Methods of generating nanoarrays and microarrays

Abstract

The methods described herein provide a means of producing an array of spatially separated proteins. The method relies on covalently attaching each protein of the plurality of proteins to a structured nucleic acid particle (SNAP), and attaching the SNAPs to a solid support.

1 .- 17 . (canceled) 
     
     
         18 . A method of forming an array of single biological entities, comprising:
 (a) providing a solid support comprising a plurality of attachment sites, wherein each attachment site comprises a covalently attached oligonucleotide, wherein the oligonucleotide comprises a self-hybridized region of internal complementarity;   (b) attaching a single biological entity to each seed oligonucleotide of a plurality of seed oligonucleotides; and   (c) hybridizing to each covalently attached oligonucleotide a seed oligonucleotide of the plurality of seed oligonucleotides to form a nucleic acid cluster, thereby providing the array of single biological entities.   
     
     
         19 . The method of  claim 18 , further comprising: (d) at each attachment site, covalently cross-linking the covalently attached oligonucleotide to the seed oligonucleotide. 
     
     
         20 . The method of  claim 19 , wherein cross-linking the covalently attached oligonucleotide to the seed oligonucleotide comprises utilizing a zero-length crosslinker. 
     
     
         21 . The method of  claim 19 , wherein cross-linking the covalently attached oligonucleotide to the seed oligonucleotide comprises utilizing a homobifunctional or heterobifunctional crosslinker. 
     
     
         22 . The method of  claim 19 , wherein covalently cross-linking the covalently attached oligonucleotide to the seed oligonucleotide comprises photoconjugating the oligonucleotide to the seed oligonucleotide. 
     
     
         23 . The method of  claim 22 , wherein photoconjugating the oligonucleotide to the seed oligonucleotide comprises irradiating the oligonucleotide and the seed oligonucleotide with ultraviolet (UV) light. 
     
     
         24 . The method of  claim 23 , wherein the oligonucleotide or the seed oligonucleotide comprises an o-nitrobenzyl functional group or a coumarin functional group. 
     
     
         25 . The method of  claim 23 , wherein the oligonucleotide or the seed oligonucleotide comprises a phenylazide, benzophenone, or phenyl-diazirine functional group. 
     
     
         26 . The method of  claim 18 , wherein the solid support comprises silicon, glass, fused silica, or quartz. 
     
     
         27 . The method of  claim 18 , wherein the solid support comprises a particle. 
     
     
         28 . The method of  claim 18 , wherein each attachment site of the plurality of attachment sites is less than 500 nanometers (nm) from any other attachment site. 
     
     
         29 . The method of  claim 18 , wherein each attachment site has a feature length of less than 300 nanometers (nm). 
     
     
         30 . The method of  claim 18 , wherein the single biological entity comprises a nucleic acid, a carbohydrate, a complex polymer, or a small molecule. 
     
     
         31 . The method of  claim 18 , wherein the single biological entity comprises a protein. 
     
     
         32 . The method of  claim 18 , further comprising isolating the single biological entity from a cell or tissue homogenate, a biological fluid, or an environmental sample. 
     
     
         33 . The method of  claim 18 , further comprising: (e) removing the nucleic acid cluster from the solid support. 
     
     
         34 . The method of  claim 33 , wherein the removing comprises chemical digestion of the nucleic acid cluster. 
     
     
         35 . The method of  claim 33 , wherein the removing comprises enzymatic digestion of the nucleic acid cluster. 
     
     
         36 . The method of  claim 18 , wherein the solid support is in a flow cell. 
     
     
         37 . The method of  claim 18 , further comprising: (f) detecting the single biological entity that is attached to the solid support via the nucleic acid cluster. 
     
     
         38 . The method of  claim 37 , wherein the detecting comprises fluorescence detection. 
     
     
         39 . The method of  claim 18 , wherein attaching the single biological entity to the seed oligonucleotide comprises covalently conjugating the single biological entity to the seed oligonucleotide. 
     
     
         40 . The method of  claim 39 , wherein covalently conjugating the seed oligonucleotide to the single biological entity comprises photoconjugating the single biological entity to the seed oligonucleotide. 
     
     
         41 . The method of  claim 40 , wherein photoconjugating the single biological entity to the seed oligonucleotide comprises irradiating the oligonucleotide and the seed oligonucleotide with ultraviolet (UV) light. 
     
     
         42 . The method of  claim 41 , wherein the single biological entity or the seed oligonucleotide comprises an o-nitrobenzyl functional group or a coumarin functional group. 
     
     
         43 . The method of  claim 42 , wherein the single biological entity or the seed oligonucleotide comprises a phenylazide, benzophenone, or phenyl-diazirine functional group. 
     
     
         44 . A method of forming an array of single biological entities, comprising:
 (a) providing a solid support comprising a plurality of attachment sites, wherein each attachment site comprises a covalently attached oligonucleotide, wherein the oligonucleotide comprises a self-hybridized region of internal complementarity;   (b) hybridizing to each oligonucleotide a seed oligonucleotide to form a nucleic acid cluster, wherein each seed oligonucleotide is covalently attached to a single biological entity; and   (c) growing each nucleic acid cluster by polymerization to form a larger polymeric molecule at each attachment site, thereby providing the array of single biological entities.