METHODS OF GENERATING NANOARRAYS AND MICROARRAYS
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.