Multivalent binding composition for nucleic acid analysis

Abstract

Multivalent binding compositions including a particle-nucleotide conjugate having a plurality of copies of a nucleotide attached to the particle are described. The multivalent binding compositions allow one to localize detectable signals to active regions of biochemical interaction, e.g., sites of protein-protein interaction, protein-nucleic acid interaction, nucleic acid hybridization, or enzymatic reaction, and can be used to identify sites of base incorporation in elongating nucleic acid chains during polymerase reactions and to provide improved base discrimination for sequencing and array based applications.

1 . A method of determining an identity of a nucleotide in a nucleic acid sequence, the method comprising:
 (a) providing a primed nucleic acid sequence comprising a nucleobase coupled to a nucleotide of said primed nucleic acid sequence at an N position, wherein said nucleobase comprises a blocking group coupled thereto;   (b) bringing said primed nucleic acid sequence into contact with a conjugated polymer-nucleotide composition under conditions sufficient to form a stable ternary complex between a nucleotide moiety of said conjugated polymer-nucleotide composition and said primed nucleic acid sequence at an N+1 position; and   (c) detecting said stable ternary complex to determine said identity of said nucleotide at said N+1 position of said primed nucleic acid sequence.   
     
     
         2 . The method of  claim 1 , wherein said conjugated polymer-nucleotide composition comprises a plurality of said nucleotide moiety conjugated to a polymer core. 
     
     
         3 . The method of  claim 2 , wherein said polymer core comprises polyethylene glycol, polypropylene glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid. 
     
     
         4 . The method of  claim 3 , wherein said polymer core is a branched. 
     
     
         5 . The method of  claim 1 , wherein said conjugated polymer-nucleotide further comprises a interaction moiety coupled to said polymer core, wherein said interaction moiety is selected from the group consisting of avidin, streptavidin, a biotin moiety, an affinity tag, an antibody or antigen-binding fragment thereof, and a receptor. 
     
     
         6 . The method of  claim 1 , wherein said conjugated polymer-nucleotide composition comprises a detectable label. 
     
     
         7 . The method of  claim 6 , wherein said detectable label comprises a fluorescent label, a fluorescence resonance energy transfer (FRET) donor, or a fluorescence resonance energy transfer (FRET) acceptor. 
     
     
         8 . The method of  claim 1 , wherein said blocking group is coupled to said nucleobase at the 3′ carbon. 
     
     
         9 . The method of  claim 1 , wherein said blocking group is selected from the group consisting of O-alkyl hydroxylamine, O-methyl, 3′-phosphorothioate, a 3′-O-malonyl, and a 3′-O-benzyl. 
     
     
         10 . The method of  claim 1 , wherein said conjugated polymer-nucleotide composition comprises a plurality of detectable labels. 
     
     
         11 . The method of  claim 10 , wherein said plurality of detectable labels comprises a fluorescent label, a fluorescence resonance energy transfer (FRET) donor, or a fluorescence resonance energy transfer (FRET) acceptor. 
     
     
         12 . The method of  claim 1 , further comprising, before (a):
 (d) amplifying a plurality of nucleic acid sequences; and   (e) contacting said plurality of nucleic acid sequences with a primer to form a library of said primed nucleic acid sequence.   
     
     
         13 . The method of  claim 12 , wherein said amplifying is performed using polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-stranded binding (SSB) protein-dependent amplification, or any combination thereof. 
     
     
         14 . The method of  claim 13 , wherein said amplifying comprises RCA. 
     
     
         15 . The method of  claim 1 , further comprising, following (b) and (c):
 (d) deblocking said nucleobase comprising said blocking group coupled thereto at said N position; and   (e) extending said primed nucleic acid sequence by incorporating a second nucleobase comprising a second blocking group coupled thereto at said N+1 position.   
     
     
         16 . The method of  claim 15 , wherein said deblocking comprises bringing said primed nucleic acid sequence into contact with a deblocking solution under conditions sufficient to remove said blocking group from said nucleobase. 
     
     
         17 . The method of  claim 15 , wherein said extending comprises bringing said primed nucleic acid sequence into contact with an extension solution under conditions sufficient to incorporate said second nucleobase into said primed nucleic acid sequence at said N+1 position. 
     
     
         18 . The method of  claim 17 , wherein said extension solution comprises:
 (i) a polymerizing enzyme that is catalytically active; and   (ii) a plurality of said second nucleobase comprising said second blocking group coupled thereto.   
     
     
         19 . The method of  claim 18 , wherein said plurality of said second nucleobase comprises a plurality types of nucleobases selected from the group consisting of adenine, cytosine, thymine, and guanine. 
     
     
         20 . The method of  claim 15 , wherein said second blocking group is selected from the group consisting of O-alkyl hydroxylamine, O-methyl, 3′-phosphorothioate, a 3′-O-malonyl, and a 3′-O-benzyl.