Nucleic acid delivery scaffolds
Disclosed herein, inter alia, are degradable nanoparticles, nanoarrays, and methods of use thereof in nucleic acid sequencing.
1. A method of sequencing a target polynucleotide, said method comprising:
(i) contacting a solid support comprising a plurality of particles with a degrading agent and decomposing said particles thereby forming a polymer film attached to solid support, wherein each particle comprises a degradable particle core and a polymer shell attached to said particle core, wherein said polymer shell comprises a first bioconjugate reactive moiety;
(ii) contacting said polymer film with an oligonucleotide comprising a second bioconjugate reactive moiety and binding said oligonucleotide to the polymer film;
(iii) hybridizing a target polynucleotide to said oligonucleotide and extending said oligonucleotide with a polymerase to form a complement of the target polynucleotide; and
(iv) sequencing said complement of the target polynucleotide.
2. The method of claim 1 , wherein said solid support comprises a plurality of wells.
3. The method of claim 2 , wherein said plurality of wells comprises one or more particles.
4. The method of claim 2 , wherein one or more particles are attached to a surface inside a well, and one or more particles are attached to a surface outside a well.
5. The method of claim 4 , further comprising removing the one or more particles attached to the surface outside a well prior to step (i).
6. The method of claim 5 , wherein removing comprises polishing the solid support with a cleaning article.
7. The method of claim 1 , wherein the solid support comprises about 1×10 5 to about 5×10 10 wells.
8. The method of claim 2 , wherein the wells are separated from each other by about 0.5 μm to about 2.0 μm.
9. The method of claim 2 , wherein the wells are separated from each other by about 0.7 μm to about 1.5 μm.
10. The method of claim 2 , wherein the wells are from about 0.2 μm to about 2 μm in diameter, and wherein the wells are about 0.5 μm to about 2 μm in depth.
11. The method of claim 1 , further comprising amplifying said complement of the target polynucleotide.
12. The method of claim 11 , wherein amplifying comprises of rolling circle amplification (RCA), exponential rolling circle amplification (eRCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), or template walking amplification.
13. The method of claim 11 , wherein amplifying comprises thermal bridge polymerase chain reaction (t-bPCR) amplification, chemical bridge polymerase chain reaction (c-bPCR) amplification, or chemical-thermal bridge polymerase chain reaction (cT-bPCR) amplification).
14. The method of claim 1 , wherein sequencing comprises sequencing by synthesis, sequencing by binding, sequencing by ligation, or pyrosequencing.
15. The method of claim 1 , wherein sequencing comprises hybridizing a sequencing primer to the target polynucleotide, or a complement thereof, and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides.
16. The method of claim 2 , wherein the solid support comprises polymerized units of alkoxysilyl methacrylate, alkoxysilyl acrylate, alkoxysilyl methylacrylamide, alkoxysilyl acrylamide, or a copolymer thereof.
17. The method of claim 2 , wherein the solid support comprises a resist, wherein the resist is a silsesquioxane resist, an epoxy-based polymer resist, poly(vinylpyrrolidone-vinyl acrylic acid) copolymer resist, an Off-stoichiometry thiol-enes (OSTE) resist, amorphous fluoropolymer resist, a crystalline fluoropolymer resist, polysiloxane resist, or a organically modified ceramic polymer resist.
18. The method of claim 1 , wherein said degradable particle core is a metal-organic framework (MOF) core.
19. The method of claim 18 , wherein said MOF core is a Isoreticular Metal-Organic Framework (IR-MOF) core, Zeolitic Imidazolate Framework (ZIF) core, Porous Coordination Network (PCN) core, Materials Institute Lavoisier (MIL) MOF core, Porous Coordination Polymer (PCP) core, or University of Oslo (UiO) MOF core.
20. The method of claim 18 , wherein said MOF core is a Zeolitic Imidazolate Framework 8 (ZIF-8) core or a UiO-66 MOF core.
21. The method of claim 1 , wherein the polymer shell comprises polyacrylamide (AAm), poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM), poly-N-isopropylacrylamide, poly N-isopropylpolyacrylamide, polyethylene glycol acrylate, methacrylate, polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), glicydyl methacrylate (GMA), glicydyl methacrylate (GMA) azide, hydroxyethylimethacrylate (HEMA), hydroxyethylacrylate (HEA), hydroxypropylmethacrylate (HPMA), polyethylene glycol methadrylate (PEGMA), polyethylene glycol acrylate (PEGA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof.
22. The method of claim 1 , wherein said degrading agent is a 0.1 to 1.0 M solution of an acid or a 0.1 to 1.0 M solution of a base.
23. The method of claim 1 , wherein said degrading agent comprises NaOH.
24. A method of sequencing a target polynucleotide, said method comprising:
(i) contacting a solid support comprising a plurality of particles with a degrading agent and decomposing said particles thereby forming a polymer film attached to solid support, wherein each particle comprises a degradable particle core and a polymer shell attached to said particle core, wherein said polymer shell comprises an oligonucleotide moiety covalently attached to said polymer shell;
(ii) hybridizing a target polynucleotide to said oligonucleotide moiety and extending said oligonucleotide with a polymerase to form a complement of the target polynucleotide; and
(iii) sequencing said complement of the target polynucleotide.
25. The method of claim 24 , wherein said solid support comprises a plurality of wells.
26. The method of claim 25 , wherein one or more particles are attached to a surface inside a well, and one or more particles are attached to a surface outside a well.
27. The method of claim 26 , further comprising removing the one or more particles attached to the surface outside a well prior to step (i).
28. The method of claim 27 , wherein removing comprises polishing the solid support with a cleaning article.
29. The method of claim 24 , wherein the solid support comprises about 1×10 5 to about 5×10 10 wells.
30. The method of claim 24 , wherein said degradable particle core is a metal-organic framework (MOF) core.