Multipart reagents having increased avidity for polymerase binding

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 .- 20 . (canceled) 
     
     
         21 . A method for hybridizing a target nucleic acid molecule to a nucleic acid molecule coupled to a hydrophilic polymer coating layer, the method comprising:
 (a) providing a surface comprising the hydrophilic polymer coating layer coupled thereto;   (b) providing at least one nucleic acid molecule that is coupled to the hydrophilic polymer coating layer, wherein the hydrophilic polymer coating layer has a water contact angle of less than 45 degrees; and   (c) bringing the at least one nucleic acid molecule coupled to the hydrophilic polymer coating layer into contact with a hybridizing composition comprising a target nucleic acid molecule at a concentration of one nanomolar or less under conditions sufficient for said target nucleic acid molecule to hybridize to the at least one nucleic acid molecule coupled to the hydrophilic polymer coating layer in 30 minutes or less.   
     
     
         22 . The method of  claim 21 , wherein said conditions are maintained at a temperature that is from about 30 degrees Celsius to 70 degrees Celsius. 
     
     
         23 . The method of  claim 21 , wherein said hydrophilic polymer coating layer comprises a plurality of said at least one nucleic acid molecules with a substantially uniform surface density. 
     
     
         24 . The method of  claim 21 , further comprising performing a nucleotide binding reaction on said surface between said at least one nucleic acid molecule and said target nucleic acid molecule. 
     
     
         25 . The method of  claim 21 , wherein the target nucleic acid molecule is present in the hybridizing composition at a concentration of 0.50 nanomolar or less. 
     
     
         26 . The method of  claim 21 , wherein the target nucleic acid molecule is present in the hybridizing composition at a concentration of 250 picomolar or less. 
     
     
         27 . The method of  claim 21 , wherein the target nucleic acid molecule is present in the hybridizing composition at a concentration of 100 picomolar or less. 
     
     
         28 . The method of  claim 21 , wherein bringing the at least one nucleic acid molecule coupled to the hydrophilic polymer coating layer into contact with the hybridization composition is performed for a time period of less than 30 minutes. 
     
     
         29 . The method of  claim 21 , further comprising hybridizing the target nucleic acid molecule to the at least one nucleic molecule coupled to the hydrophilic polymer coating layer at a hybridization efficiency that is increased as compared to a comparable hybridization reaction performed for 120 minutes at 90 degrees Celsius for 5 minutes followed by cooling for 120 minutes to reach a final temperature of 37 degrees Celsius in a buffer comprising saline-sodium citrate. 
     
     
         30 . The method of  claim 21 , further comprising hybridizing the target nucleic acid molecule to the at least one nucleic acid molecule with a hybridization stringency of at least 80%. 
     
     
         31 . The method of  claim 21 , wherein the hydrophilic polymer coating layer exhibits a level of non-specific Cyanine 3 dye absorption of less than about 0.25 molecules per square micrometer. 
     
     
         32 . The method of  claim 21 , wherein the hybridization composition further comprises:
 (a) at least one organic solvent that is polar and aprotic; and   (b) a pH buffer.   
     
     
         33 . The method of  claim 32 , wherein the at least one organic solvent has a dielectric constant of no greater than about 115 as measured at 68 degrees Fahrenheit. 
     
     
         34 . The method of  claim 32 , wherein the at least one organic solvent comprises at least one functional group selected from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. 
     
     
         35 . The method of  claim 34 , wherein the at least one organic solvent comprises formamide. 
     
     
         36 . The method of  claim 32 , wherein the at least one organic solvent is miscible with water. 
     
     
         37 . The method of  claim 32 , wherein the at least one organic solvent is at least about 5% by volume based on the total volume of the hybridizing composition. 
     
     
         38 . The method of  claim 37 , wherein the at least one organic solvent is at most about 95% by volume based on the total volume of the hybridizing composition. 
     
     
         39 . The method of  claim 32 , wherein the pH buffer is at most about 90% by volume of the total volume of the hybridizing composition. 
     
     
         40 . The method of  claim 32 , wherein the pH buffer comprises 2-(N-morpholino)ethanesulfonic acid, acetonitrile, 3-(N-morpholino)propanesulfonic acid, methanol, or a combination thereof. 
     
     
         41 . The method of  claim 32 , wherein the pH buffer further comprises a second organic solvent. 
     
     
         42 . The method of  claim 32 , wherein the pH buffer is present in the hybridizing composition in an amount that is effective to maintain the pH of the hybridizing composition in a range of about 3 to about 10. 
     
     
         43 . The method of  claim 21 , wherein the hybridizing composition further comprises a molecular crowding agent. 
     
     
         44 . The method of  claim 43 , wherein the molecular crowding agent is selected from the group consisting of polyethylene glycol, dextran, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, hydroxybutyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose, and hydroxyl methyl cellulose, and any combination thereof. 
     
     
         45 . The method of  claim 44 , wherein the molecular crowding agent is polyethylene glycol. 
     
     
         46 . The method of  claim 45 , wherein the molecular crowding agent has a molecular weight in the range of about 5,000 to 40,000 Daltons. 
     
     
         47 . The method of  claim 43 , wherein an amount of the molecular crowding agent is at least about 5% by volume based on the total volume of the hybridizing composition. 
     
     
         48 . The method of  claim 47 , wherein an amount of the molecular crowding agent at most about 50% by volume based on the total volume of the hybridizing composition. 
     
     
         49 . The method of  claim 21 , wherein the at least one nucleic acid molecule coupled to the hydrophilic polymer coating layer is coupled to the hydrophilic polymer coating layer through covalent bonding. 
     
     
         50 . The method of  claim 21 , wherein the hydrophilic polymer coating layer comprises at least one dendrimer.