiment, the surface of the substrate the support to suit the application for which it is intended or comprises glass . In some embodiments , the surface of the to which it may be put. substrate can comprise both functionalized silane coated Where metals are used as substrates or as pads on a regions and inert regions. In some embodiments , the inert 50 substrate , this may be because of the desired application : the regions are selected from glass regions, metal regions , mask conductivity of metals can allow modulation of the electric regions and interstitial regions, or combinations thereof. In field in DNA - based sensors . In this way, DNA mismatch one embodiment , the inert regions comprise glass. discrimination may be enhanced , the orientation of immo In any of the embodiments described herein , QC markers bilized oligonucleotide molecules can be affected , or DNA can be included in the polymer and / or primer structures . 55 hybridization kinetics can be accelerated . In any embodiments described herein , the polymer or In some embodiments , the substrate is silica -based but the grafted polymer may be applied to the surface of the shape of the substrate employed may be varied in accor substrate via various surface application techniques known dance with the application for which the present application to one skilled in the art , for example , spin coating , spray is practiced . Generally , however , slides of support material, coating , dip coating , ink jet coating , etc . 60 such as silica , e . g . fused silica , are of particular utility in the Substrates Materials and Design preparation and subsequent integration of molecules . Of In some embodiments , substrates used in the present particular use in the practice of the present application are application include silica - based substrates , such as glass , fused silica slides sold under the trade name SPECTRA fused silica and other silica -containing materials . In some SILTM . This notwithstanding, it will be evident to the skilled embodiments, silica -based substrates can also be silicon , 65 person that the present application is equally applicable to silicon dioxide , silicon nitride , silicone hydrides . In some other presentations of substrate ( including silica -based sup embodiments , substrates used in the present application ports ) , such as beads, rods and the like . US 9 ,815 ,916 B2 45 46 In some embodiments , the surface of the substrate com species of attached primer are often referred to as bridge prises both functional molecules -coated regions and inert amplification because double stranded amplicons form a regions with no coatings . In some such embodiments , the bridge - like structure between the two attached primers that functionalized molecule coatings are hydrogel or polymer flank the template sequence that has been copied . Exemplary coatings . The functionalmolecules - coated regions can com - 5 reagents and conditions that can be used for bridge ampli prise reactive sites, and thus , can be used to attach molecules fication are described , for example , in U . S . Pat. No. 5 ,641 , through chemical bonding or other molecular interactions. 658 ; U . S . Patent Publ. No. 2002 /0055100 ; U . S . Pat . No. In some embodiments , the functional molecules -coated 7 , 115 ,400 ; U . S . Patent Publ. No. 2004 /0096853 ; U . S . Patent regions ( e . g . reactive features, pads, beads, posts or wells ) Publ. No . 2004 /0002090 ; U . S . Patent Publ. No . 2007 / and the inert regions ( referred to as interstitial regions ) can 10 0128624 ; and U . S . Patent Publ. No . 2008 /0009420 , each of alternate so as to form a pattern or a grid . Such patterns can which is incorporated herein by reference in its entirety . be in one or two dimensions. In some embodiments , the inert PCR amplification can also be carried out with one of the regions can be selected from glass regions , metal regions , amplification primers attached to a polymer coating and the mask regions, or combinations thereof. Alternatively these second primer in solution . An exemplary format that uses a materials can form reactive regions. Inertness or reactivity 15 combination of one attached primer and soluble primer is will depend on the chemistry and processes used on the emulsion PCR as described , for example , in Dressman et al. , substrate . In one embodiment, the surface comprises glass Proc. Natl. Acad. Sci . USA 100 :8817 -8822 (2003 ), WO regions . In another embodiment, the surface comprises 05 / 010145 , or U . S . Patent Publ . Nos . 2005 /0130173 or metal regions. In still another embodiment, the surface 2005 /0064460 , each of which is incorporated herein by comprises mask regions. In some embodiments of the com - 20 reference . Emulsion PCR is illustrative of the format and it positions described herein , the substrate can be a bead . will be understood that for purposes of the methods set forth Non - limiting exemplary substrate materials that can be herein the use of an emulsion is optional and indeed for coated with a polymer of the present disclosure or that can several embodiments an emulsion is not used . Furthermore , otherwise be used in a composition or method set forth primers need not be attached directly to substrate or solid herein are described in U . S . Pat. Nos . 8 , 778 , 848 and 8 , 778 , 25 supports as set forth in the ePCR references and can instead 849 , each of which is incorporated herein by reference . be attached to a polymer coating as set forth herein . In some embodiments , a substrate described herein forms RCA techniques can be modified for use with a method , at least part of a flow cell or is located in a flow cell . In some composition or apparatus of the present disclosure . Exem such embodiments, the flow cells further comprise poly - plary components that can be used in an RCA reaction and nucleotides attached to the surface of the substrate via the 30 principles by which RCA produces amplicons are described , functional molecules coating, for example , a polymer coat for example , in Lizardi et al. , Nat. Genet. 19 :225 - 232 ( 1998 ) ing. In some embodiments , the polynucleotides are present and US 2007 /0099208 A1, each of which is incorporated in the flow cells in polynucleotide clusters , wherein the herein by reference . Primers used for RCA can be in solution polynucleotides of the polynucleotide clusters are attached or attached to a polymer coating . to a surface of the flow cell via the polymer coating . In such 35 MDA techniques can be modified for use with a method , embodiments , the surface of the flow cell body to which the composition or apparatus of the present disclosure . Some polynucleotides are attached is considered the substrate . In basic principles and useful conditions for MDA are other embodiments , a separate substrate having a polymer described , for example , in Dean et al. , Proc. Natl. Acad . Sci. coated surface is inserted into the body of the flow cell . In USA 99 :5261 -66 (2002 ) ; Lage et al. , Genome Research preferred embodiments, the flow cell is a flow chamber that 40 13 : 294 - 307 (2003 ) ; Walker et al . , Molecular Methods for is divided into a plurality of lanes or a plurality of sectors , Virus Detection , Academic Press , Inc ., 1995 ; Walker et al. , wherein one or more of the plurality of lanes or plurality of Nucl. Acids Res. 20 : 1691 - 96 ( 1992 ) ; U . S . Pat. No . 5 , 455 , sectors comprises a surface that is coated with a covalently 166 ; U .S . Pat. No . 5 , 130 ,238 ; and U .S . Pat . No . 6 ,214 , 587 , attached polymer coating described herein . In some embodi - each of which is incorporated herein by reference . Primers ments of the flow cells described herein , the attached poly - 45 used for MDA can be in solution or attached to a polymer nucleotides within a single polynucleotide cluster have the coating . same or similar nucleotide sequence . In some embodiments In particular embodiments a combination of the above of the flow cells described herein , the attached polynucle - exemplified amplification techniques can be used . For otides of different polynucleotide clusters have different or example , RCA and MDA can be used in a combination nonsimilar nucleotide sequences . Exemplary flow cells and 50 wherein RCA is used to generate a concatameric amplicon substrates for manufacture of flow cells that can be used in in solution ( e . g . using solution -phase primers ) . The ampli method or composition set forth herein include , but are not con can then be used as a template for MDA using primers limited to , those commercially available from Illumina , Inc . that are attached to a polymer coating . In this example , ( San Diego , Calif . ) or described in US 2010 /0111768 A1 or amplicons produced after the combined RCA and MDA US 2012 /0270305 , each of which is incorporated herein by 55 steps will be attached to the polymer coating reference . In some embodiments , the functionalized hydrogel or Sequencing Application polymer - coated substrate described herein can be used in a A composition , apparatus or method set forth herein can method for determining a nucleotide sequence of a poly be used with any of a variety of amplification techniques. nucleotide . In such embodiments , the method can comprise Exemplary techniques that can be used include , but are not 60 the steps of ( a ) contacting a polynucleotide polymerase with limited to , polymerase chain reaction ( PCR ) , rolling circle polynucleotide clusters attached to a surface of a substrate amplification (RCA ) , multiple displacement amplification via any one of the polymer or hydrogel coatings described (MDA ), or random prime amplification (RPA ). In particular herein ; (b ) providing nucleotides to the polymer - coated embodiments , one or more primers used for amplification surface of the substrate such that a detectable signal is can be attached to a polymer coating . In PCR embodiments , 65 generated when one or more nucleotides are utilized by the one or both of the primers used for amplification can be polynucleotide polymerase ; ( c ) detecting signals at one or attached to a polymer coating . Formats that utilize two more polynucleotide clusters; and ( d ) repeating steps ( b ) and US 9 ,815 ,916 B2 47 48 ( c ), thereby determining a nucleotide sequence of a poly - is incorporated herein by reference in its entirety ). In pyro nucleotide present at the one or more polynucleotide clus sequencing , released PPi can be detected by being immedi ters . ately converted to adenosine triphosphate ( ATP ) by ATP Nucleic acid sequencing can be used to determine a sulfurylase , and the level of ATP generated can be detected nucleotide sequence of a polynucleotide by various pro - 5 via luciferase - produced photons . Thus , the sequencing reac cesses known in the art . In a preferred method , sequencing - tion can be monitored via a luminescence detection system . by -synthesis ( SBS ) is utilized to determine a nucleotide Excitation radiation sources used for fluorescence based sequence of a polynucleotide attached to a surface of a detection systems are not necessary for pyrosequencing substrate via any one of the polymer coatings described procedures. Useful fluidic systems, detectors and procedures herein . In such process , one or more nucleotides are pro - 10 that can be used for application of pyrosequencing to arrays vided to a template polynucleotide that is associated with a of the present disclosure are described , for example , in WO polynucleotide polymerase . The polynucleotide polymerase 12 /058096 A1, US 2005 / 0191698 A1 , U . S . Pat. No. 7 , 595 , incorporates the one or more nucleotides into a newly 883 , and U . S . Pat. No. 7 ,244 ,559 , each of which is incor synthesized nucleic acid strand that is complementary to the porated herein by reference in its entirety . polynucleotide template . The synthesis is initiated from an 15 Sequencing -by - ligation reactions are also useful includ oligonucleotide primer that is complementary to a portion of ing , for example , those described in Shendure et al . Science the template polynucleotide or to a portion of a universal or 309: 1728 - 1732 (2005 ) ; U . S . Pat. No . 5 ,599 ,675 ; and U . S . non -variable nucleic acid that is covalently bound at one end Pat . No. 5 ,750 ,341 , each of which is incorporated herein by of the template polynucleotide . As nucleotides are incorpo reference in its entirety . Some embodiments can include rated against the template polynucleotide, a detectable signal 20 sequencing - by -hybridization procedures as described , for is generated that allows for the determination of which example , in Bains et al. , Journal of Theoretical Biology nucleotide has been incorporated during each step of the 135 ( 3 ), 303 - 7 ( 1988 ) ; Drmanac et al ., Nature Biotechnology sequencing process . In this way , the sequence of a nucleic 16 , 54 - 58 ( 1998 ) ; Fodor et al. , Science 251( 4995 ) , 767 - 773 acid complementary to at least a portion of the template ( 1995 ) ; and WO 1989/ 10977 , each of which is incorporated polynucleotide can be generated , thereby permitting deter - 25 herein by reference in its entirety . In both sequencing -by mination of the nucleotide sequence of at least a portion of ligation and sequencing -by -hybridization procedures , the template polynucleotide . nucleic acids that are present at sites of an array are Flow cells provide a convenient format for housing an subjected to repeated cycles of oligonucleotide delivery and array that is produced by the methods of the present disclo detection . Fluidic systems for SBS methods as set forth sure and that is subjected to a sequencing -by - synthesis 30 herein or in references cited herein can be readily adapted (SBS ) or other detection technique that involves repeated for delivery of reagents for sequencing -by - ligation or delivery of reagents in cycles . For example, to initiate a first sequencing -by -hybridization procedures . Typically , the oli SBS cycle , one or more labeled nucleotides, DNA poly - gonucleotides are fluorescently labeled and can be detected merase , etc . , can be flowed into / through a flow cell that using fluorescence detectors similar to those described with houses a nucleic acid array made by methods set forth 35 regard to SBS procedures herein or in references cited herein . Those sites of an array where primer extension herein . causes a labeled nucleotide to be incorporated can be Some embodiments can utilize methods involving the detected . Optionally, the nucleotides can further include a real- time monitoring of DNA polymerase activity . For reversible termination property that terminates further example , nucleotide incorporations can be detected through primer extension once a nucleotide has been added to a 40 fluorescence resonance energy transfer ( FRET) interactions primer. For example , a nucleotide analog having a reversible between a fluorophore - bearing polymerase and y -phosphate terminator moiety can be added to a primer such that labeled nucleotides , or with zeromode waveguides ( ZMWs) . subsequent extension cannot occur until a deblocking agent Techniques and reagents for FRET -based sequencing are is delivered to remove the moiety . Thus , for embodiments described , for example , in Levene et al . Science 299 , 682 that use reversible termination , a deblocking reagent can be 45 686 ( 2003 ) ; Lundquist et al. Opt . Lett. 33 , 1026 - 1028 delivered to the flow cell (before or after detection occurs ) . ( 2008 ) ; Korlach et al. Proc. Natl. Acad . Sci. USA 105 , Washes can be carried out between the various delivery 1176 - 1181 ( 2008 ) , the disclosures of which are incorporated steps. The cycle can then be repeated n times to extend the herein by reference in its entirety . primer by n nucleotides, thereby detecting a sequence of Some SBS embodiments include detection of a proton length n . Exemplary SBS procedures, fluidic systems and 50 released upon incorporation of a nucleotide into an exten detection platforms that can be readily adapted for use with sion product. For example , sequencing based on detection of an array produced by the methods of the present disclosure released protons can use an electrical detector and associated are described , for example , in Bentley et al. , Nature 456 : techniques that are commercially available from Ion Torrent 53 -59 (2008 ) , WO 04 /018497 ; U . S . Pat. No . 7 , 057 ,026 ; WO (Guilford , Conn ., a Life Technologies subsidiary) or 91 /06678 ; WO 07 / 123744 ; U . S . Pat. No. 7 , 329 ,492 ; U . S . 55 sequencing methods and systems described in US 2009/ Pat. No . 7, 211, 414 ; U . S . Pat. No . 7 ,315 , 019 ; U . S . Pat . No . 0026082 A1; US 2009 / 0127589 A1; US 2010 /0137143 A1; 7 ,405 ,281 , and US 2008 /0108082 , each of which is incor - or US 2010 /0282617 A1, each of which is incorporated porated herein by reference in its entirety . herein by reference in its entirety . Other sequencing procedures, including for example Another useful application for an array of the present those that use cyclic reactions , can be used , such as pyro - 60 disclosure , for example , having been produced by a method sequencing . Pyrosequencing detects the release of inorganic set forth herein , is gene expression analysis . Gene expres pyrophosphate (PPI ) as particular nucleotides are incorpo - sion can be detected or quantified using RNA sequencing rated into a nascent nucleic acid strand (Ronaghi , et al ., techniques, such as those, referred to as digital RNA Analytical Biochemistry 242 (1 ) , 84 - 9 ( 1996 ); Ronaghi, sequencing. RNA sequencing techniques can be carried out Genome Res. 11 (1 ) , 3 - 11 (2001 ) ; Ronaghi et al. Science 65 using sequencing methodologies known in the art such as 281 ( 5375 ) , 363 (1998 ) ; U . S . Pat. No . 6 ,210 ,891 ; U . S . Pat. those set forth above. Gene expression can also be detected No. 6 ,258 ,568 and U . S . Pat. No . 6 ,274 ,320 , each of which or quantified using hybridization techniques carried out by US 9 ,815 ,916 B2 49 50 direct hybridization to an array or using a multiplex assay , -continued the products of which are detected on an array . An array of N3 the present disclosure , for example , having been produced Acrylamide / N - ( 2 - aminoethyl ) by a method set forth herein , can also be used to determine 5 NH methacrylamide genotypes for a genomic DNA sample from one or more hydrochloride/ H20 individual . Exemplary methods for array - based expression Vazo56 , 3 h , 45° C . IPHA . HCI and genotyping analysis that can be carried out on an array NaOH of the present disclosure are described in U . S . Pat . No . 10 7 ,582 ,420 ; 6 , 890 , 741 ; 6 , 913 ,884 or 6 ,355 ,431 or U . S . Pat . NHNH Pub. Nos . 2005/ 0053980 A1; 2009/ 0186349 Al or US 2005 /0181440 A1 , each of which is incorporated herein by reference in its entirety . 15 In some embodiments of the above - described method AZAPA (M1 ) which employ a flow cell , only a single type of nucleotide is present in the flow cell during a single flow step . In such embodiments , the nucleotide can be selected from the group consisting of dATP , dCTP, dGTP, HTTP and analogs thereof. " In other embodiments of the above- described method which employ a flow cell, a plurality of different types of nucleo tides are present in the flow cell during a single flow step . In such methods, the nucleotides can be selected from DATP , 25 CTP, dGTP, DTTP and analogs thereof. 25 Determination of the nucleotide or nucleotides incorpo rated during each flow step for one or more of the poly nucleotides attached to the polymer coating on the surface of the substrate present in the flow cell is achieved by detecting 30 a signal produced at or near the polynucleotide template . In N3 some embodiments of the above -described methods, the NH detectable signal comprises and optical signal. In other embodiments , the detectable signal comprises a non -optical signal. In such embodiments, the non - optical signal com NH, HCl prises a change in pH at or near one or more of the polynucleotide templates . A NH NH 0 EXAMPLES 40 NH Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims. 45 L - PAMAZA Example 1 Scheme 1 illustrates a synthetic scheme for the prepara tion of poly -acrylamide - co - AZAPA -co - aminoethylacrylam Scheme 1 . Synthesis of Orthogonally Bifunctionalized Polyacrylamides ide ( L -PAmAzA ). In the first step , N - ( 5 - azidoacetamidyl pentyl ) acrylamide ( AZAPA ) was synthesized from reacting - Br N - ( 5 - bromoacetamidylpentyl) acrylamide (BrAPA ) with NH sodium azide in DMF at 35° C . for 2 hours . Then , L - PA 55 mAzA was synthesized using an AIBN -type polymer initia tion system (Vazo56 ) by reacting AzAPA with acrylamide NaN3, DMF and N -( 2 - aminoethyl) methacrylamide HCl. The resulting 2 h , 35° C . L -PAmAzA has the three recurring units in the molar ratio x : y : z of about 90 % to about 5 % to about 5 % . NH In addition , crosslinking of the L -PAmAzA was achieved by the introduction of N ,N -methylenebisacrylamide mono 65 mers into the polymerization reaction , which resulted in a BrAPA crosslinked polymer (XL - PAMAZA ) with exemplary struc ture shown as follows : US 9 , 815, 916 B2 5 52 - continued M4 N3 N N NH

NH , HCl TABLE 1

Polymer M1 (mol % ) M2 (mol % ) M3a /b (mol % ) M4 (mol % )

15 a : 90 O

thithyay o N a : 90 1

= w 10a a : 8585 x :y : z = ~ 90 : - 5 :25 uaaauna b : 90 = N HN 20 a : 90 au a : 90 0 . 5 - In order to demonstrate the orthogonal reactivity of the XL - PAMAZA 25 bifunctional polyacrylamides , the coating performance of three new polyacrylamides in Table 1 ( Polymer 1 , “ P1 ” ; A series of linear polyacrylamides bearing orthogonal Polymer 4 , “ P4” ; Polymer 6 , “ P6 " ) containing 5 mol % functionalities were prepared using the thermal initiator aminoethyl functionality on epoxy monolayer surface was Vazo56 following the similar procedure described above . 30 assessed against the standard norbornene monolayer surface . The reaction time was about 1 . 5 hours to about 3 hours , Polymer 1 and Polymer 6 are L - PAmAzA and XL - PAmAzA followed by a purification step through precipitation into with the structures illustrated above. The simplified structure MeCN . Table 1 below summarizes the amounts of the , of Polymer 4 is shown below . monomers for the polymerization reactions . Polymer 4

NH N3 B , •2HCI NH HN N •2HCI ANv = XANHNH2 , NH NH, HCl Vazo56 #

M2 NH , HCl N 0 NH 0 NH

O NH

V u N - ( 2 - aminoethyl) HN metharcrylamide hydrochloride

8 x A x :y :z = ~ 90 :~ 5 :~ 5

Standard PAZAM polymer was used as control. The flow M3a : R = H 65 cell layout for the norbornene monolayer surface and the M3b : R = Me epoxy monolayer surface are summarized in Table 2 and Table 3 respectively . US 9 ,815 ,916 B2 53 54 TABLE 2 Polymer coupling Polymer Temperature coupling Vol. std . [Final [P5 /P7 ]/ Channel (°C . ) time (min ) PAZAM (UL ) Polymer PAZAM ]/ w /v % UM 81 420 PAZAM 0 . 5 ao control 420 P1 0 .5

8 420 P4 0 . 5 420 P6 0 . 5 O 88888888 420 PAZAM 0 . 5 control OvaAWNIE 60 420 Pi 0 .5 60 420 P4 0. 5 O 60 420 P6 0 .5

TABLE 3 Polymer coupling Polymer temperature coupling Vol. std . [Final [P5 /P7 ]/ Channel (°C . ) time (min ) PAZAM (UL ) Polymer PAZAM ]/ w /v % UM 420 PAZAM 0 . 5 control 60 420 P1 0 .5 |??? 420 P4 0 .5 420 P6 0 . 5 888888881 88888888 420 PAZAM 0 . 5 control 60 420 P1 0 . 5 420 P4 0 .5 60 420 P6 0 .5 ?

HiSeq substrates ( provided by ILLUMINA , San Diego , TABLE 4 - continued Calif . ) were used for this initial screening and the CVD process was performed using a desiccator. The bifunctional % Intensity change, % Surface polyacrylamides polymers reacted with norbornene via 35 - Lanes QC1 - > QC3 Loss Polymer strain -promoted azide click reaction to covalently bond to 0 % 0 % P4 - 3 % 3 % P6 the norbornene monolayer surface at 60° C . Similarly , 13 % - 13 % PAZAM bifunctional polyacrylamides polymers were coated onto the control epoxy monolayers via epoxy ring opening reaction with Nauw - 2 % 2 % P1 amine functional groups which results in covalent bonding 40 - 6 % 6 % P4 of the polymers to the surface . Two QC metrics were used - 5 % 5 % P6 to measure the success of the method . Both QC1 and QC3 1223143 utilize green laser, with PMT at 450V and filter emission at 555 BP. The TET OC oligo mix for QC1 is 1 . 6 mM : 100 mL TABLE 5 oligos at 16 uM + 0 . 9 mL HT1. The TET QC oligo mix for % Intensity change , % Surface QC3 is 0 .6 mM ( each ) : 35 mL oligos at 16 uM + 0 . 9 mL HT1. Loss Polymer The Typhoon florescence image of the polymers coated flow Lanes QC1 - > QC3 84 % - 84 % PAZAM cell and the related chart ofmedian Typhoon intensity of the control polymers on the norbornene silane monolayer surface for 2 31 % - 31 % P1 TET QC1 and TET QC3 are illustrated in FIGS . 1A , 1B , 1C30 11 % - 11 % P4 and 1D respectively . The Typhoon florescence image of the AWN 13 % - 13 % P6 84 % - 84 % PAZAM polymers coated flow cell and the related chart of median control Typhoon intensity of the polymers on the epoxy silane 32 % - 32 % P1 monolayer surface for TET QC1 and TET QC3 are illus 12 % - 12 % P4 trated in FIGS. 2A , 2B , 2C and 2D respectively . 55 Ova 24 % - 24 % XL - PAAm3 TET QC measurements for the norbornene surface and the epoxy surface are summarized in Table 4 and Table 5 The results from the above noted pair of flow cells respectively . provided evidence supporting the use of orthogonal reactiv 60 ity of the polyacrylamide materials to support Sequencing TABLE 4 by - Synthesis . First, all the azido - functionalized materials % Intensity change , % Surface tested were capable of adhering securely to a norbornene Lanes QC1 - > QC3 Loss Polymer surface . This means that the azide incorporation into the polymer structure was such that a stable surface could be = 11 % - 11 % PAZAM control 65 obtained , as measured by a thermal stress test . Second , all of N - 1 % 1% P1 the amine - functionalized polymers were capable of coating the epoxy surface that was generated by the use of a US 9 ,815 ,916 B2 55 56 desiccator . The surface primer densities were approximately 20 - 30 k . In these experiments , the control polymer ( i. e ., the Scheme 2. 2 . standard PAZAM ) , which contained no amine functionality , NH2+ HCl showed the largest surface loss after the thermal stress test. This is the expected result . The bifunctionalized polyacry lamide polymers P1, P4 and P6 , each with 5 % amine functionality , showed reasonable surface stability . The O NH results indicated that these polyacrylamide coated surfaces were robust ( surface losses ranging from about 20 % to 30 % 10 after subjecting the polymer coated surface to the standard C12H255 ' s OH - DMSO , Vazo56 Stress test ) . The results of TET QC signal changes of the norbornene monolayer surface and the epoxy monolayer surface are shown in FIG . 3A and FIG . 3B respectively . Of 13 the three bifunctionalized polyacrylamides tested , Polymer 4 demonstrates the best surface robustness. The orthogonal polyacrylamides prepared by the proce dure described above are generally random copolymers. It 20 may be desirable to separate different functional parts of the polymer architecture , for example , separating all the azide NH2 functional groups from all the amine functional groups to different segments of the polymer chain . This alternative synthesis is readily achievable using controlled radical 25 polymerization (CRP ) methods ( e . g . , RAFT, ATRP ) . NH Scheme 2 . 1 and 2 . 2 demonstrate two synthetic routes for preparing a block copolymer AEMA - b - AZAPA (Polymer 7 ). OH C2H2C12H25Ss s1 AZAPA 3030 Color Litty Scheme 2 . 1 . NH2

35 O NH

40 NH

NH NH2

45

O NH NH NH

AZAPA HO 50 min minu NC O = AEMA - b - AZAPA N3 Polymer 7 55 The coating performance of a block copolymer AEMA b - AzAPA prepared by a RAFT technique according to NH2 Scheme 2 . 2 was compared to that of a random copolymer Polymer 4 on epoxy silane monolayer surface . The CVD NH process was performed on the flow cell using a desiccator in an oven at 60° C . and the flow cell was incubated overnight. The flow cell layout is summarized in Table 6 . The coupling reaction between the amino functional groups of Polymer 7 mots 65 ma and Polymer 4 with the epoxy surface was performed at 60° C . for an hour. US 9 ,815 , 916 B2 57 58 TABLE 6 Polymer Polymer/ Epoxy Vol. of polymer coupling temp. surface coupling used for Approx . Channel ( C . ) time (min ) coating (ul ) Polymer (polymer ]/ w /v % [P5 / P7 ]/ UM

g 8 450 Polymer 7 0 . 3 * ?

8 N ; 50 Polymer 7 0 . 3 * ?

8 w ; 450 Polymer 7 0 . 3 * ? A ; 8 450 Polymer 7 0 . 3 * ? 8 a ; 450 Polymer 4 0 . 5 ? 8 ; Polymer 4 0 . 5 ? a 450 8 ; 450 Polymer 4 0 . 5 ? v

o ; 8 450 Polymer 4 0 . 5 ? * The solids content of this batch , as measured by RI, was very high (4 . 9 % Brix @ 0 . 3 % w /v ) 20 Two QC metrics ( QC1 and QC3 ) were used to measure TABLE 7 . the success of the method . The Typhoon florescence image % Intensity change , % Surface of the polymers coated flow cell and the related chart of Lanes Polymer QC1- > QC3 Loss median Typhoon intensity of the polymers on the epoxy 25 Polymer 7 4 % - 4 % Polymer 7 4 % - 4 % silane monolayer surface for TET QC1 and TET QC3 are Polymer 7 3 % - 3 % Polymer 7 2 % - 2 % illustrated in FIGS. 4A , 4B , 4C and 4D respectively . The Polymer 4 12 % - 12 % results of TET QC signal change are shown in FIG . 4E . TET OvaAWNA Polymer 4 13 % - 13 % 30 Polymer 4 13 % - 13 % QC measurements for the epoxy surface are summarized in Polymer 4 14 % - 14 % Table 7 below . Both materials yielded stable surfaces as measured by TET QC performed after a thermal Stress Test . In each case , the coatings were very uniform . Example 2

Scheme 3 .

HNO HNO HN H?N So HNCo HN Co NH2 DNA copolymer NH N N

Coat

Silanize Wuhan 0 - si- 0 mm. US 9 ,815 ,916 B2 59 60 - continued

ttttttHNHNO H3N20 up to

HN Y

wan20 - si - 0 ,

Scheme 3 illustrates a flow chart of substrate preparation by immobilizing a DNA copolymer to a silanized substrate - continued surface . First, DNA copolymer is formed by reacting alkyne functionalized primers with acrylamide, azido -acrylamide 30 - 0 - USAP - 0 Base and amino -acrylamide monomers to form a pre - grafted ternary copolymer (DNA copolymer ) . The substrate surface is first treated with a silane comprising epoxy groups. Then the DNA copolymer is immobilized to the substrate surface by reacting the primary amino groups of the polymer with os the epoxy groups of the silane . The architecture of the DNA 35 copolymer prepared by this process may be modified by JomoPrimer addition of other monomers , for example , N ,N -methylen Primer = P5 or P7 ebisacrylamide can be added to introduce crosslinking in a defined manner , or inimers ( or monomer - initiators ) can be NN added to introduce branching points in a defined manner. 40 Tz- Am Controlled polymerization techniques such as RAFT, ATRP, or NMP may also be used to create block copolymer N - N r . t. , < 1 h structures separating out the functional parts of the polymer to be more effective , if needed . Example 3 45 111 mit Scheme 4 . Oligo Reaction with Tetrazine - Functionalized Polymer

50

ON NHO NH 55 tothe Tz - Am H mu NH 60 Scheme 4 illustrates the reaction between bicyclo [6 .1 .0 ] non - 4 - yne (“ BCN ” ) functionalized P5 or P7 primers with tetrazine modified acrylamide polymer (“ Tz - Am ” ) to form a grafted polymer . This catalyst - free, strain promoted click Tz - Am reaction can be performed at room temperature and it is 65 compatible with aqueous environment. The resulting grafted polymer can be purified using a number of methods, e . g . precipitation or tangential flow filtration (“ TFF ” ) etc . Other US 9 ,815 ,916 B2 61 62 non - limiting possible polymer backbones that can be used in this process include polyacrylates or polyphosphazenes. - continued

Scheme 5 . Attachment of Pre - grafted Tetrazine Polymer to Surface

Tz - Am

Tz - Am NH

i

såär man min wan min Scheme 5 illustrates the attachment of the pre - grafted tetrazine acrylamide polymer to norbornene functionalized surface of a substrate . The norbornene silanized surface is a 30 standard part of the NextSeq? platform of Illumina. Alter natively, tetrazine functionalized polymer and BCN primers may be attached to the substrate surface in situ instead of forming the grafted polymer. To assess the feasibility of this approach , initial experi 35 ments were carried out using a model system in a small scale solution reaction (Scheme 6 ).

Scheme 6 .

H

V= N N + ? ? V? Nb

Tz - Am mn Tz - BiPy

??

mi Nb - Tz - BiPy 65 Scheme 6 demonstrates the reaction between norbornene (Nb ) and a commercially available bipyridyl tetrazine (BiPy ) at 1 : 1 mole ratio . The reaction was carried out at US 9 ,815 ,916 B2 63 64 room temperature in an NMR tube , using CDC1z as solvent -continued with mild agitation . A NMR spectrum of the reaction mix ture was taken at three different time points , one at the beginning of the reaction (t = 0 ) , one at 15 minutes and one 5 HHH at 60 minutes. The NMR spectra showed that the peak of the two alkene hydrogens of norbornene (with chemical shift at HO . ?? about 5 . 8 ppm ) was disappearing and became almost invis ible after one hour ( See FIG . 5 ) . This indicates the rapid 10 kinetics of the reaction between tetrazine and norbornene . ?? NH NH

Scheme 7 .

N - N -N2 MeOH , It NEN 20 Scheme 8 illustrates the preparation of a pre - grafted poly (glycidyl methacrylate ) comb polymer by reacting the ?? glycidyl ether groups of the poly ( glycidyl methacrylate ) with the amino groups of the functionalized primers and N - N amino - PEG -azide . This grafted polymer can be attached to a standard norbornene surface via catalyst - free, strain pro moted click reaction between the side chain azido groups of the polymer and the norbornenes . A number of commer cially available amino azides can be used and the azido groups may also be replaced with other orthogonal func tional groups. Example 5 Illlll Scheme 9 . Preparation of Pre - grafted Poly( Glycidyl Methacrylate ) ?? 35 Boc In a separate experiment , Scheme 7 demonstrates a facile NH strain promoted [ 4 + 2 ] cycloaddition of cyclooctyne ( 10 mM ) with a bisphenyl substituted 1, 2, 4, 5 - tetrazine (1 mM ). * The reaction was carried out at room temperature in dried MeOH . FIG . 6 shows the pattern of UV - vis absorption decrease of cyclooctyne which indicates the reaction was nearly completed after only 9 minutes . See W . Chen , D . 45Hot L NH2 Wang , C . Dai , D . Hamelberg B . Wang , Chem . Commun ., 2012, 48 , 1736 - 1738 . 50 HHH Example 4 0 0 0 0 0 TO ??HO HOHO

Scheme 8 . Preparation of Pre - grafted Poly (Glycidyl Methacrylate ) 55 AL OH ?? ??

HN M i krofonNH - ??? NH2 US 9 ,815 , 916 B2 65 66 Scheme 9 illustrates the preparation of a pre - grafted a synthetic route utilizing the cyclic hexachlorophosphazene poly ( glycidyl methacrylate ) comb polymer by reacting the core for the construction of modified acrylamide monomers. glycidyl groups of the poly ( glycidyl methacrylate ) with the amino groups of the functionalized primers and amino - PEG Boc -amide . This grafted polymer is then subject to Boc - 5 Scheme 10 . 1 . deprotection to generate the primary amino functionalized side chain , which be attached to a glycidyl or epoxy func tionalized surface . NH Example 6 10 ci ci R - HN FIG . 7 illustrates the possible surface chemistry of a N N pre -grafted dendrimer with oligonucleotides bonded exter CI RULLR nal surface . The origin point of the dendrimer can be e functionalized with an azido group for direct surface attach - 15 al v = RN R ment. Alternatively , the azido group can react with an alkyne group in the center point of a second dendrimer , wherein the second dendrimer has substrate attachment groups “ A ” NH covered external surface to create a Janus type particle for self- assembly . 20 R mun HN Example 7 Scheme 10 . 2 and 10 . 3 demonstrate the synthesis of two Orthogonal polymers with backbone polyphosphazene scaffolds for subsequent polymer attach can also be used in the present application . Polyphospha - 25 ment. Several polyphosphazene syntheses have been zenes can serve as linear scaffolds for possible branching of reported by Qiu et al. , Nanotechnology , 18 ( 2007 ) 475 - 602 the polymer architecture , building dendronized polymers , or and Cheng et al. , Journal of Polymer Science , Part A : for subsequent polymer attachment. Scheme 10 . 1 illustrates Polymer Chemistry , 2013 , 51, 1205 - 1214 .

Scheme 10 . 2 .

HNHN

IN TT L CL Propargylamine 'N = * C12H255 CL N evelynO HN CNC NH

SC12H25 RR NEN

-Z -A ante

2 R = Z ' s SC12H25 theZ- time US 9 ,815 ,916 B2 67 US a15916 68 Scheme 10 . 3 . CICI Ri Ri OH N Cl RII N = Ci ZA Ri [Reagent ] ????S F R2 R2 ?? C12H25S s - NH2 R * F Y FR F RR S - O ' S SC12H25

Rj = min Nz RR2 == minn1 NH Scheme 10 . 4 illustrates two possible routes to prepare linear polydichlorophosphazene (PDCP ) backbone . Route 1 30 -continued is the anionic controlled polymerization . Route 2 is the ring CF3 opening reaction of hexachlorophosphazene . Route 1 is preferred with potential access to linear , cyclo - linear and F3C - 0 cross - linked polymer architecture , as well as the possibilitybility otp = NTTMS- TMS to introduce cross - linking . 35 O .

Scheme 10. 4 CF3 Route 1 40 FzCO TMS — N3 + PCls + D 45 CF3 Route 2 cici PEN 50 ci Controlled No control F2C7 TMS 1 +PN NEP - c1

SEQUENCE LISTING

< 160 > NUMBER OF SEO ID NOS : 4

V< 210 > SEQ ID NO 1 < 211 > LENGTH : 29 V< 212 > TYPE : DNA < 213P > ORGANISM : Artificial Sequence V< 220N > FEATURE : < NNNNN223N > OTHER INFORMATION : Artifically Synthesized Primer US 9 ,815 ,916 B2 69 70 - continued

< 400 > SEQUENCE : 1 aatgatacgg cgaccaccga gauctacac 29

< 210 > SEQ ID NO 2 < 211 > LENGTH : 24 < 212 > TYPE : DNA ?< 213 > ORGANISM : Artificial Sequence ?< 220NNEPP > FEATURE : ?< NNNN223 > OTHER INFORMATION : Artifically Synthesized Primer < 400 > SEQUENCE : 2 caagcagaag acggcatacg agat 24

< 210 > SEQ ID NO 3 ?.< 211 > LENGTH : 20 ?< 212 > TYPE : DNA ?< 213 > ORGANISM : Artificial Sequence ?< 220 > FEATURE : ?< 223 > OTHER INFORMATION : Artifically Synthesized Primer < 400 > SEQUENCE : 3 aatgatacgg cgaccaccga 20

< 210 > SEQ ID NO 4 < 211 > LENGTH : 21 ?< 212 > TYPE : DNA ?< 213 > ORGANISM : Artificial Sequence

? 20 > FEATURE :

?< 223 > OTHER INFORMATION : Artifically Synthesized Primer < 400 > SEQUENCE : 4 caagcagaag acggcatacg a 21

40 What is claimed is : wherein : 1 . A polymer for surface functionalization , comprising a each R14 , R20 , R1b and R25 is independently selected from recurring unit of Formula ( I ) and a recurring unit of Formula the group consisting of hydrogen , optionally substi ( II ) : tuted alkyl, and optionally substituted phenyl; 45 each R3a and R3b is independently selected from the group consisting of hydrogen , optionally substituted alkyl, optionally substituted phenyl , and optionally substi -? tuted C7- 14 aralkyl ; and 50 each L ' and L² is independently selected from the group N - R3a consisting of an optionally substituted alkylene linker and an optionally substituted heteroalkylene linker. 2 . The polymer of claim 1 , wherein Rla is hydrogen or Rla 55 optionally substituted alkyl and each of R20 and R3a is R20 hydrogen . NH2 (11 ) 3. The polymer of claim 1 , wherein R 16 is hydrogen or optionally substituted alkyl and each of R25 and R36 is hydrogen . N — R35 60 4 . The polymer of claim 1 , wherein L2 is an optionally substituted alkylene. 5 . The polymer of claim 1 , wherein L ' is an optionally RIO substituted alkylene. R 25 65 6 . The polymer of claim 1 , wherein the recurring unit of Formula (I ) is represented by Formula ( Ia ) and Formula (II ) is represented by Formula ( IIa ): US 9, 815 ,916 B2 72 ( la ) (IIIa )

?? RTU ( IIIb ) Rla R 45 RSS W R65RO R7R70 ( IIa ) NH2 wherein each R4a , R45 , and R5b is independently hydrogen or C1- 3 alkyl; and each R5a , Róa , Rób, and R75 is independently hydrogen , 20 optionally substituted C1- 6 alkyl, or optionally substi NH tuted phenyl. 12 . The polymer of claim 11, wherein each R4a , R45 , and R5 is independently hydrogen or methyl. 13. The polymer of claim 11 , wherein R6 and R7b are RIO 25 both hydrogen . 14 . The polymer of claim 11 , wherein at least one of R5a or Røa is hydrogen . wherein Rla is hydrogen or methyl and R15 is hydrogen or 15 . The polymer of claim 11 , wherein at least one of R5a methyl. or Røa is methyl. 7 . The polymer of claim 1 , wherein L ' is an optionally 30 16 . The polymer of claim 11 , wherein the one or more substituted heteroalkylene . recurring units of Formula ( IIIa ) are represented by ( IIIal ) , 8 . The polymer of claim 7 , wherein Ll is (CH . ) – (Ma2 ) , or (nas ) : NH — (CH2 )n - optionally substituted with one or more oxo , and wherein each m and n is independently an integer from 35 ( IIIal) 1 to 10 . O NH2 9 . The polymer of claim 1 , wherein the recurring unit of Formula ( I) is represented by Formula ( Ib ) : ( IIIa2 ) (lb ) Y N3, NH iti NH2 ( IIIa3 )

NH 17. The polymer of claim 11 , wherein the one or more recurring units of Formula ( IIIb ) are represented by ( IIIb1) : R la 55 ( IIIb1) wherein Rla is hydrogen or methyl. 10 . The polymer of claim 1 , further comprising one or more recurring units selected from the group consisting of ou N NH polyacrylamides, polyacrylates, polyurethanes , polysilox anes , silicones, polyacroleins, polyphosphazenes, polyiso cyanates , poly -ols , and polysaccharides, and combinations thereof. 18 . A substrate having a first surface comprising a poly 11 . The polymer of claim 10 . further comprising one or mer of claim 1 covalently bonded thereto . more recurring units of Formula ( IIIa ) or ( IIIb ) or both : * * * * *