Photocrosslinked oligo-mediated cleavage

Abstract

Provided herein are compositions and methods for spatial profiling of biomolecules in a sample. Methods comprise photocrosslinked oligo-mediated cleavage (POMC), utilizing molecular proximity recording to generating spatial inference data without the need for indexing probe locations. Processes include disclosed herein generating concatemers of docking oligonucleotides, photo crosslinking oligonucleotides to generate cleavage sites, and generating spatial information from generated fragments.

Description

PCT PATENT APPLICATIONPHOTOCROSSLINKED OLIGO-MEDIATED CLEAVAGECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 623,973 filed January 23, 2024, which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ST .26 xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on January- 13, 2025, is named 205590-708601_SL.xml and is 6,475 bytes in size.BACKGROUND

[0003] Understanding how molecules are arranged within cells, and how cells are arranged within tissues, is important to interpreting, predicting, and engineering biological states. Advancements in microscopy have allowed for high-resolution reconstructions of subcellular structures, but the composition of biomolecules within such structures remains difficult to capture. Thus, there is an unmet need for obtaining greater detail with regard to biomolecule presence within tissue. Additionally, there is an opportunity for creating efficiencies in such analyses.BRIEF SUMMARY

[0004] Provided herein are methods of biological information generation, the methods comprising: a) immobilizing a concatemer onto a biological sample, wherein the concatemer is a nucleic acid and comprises two or more copies of a first docking oligonucleotide comprising: a barcode sequence; and a flanking region, b) contacting the biological sample with at least one cleaving oligonucleotide, wherein the cleaving oligonucleotide comprises a region complementary to a region on the first docking oligonucleotide, and wherein the first docking oligonucleotide and the cleaving oligonucleotide comprise at least one of each independently: a photocrosslinking agent, and at least one restriction site; c) illuminating predefined regions of the sample to activate the photocrosslinking agent, thereby binding the first docking oligonucleotide and the cleaving oligonucleotide to generate a photocrosslinked oligonucleotide complex; d) contacting the photocrosslinked oligonucleotide complex with an endonuclease, wherein the endonuclease cleaves the photocrosslinked oligonucleotide complex at one of the at least one restriction site, thereby generating a duplexed fragment of the photocrosslinked oligonucleotide complex, andwherein the duplexed fragment comprises the barcode sequence; e) polymerizing the duplexed fragment using cross-junction synthesis to generate a contiguous nucleic acid comprising copy of a region from each of the first docking oligonucleotide comprising the barcode sequence and the cleaving oligonucleotide; f) sequencing the contiguous nucleic acid, wherein an association of barcode sequence and cleaving oligonucleotide provides biological information. Further provided herein are methods, wherein the biological sample comprises a population of cells, a tissue sample, or a protein. Further provided herein are methods, wherein the biological sample is fixed on a surface. Further provided herein are methods, wherein the docking oligonucleotide and the cleaving oligonucleotide each independently comprise ribonucleotides, deoxyribonucleotides, or any variation or combination thereof. Further provided herein are methods, wherein the concatemer is generated by rolling circle amplification (RCA), strand-displacement amplification (SDA), Nicking Enzyme Amplification Reaction (NEAR), exponential amplification reaction (EXPAR), enzymatic oligonucleotide synthesis via internal inosine modification and exonuclease V, nick translation, Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Multiple Displacement Amplification (MDA), Ligase Chain Reaction (LCR), Recombinase Polymerase Amplification (RPA), Ramification amplification method (RAM), Nucleic Acid Sequence-Based Amplification (NASBA), signal amplification by exchange reaction (SABER), or primer exchange reaction (PER). Further provided herein are methods, wherein the concatemer comprises at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000. at least 10,000 copies of the first docking oligonucleotide. Further provided herein are methods, wherein the barcode sequence is from about 3 to about 30 nucleotides. Further provided herein are methods, wherein the first docking oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, or more than 1000 nucleotides. Further provided herein are methods, wherein the cleaving oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, from about 1000 to about 1500, from about 1500 to about 2000, from about 2000 to about 2500, from about 2500 to about 3000, from about 3500 to about 4000, or more than 4000 nucleotides. Further provided herein are methods, wherein the photocrosslinking agent is located from about 2 to about 12 bases from the restriction site. Further provided herein are methods, wherein the photocrosslinking agent is located about 4 bases from the restriction site. Further provided herein are methods, wherein the restriction site is from about 5 to about 20 bases from the 5’ end of the cleaving oligonucleotide.Further provided herein are methods, wherein the restriction site is about 12 bases from the 5?end of the cleaving oligonucleotide.

[0005] Further provided to methods described herein are methods, wherein the immobilizing is by affinity binding, conjugation, incorporation into a hydrogel, crosslinking, or photo-crosslinking. Further provided herein are methods, wherein the immobilizing is at random locations throughout the biological sample. Further provided herein are methods, wherein the immobilizing is at specific targets in the biological sample. Further provided herein are methods, wherein the cleaving oligonucleotide further comprises a modification. Further provided herein are methods, wherein the modification comprises a photolinker, a fluorophore, a photo-cleavable spacer, a protein, a small molecule, a lipid, a nanoparticle, a lipid nanoparticle, or any combination thereof. Further provided herein are methods, wherein the modification comprises a modification to affect tethering, diffusion, or localization of an oligonucleotide. Further provided herein are methods, wherein the modification to affect tethering of an oligonucleotide comprises a base modified with an amino group, acrylic phosphoramidite, an alkyne, an azido, chloroacetamide, vinylsulfonamide, an aliphatic aldehyde, or any combination thereof. Further provided herein are methods, wherein the modification to affect diffusion comprise conjugation with additional nucleic acid strands, peptide, dextran, a polyethylene glycol (PEG), a short strand polymer, or any combination thereof. Further provided herein are methods, wherein the short strand polymer comprises a poly(glycerol) (PG), poly(oxazoline) (POX), poly(hydroxypropyl methacrylate) (PGPMA), poly(2-hydroxy ethyl methacrylate) (PHEMA), poly(N-(2- hydroxypropyl) methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N- acryloylmorpholine) (PAcM), or any combination thereof. Further provided herein are methods, wherein the modification to affect localization comprises a hybridization sequence directed to endogenous RNA, DNA, or cDNA. Further provided herein are methods, wherein the modification to affect localization comprises a conjugation to a biotin, a protein, an antibody or fragment thereof, a nanobody, a lipid, a nanoparticle, a lipid nanoparticle, other affinity reagent, or any combination thereof.

[0006] Further provided to methods described herein are methods, wherein the cleaving oligonucleotide further comprises a primer at the 3’ end of the cleaving oligonucleotide. Further provided herein are methods, further comprising hybridizing the primer to a complementary' domain on an mRNA in the sample. Further provided herein are methods, further comprising contacting the primer with a reverse transcriptase, thereby extending the primer to generate a complementary copy of a region of the mRNA. Further provided herein are methods, wherein the hybridizing step occurs prior to the illuminating step. Further provided herein are methods, wherein the hybridizing step occurs after the illuminating step. Further provided herein aremethods, further comprising hybridizing the primer to a second docking oligonucleotide, wherein the second docking oligonucleotide comprises a barcode sequence. Further provided herein are methods, further comprising contacting the primer with a polymerase, thereby extending the primer to generate a duplexed region comprising a region of the cleaving oligonucleotide and the second docking oligonucleotide. Further provided herein are methods, wherein the duplexed region further comprises a crosslinking agent. Further provided herein are methods, further comprising activating the crosslinking agent to generate a covalently bound duplex. Further provided herein are methods, wherein the activating is in the illuminating step. Further provided herein are methods, wherein the activating is in a second illuminating step.

[0007] Further provided to methods described herein are methods, further comprising controlling a diffusion factor of the sample, wherein the diffusion factor comprises a viscosity, a time, a temperature, a presence of crowding agents, a pH, an electric field, physical features, or any combination thereof. Further provided herein are methods, wherein the viscosity is greater than 1 cP. Further provided herein are methods, wherein the viscosity is from about 1 to about 10 cP, from about 10 to about 20 cP, from about 20 to about 30 cP, from about 30 to about 40 cP, from about 40 to about 50 cP, from about 50 to about 60 cP, from about 60 to about 70 cP, from about 70 to about 80 cP, from about 80 to about 90 cP, from about 100 to about 150 cP, from about 150 to about 300 cP. from about 300 to about 500 cP, from about 500 to about 1000 cP, from about 1000 to about 2000 cP, from about 2000 to about 3000 cP.

[0008] Further provided to methods described herein are methods, wherein the cleaving oligonucleotide further comprises one or more unique molecular modifiers (UMI). Further provided herein are methods, wherein the UMI comprises one or more noncanonical bases, universal bases, other bases which a polymerase will pair randomly or incorrectly, or any combination thereof.

[0009] Further provided to methods described herein are methods, wherein the endonuclease is a Type I, Type II, Type IIS, Type IIG, Type III, Type IV, or Type V endonuclease, or a Nickase. Further provided herein are methods, wherein the endonuclease comprises BtsI, Nb.BtsI, BbvCI, EcoRI, EcoRII, BamHI, Hindlll, TaqI, Notl, HinFI, Sau3AI, PvuII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I, Kpnl , PstI, SacI, Sall, Seal , Spel, SphI, Stul, Xbal, Alwl, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, BsmI, Nb.BsmI, BsmAI, Nt.BsmAI, BssSI, Nb.BssSI, Nt.BstNBI, BtsI, Nb.BtsI, or any combination thereof.

[0010] Further provided to methods described herein are methods, wherein the endonuclease is a Cas enzyme. Further provided herein are methods, wherein the Cas enzyme comprises a Type I, Type II, Type III, or Type IV Cas endonuclease. Further provided herein are methods, wherein theCas enzyme is a Cas9 enzyme. Further provided herein are methods, further providing a guide RNA specific for a sequence on the docking oligonucleotide or the cleaving oligonucleotide.

[0011] Further provided to methods described herein are methods, wherein the contacting the biological sample with an endonuclease step is before the illuminating step.

[0012] Further provided to methods described herein are methods, wherein the contacting the biological sample with at least one cleaving oligonucleotide comprises contacting the biological sample with more than one cleaving oligonucleotide, wherein each cleaving oligonucleotide comprises a region complementary to a different region on the docking oligonucleotide.

[0013] Further provided to methods described herein are methods, wherein the photocrosslinking agent comprises a photoreactive nucleobase. Further provided herein are methods, wherein the photoreactive nucleobase comprises 3-cyanovinylcarbazole phosphoramidite (CNVK) or 5- phenylethynyl-2'-deoxyuridine (PhedU).

[0014] Further provided to methods described herein are methods, wherein the polymerizing comprises depositing a DNA polymerase or an RNA polymerase on the sample. Further provided herein are methods, wherein the DNA polymerase comprises a Phi29 DNA Pol, an E. coli Pol I, an E. coli Pol II, an E. coli Pol III, an E. coli Pol IV, an E. coli Pol V, a T4 DNA Pol, a Bsm DNA Pol I, a Bst DNA Pol, a Reverse Transcriptase, or any combination thereof. Further provided herein are methods, wherein the RNA polymerase comprises T7. T3. SP6, or any combination thereof.

[0015] Further provided to methods described herein are methods, further comprising analyzing a frequency of association of barcode sequence and cleaving nucleic acid to generate spatial mapping of the biological sample.

[0016] Provided herein are methods of biological information generation, the methods comprising: a) immobilizing a first docking oligonucleotide template onto a biological sample, wherein the first docking oligonucleotide template comprises: a barcode sequence; and a flanking region, b) amplifying the first docking oligonucleotide template to generate a concatemer, wherein the concatemer comprises two or more copies of a first docking oligonucleotide; c) contacting the biological sample with at least one cleaving oligonucleotide, wherein the cleaving oligonucleotide comprises a region complementary to a region on the first docking oligonucleotide, wherein the first docking oligonucleotide and the cleaving oligonucleotide comprise at least one of each independently: a photocrosslinking agent, and at least one restriction site; d) illuminating predefined regions of the sample to activate the photocrosslinking agent, thereby binding the first docking oligonucleotide and the cleaving oligonucleotide to generate a photocrosslinked oligonucleotide complex; e) contacting the photocrosslinked oligonucleotide complex with an endonuclease, wherein the endonuclease cleaves the photocrosslinked oligonucleotide complex at one of the at least one restriction site, thereby generating a duplexed fragment of thephotocrosslinked oligonucleotide complex, and wherein the duplexed fragment comprises the barcode sequence; f) polymerizing the duplexed fragment using cross-junction synthesis to generate a contiguous nucleic acid comprising copy of a region from each of the first docking oligonucleotide comprising the barcode sequence and the cleaving oligonucleotide; g) sequencing the contiguous nucleic acid, wherein an association of barcode sequence and cleaving oligonucleotide provides biological information. Further provided herein are methods, wherein the biological sample comprises a population of cells, a tissue sample, or a protein. Further provided herein are methods, wherein the biological sample is fixed on a surface. Further provided herein are methods, wherein the docking oligonucleotide and the cleaving oligonucleotide each independently comprise ribonucleotides, deoxyribonucleotides, or any variation or combination thereof. Further provided herein are methods, wherein the concatemer is generated by rolling circle amplification (RCA), strand-displacement amplification (SDA), Nicking Enzyme Amplification Reaction (NEAR), exponential amplification reaction (EXPAR), enzymatic oligonucleotide synthesis via internal inosine modification and exonuclease V. nick translation. Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Multiple Displacement Amplification (MDA), Ligase Chain Reaction (LCR), Recombinase Polymerase Amplification (RPA), Ramification amplification method (RAM), Nucleic Acid Sequence-Based Amplification (NASBA), signal amplification by exchange reaction (SABER), or primer exchange reaction (PER). Further provided herein are methods, wherein the concatemer comprises at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000 copies of the first docking oligonucleotide. Further provided herein are methods, wherein the first docking oligonucleotide is immobilized to a splint sequence in a sample. Further provided herein are methods, wherein the splint sequence is a region of genomic DNA, mRNA, cDNA, or other nucleic acid in a sample. Further provided herein are methods, wherein the barcode sequence is from about 3 to about 30 nucleotides. Further provided herein are methods, wherein the first docking oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, or more than 1000 nucleotides. Further provided herein are methods, wherein the cleaving oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, from about 1000 to about 1500, from about 1500 to about 2000. from about 2000 to about 2500, from about 2500 to about 3000, from about 3500 to about 4000, or more than 4000 nucleotides. Further provided herein are methods, wherein the photocrosslinking agent is located from about 2 to about 12 bases from the restriction site. Further provided herein are methods,wherein the photocrosslinking agent is located about 4 bases from the restriction site. Further provided herein are methods, wherein the restriction site is from about 5 to about 20 bases from the 5’ end of the cleaving oligonucleotide. Further provided herein are methods, wherein the restriction site is about 12 bases from the 5’ end of the cleaving oligonucleotide.

[0017] Further provided to methods described herein are methods, wherein the immobilizing is by an affinity binding, conjugation, incorporation into a hydrogel, crosslinking, or photocrosslinking. Further provided herein are methods, wherein the immobilizing is at random locations throughout the biological sample. Further provided herein are methods, wherein the immobilizing is at specific targets in the biological sample.

[0018] Further provided to methods described herein are methods, wherein the cleaving oligonucleotide further comprises a modification. Further provided herein are methods, wherein the modification comprises a photolinker, a fluorophore, a photo-cleavable spacer, a protein, a small molecule, a lipid, a nanoparticle, a lipid nanoparticle, or any combination thereof. Further provided herein are methods, wherein the modification comprises a modification to affect tethering, diffusion, or localization of an oligonucleotide. Further provided herein are methods, wherein the modification to affect tethering of an oligonucleotide comprises a base modified with an amino group, acrylic phosphoramidite, an alky ne, an azido, chloroacetamide, vinylsulfonamide, an aliphatic aldehyde, or any combination thereof. Further provided herein are methods, wherein the modification to affect diffusion comprise conjugation with additional nucleic acid strands, peptide, dextran, a polyethylene glycol (PEG), a short strand polymer, or any combination thereof. Further provided herein are methods, wherein the short strand polymer comprises a poly(glycerol) (PG), poly(oxazoline) (POX), poly (hydroxy propyl methacrylate) (PGPMA), poly(2-hydroxy ethyl methacrylate) (PHEMA). poly(N-(2- hydroxypropyl) methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N- acryloylmorpholine) (PAcM), or any combination thereof. Further provided herein are methods, wherein the modification to affect localization comprises a hybridization sequence directed to endogenous RNA, DNA. or cDNA. Further provided herein are methods, wherein the modification to affect localization comprises a conjugation to a biotin, a protein, an antibody or fragment thereof, a nanobody, a lipid, a nanoparticle, a lipid nanoparticle, other affinity7reagent, or any combination thereof.

[0019] Further provided to methods described herein are methods, wherein the cleaving oligonucleotide further comprises a primer at the 3’ end of the cleaving oligonucleotide. Further provided herein are methods, further comprising hybridizing the primer to a complementary domain on an mRNA in the sample. Further provided herein are methods, further comprising contacting the primer with a reverse transcriptase, thereby extending the primer to generate acomplementary copy of a region of the mRNA. Further provided herein are methods, wherein the hybridizing step occurs prior to the illuminating step. Further provided herein are methods, wherein the hybridizing step occurs after the illuminating step. Further provided herein are methods, further comprising hybridizing the primer to a second docking oligonucleotide, wherein the second docking oligonucleotide comprises a barcode sequence. Further provided herein are methods, further comprising contacting the primer with a polymerase, thereby extending the primer to generate a duplexed region comprising a region of the cleaving oligonucleotide and the second docking oligonucleotide. Further provided herein are methods, wherein the duplexed region further comprises a crosslinking agent Further provided herein are methods, further comprising activating the crosslinking agent to generate a covalently bound duplex. Further provided herein are methods, wherein the activating is in the illuminating step. Further provided herein are methods, wherein the activating is in a second illuminating step.

[0020] Further provided to methods described herein are methods, further comprising controlling a diffusion factor of the sample, wherein the diffusion factor comprises a viscosity, a time, a temperature, a presence of crowding agents, a pH, an electric field, physical features, or any combination thereof. Further provided herein are methods, wherein the viscosity is greater than 1 cP. Further provided herein are methods, wherein the viscosity is from about 1 to about 10 cP, from about 10 to about 20 cP, from about 20 to about 30 cP, from about 30 to about 40 cP, from about 40 to about 50 cP, from about 50 to about 60 cP, from about 60 to about 70 cP, from about 70 to about 80 cP, from about 80 to about 90 cP, from about 100 to about 150 cP, from about 150 to about 300 cP, from about 300 to about 500 cP, from about 500 to about 1000 cP, from about 1000 to about 2000 cP, from about 2000 to about 3000 cP.

[0021] Further provided to methods described herein are methods, wherein the cleaving oligonucleotide further comprises one or more unique molecular modifiers (UMI). Further provided herein are methods, wherein the UMI comprises one or more noncanonical bases, universal bases, other bases which a polymerase will pair randomly or incorrectly, or any combination thereof.

[0022] Further provided to methods described herein are methods, wherein the endonuclease is a Type I, Type II, Type IIS, Type IIG, Type III, Type IV, or Type V endonuclease, or a Nickase. Further provided herein are methods, wherein the endonuclease comprises BtsI, Nb.BtsI, BbvCI, EcoRI, EcoRII, BamHI, Hindlll, TaqI, Notl, HinFI, Sau3AI, PvuII, Smal, Haelll, Hgal, Alul, EcoRV. EcoP151. Kpnl , Pstl, Sacl, Sall. Seal , Spel, Sphl, Stul. Xbal, Alwl, Nt.Alwl, Nb.BbvCl, Nt.BbvCI, BsmI, Nb.BsmI, BsmAI, Nt.BsmAI, BssSI, Nb.BssSI, Nt.BstNBI, BtsI, Nb.BtsI, or any combination thereof.

[0023] Further provided to methods described herein are methods, wherein the endonuclease is a Cas enzyme. Further provided herein are methods wherein the Cas enzy me comprises a Type I, Type II, Type III, or Type IV Cas endonuclease. Further provided herein are methods wherein the Cas enzyme is a Cas9 enzyme. Further provided herein are methods further providing a guide RNA specific for a sequence on the docking oligonucleotide or the cleaving oligonucleotide.

[0024] Further provided to methods described herein are methods, wherein the contacting the biological sample with an endonuclease step is before the illuminating step.

[0025] Further provided to methods described herein are methods wherein the contacting the biological sample with at least one cleaving oligonucleotide comprises contacting the biological sample with more than one cleaving oligonucleotide, wherein each cleaving oligonucleotide comprises a region complementary' to a different region on the docking oligonucleotide.

[0026] Further provided to methods described herein are methods wherein the photocrosslinking agent comprises a photoreactive nucleobase. Further provided herein are methods, wherein the photoreactive nucleobase comprises 3-cyanovinylcarbazole phosphoramidite (CNVK) or 5- pheny 1 ethyny 1-2 '-deoxy uri dine (PhedU) .

[0027] Further provided to methods described herein are methods, wherein the polymerizing comprises adding a DNA polymerase or an RNA polymerase to the sample. Further provided herein are methods, wherein the DNA polymerase comprises a Phi29 DNA Pol, an E. coli Pol I, an E. coli Pol II, an E. coli Pol III, an E. coli Pol IV, an E. coli Pol V, a T4 DNA Pol, a Bsm DNA Pol I, a Bst DNA Pol, a Reverse Transcriptase, or any combination thereof. Further provided herein are methods, wherein the RNA polymerase comprises T7, T3, SP6, or any combination thereof.

[0028] Further provided to methods described herein are methods, further comprising analyzing a frequency of association of barcode sequence and cleaving nucleic acid to generate spatial mapping of the biological sample.

[0029] Provided herein are compositions, the compositions comprising: a) a concatemer, wherein the concatemer is a nucleic acid and comprises two or more copies of a first docking oligonucleotide comprising: a barcode sequence; and a flanking region, b) at least one cleaving oligonucleotide, wherein the cleaving oligonucleotide comprises a region complementary to a region on the first docking oligonucleotide, wherein the first docking oligonucleotide and the cleaving oligonucleotide comprise at least one of each independently: a photocrosslinking agent, and at least one restriction site; and c) an endonuclease, wherein the endonuclease recognizes the at least one restriction site. The composition of claim 109. wherein the docking oligonucleotide and the cleaving oligonucleotide each independently7comprise ribonucleotides, deoxyribonucleotides, or any variation or combination thereof. Further provided herein are compositions, wherein the concatemer comprises at least 10, at least 50, at least 100, at least 200,at least 300, at least 400. at least 500, at least 600, at least 700. at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000 copies of the first docking oligonucleotide. Further provided herein are compositions, wherein the barcode sequence is from about 3 to about 30 nucleotides. Further provided herein are compositions, wherein the first docking oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, or more than 1000 nucleotides. Further provided herein are compositions, wherein the cleaving oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, from about 1000 to about 1500, from about 1500 to about 2000, from about 2000 to about 2500, from about 2500 to about 3000, from about 3500 to about 4000, or more than 4000 nucleotides. Further provided herein are compositions, wherein the photocros slinking agent is located from about 2 to about 12 bases from the restriction site. Further provided herein are compositions, wherein the photocrosslinking agent is located about 4 bases from the restriction site. Further provided herein are compositions, wherein the restriction site is from about 5 to about 20 bases from the 5’ end of the cleaving oligonucleotide. Further provided herein are compositions, wherein the restriction site is about 12 bases from the 5’ end of the cleaving oligonucleotide. Further provided herein are compositions, wherein the cleaving oligonucleotide further comprises a modification. Further provided herein are compositions, wherein the modification comprises a photolinker, a fluorophore, a photo-cleavable spacer, a protein, a small molecule, a lipid, a nanoparticle, a lipid nanoparticle, or any combination thereof. Further provided herein are compositions, wherein the modification comprises a modification to affect tethering, diffusion, or localization of an oligonucleotide. Further provided herein are compositions, wherein the modification to affect tethering of an oligonucleotide comprises a base modified with an amino group, acrylic phosphorami di te, an alkyne, an azido, chloroacetamide, vinylsulfonamide, an aliphatic aldehyde, or any combination thereof. Further provided herein are compositions, wherein the modification to affect diffusion comprise conjugation with additional nucleic acid strands, peptide, dextran, a polyethylene glycol (PEG), a short strand polymer, or any combination thereof. Further provided herein are compositions, wherein the short strand polymer comprises a poly(glycerol) (PG), poly(oxazoline) (POX), poly(hydroxypropyl methacrylate) (PGPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2- hydroxy propyl) methacrylamide) (HPMA). poly(vinylpyrrohdone) (PVP). poly(N.N-dimethyl acrylamide) (PDMA), poly(N- acryloylmorpholine) (PAcM), or any combination thereof. Further provided herein are compositions, wherein the modification to affect localization comprises a hybridization sequence directed to endogenous RNA, DNA, or cDNA. Further provided herein are compositions, whereinthe modification to affect localization comprises a conjugation to a biotin, a protein, an antibody or fragment thereof a nanobody, a lipid, a nanoparticle, a lipid nanoparticle, other affinity reagent, or any combination thereof.

[0030] Further to compositions described herein are compositions, wherein the cleaving oligonucleotide further comprises a primer at the 3’ end of the cleaving oligonucleotide. Further provided herein are compositions, wherein the primer further comprises a photocrosslinking agent.

[0031] Further to compositions described herein are compositions, wherein the cleaving oligonucleotide further comprises one or more unique molecular modifiers (UMI). Further provided herein are compositions, wherein the UMI comprises one or more noncanonical bases, universal bases, other bases which a polymerase will pair randomly or incorrectly, or any combination thereof.

[0032] Further to compositions described herein are compositions, wherein the endonuclease is a Type I, Type II, Type IIS, Type IIG, Type III, or Type IV endonuclease, Type V, or a Nickase. Further provided herein are compositions, wherein the endonuclease comprises BtsI, Nb.BtsI, BbvCI, EcoRI, EcoRII, BamHI, HmdIII, TaqI, Notl, HinFI, Sau3AI, PvuII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I, Kpnl , PstI, Sad, Sall, Seal , Spel, SphI, Stul, Xbal, Alwl, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, BsmI, Nb.BsmI, BsmAI, Nt.BsmAI, BssSI, Nb.BssSI, Nt.BstNBI, BtsI, Nb.BtsI, or any combination thereof.

[0033] Further to compositions described herein are compositions, wherein the endonuclease is a Cas enzyme. Further provided herein are compositions, wherein the Cas enzyme comprises a Type I, Type II, Type III, or Type IV Cas endonuclease. Further provided herein are compositions, wherein the Cas enzyme is a Cas9 enzyme. Further provided herein are compositions, further providing a guide RNA specific for a sequence on the docking oligonucleotide or the cleaving oligonucleotide.

[0034] Further to compositions described herein are compositions, wherein the photocrosslinking agent comprises a photoreactive nucleobase. Further provided herein are compositions, wherein the photoreactive nucleobase comprises 3-cyanovinylcarbazole phosphoramidite (CNVK.) or 5- phenylethynyl-2'-deoxyuridine (PhedU).

[0035] Further to compositions described herein are compositions, further comprising a DNA polymerase or an RNA polymerase. Further provided herein are compositions, wherein the DNA polymerase comprises a Phi29 DNA Pol, an E. coli Pol I, an E. coli Pol II, an E. coli Pol III, an E. coli Pol IV, an E. coli Pol V. a T4 DNA Pol. a Bsm DNA Pol I, a Bst DNA Pol, a Reverse Transcriptase, or any combination thereof. Further provided herein are compositions, wherein the RNA polymerase comprises T7, T3, SP6, or any combination thereof.

[0036] Provided herein are kits comprising a composition as described herein.

[0037] Provided herein are libraries of nucleic acids comprising: a population of docking oligonucleotides, wherein the population of docking oligonucleotides comprises: a barcode sequence; and a flanking region; and a population of cleaving oligonucleotides, wherein the population of cleaving oligonucleotides comprises at least one region complementary' to a region on a docking oligonucleotide in the population of docking oligonucleotides, wherein the population of docking oligonucleotides and the population of cleaving oligonucleotides comprise at least one independently: a photocrosslinking agent, and at least one restriction site. Further provided herein are libraries of nucleic acids, wherein the population of cleaving oligonucleotides further comprises a plurality of regions complementary to more than one docking oligonucleotide. Further provided herein are libraries of nucleic acids, wherein the population of cleaving oligonucleotides further comprises a 3’ primer. Further provided herein are libraries of nucleic acids, further comprising a population of concatemers, wherein each concatemer comprises at least two copies of a docking oligonucleotide of the population of docking oligonucleotides.

[0038] Provided herein are libraries of nucleic acids, wherein the library of nucleic acids comprises duplexed fragments as described herein.

[0039] Provided herein are libraries of nucleic acids, wherein the library' of nucleic acids comprises contiguous nucleic acids as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The novel features of the invention are set forth w ith particularity7in the appended claims. A better understanding of the features and advantages of the present invention w ill be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0041] FIGURE 1 illustrates the basic concept of photocrosslinked oligo-mediated cleavage (POMC), comprising photocrosslinking cleaving and docking strands to form a double-stranded region followed by enzymatic treatment to cleave oligonucleotides at a restriction site.

[0042] FIGURE 2A is an illustration of cells (top panel) contacted with non-photocrosslinking concatemers and released by cleaving with endonuclease, showing duplex products released across the entire area of sample (bottom panel).

[0043] FIGURE 2B is an illustration of cells (top panel) contacted with concatemers comprising a photocrosslinking agent. Following activation of photocrosslinking agent in ROIs and removal of oligos not covalently bound, duplexes are only released from ROIs (bottom panel).

[0044] FIGURE 3 illustrates exemplary' docking oligonucleotides comprising a unique modifier domain either 3’ or 5’ of the barcode sequence.

[0045] FIGURE 4 illustrates reverse transcription of cDNA from endogenous mRNA in a sample by extension of a cleaving oligonucleotide from a polyT primer.

[0046] FIGURES 5A-5C illustrate exemplary7variations in positioning of the cleaving oligo relative to the barcode, and different restriction sites resulting in release: FIGURE 5A illustrates a matrix with a single double-stranded restriction site between the crosslinking site and a barcode on the docking strand concatemer and the resulting duplexed fragment comprising the docking oligonucleotide barcode and the cleaving oligonucleotide primer; FIGURE 5B illustrates a matrix with a single single-stranded restriction site between the crosslinking site and a barcode on the docking strand concatemer and the resulting duplexed fragment comprising the docking oligonucleotide barcode and the cleaving oligonucleotide barcode; FIGURE 5C illustrates a matrix with a single stranded restriction site 3’ of the barcode on the docking oligonucleotide and a double-stranded restriction site 5’ of the barcode on the docking oligonucleotide.

[0047] FIGURES 6A-6E illustrate steps in crosslinking cleaving oligonucleotides to a concatemer comprising docking oligonucleotides (FIGS. 6A-6B). cleaving the duplex with an endonuclease (FIG. 6C), generating cDNA from an endogenous mRNA by priming with the released duplex (FIG. 6D), and polymerizing the complex to a continuous nucleic acid by crossjunction synthesis (FIG. 6E).

[0048] FIGURES 7A-7D illustrate steps in barcode association by a bridging oligonucleotide and cross-junction synthesis wherein a cleaving oligonucleotide crosslinks with a first docking oligonucleotide (FIG. 7A), a primer on the cleaving oligonucleotide hybridizes and extends on a second docking oligonucleotide (FIG. 7B), restriction endonucleases cleave the duplexed oligonucleotides (FIG. 7C), and the barcodes are copied to a single nucleic acid by cross-junction synthesis (FIG. 7D).

[0049] FIGURES 8A-8G illustrate a workflow incorporating POMC for spatial inference: FIG. 8A illustrates priming cDNA generation with a cleaving oligonucleotide comprising CNVK; FIG. 8B illustrates generating barcoded docking oligonucleotide concatemers using RCA; FIG. 8C illustrates crosslinking cDNA / cleaving oligonucleotide to a first concatemer by activation of CNVK; FIG. 8D illustrates a bridge cleaving oligonucleotide comprising CNVK; FIG. 8E illustrates activating CNVK in the bridge strands to crosslink to the first concatemers; FIG. 8F illustrates extending the bridge strand 3‘ ends to hybridize with the second concatemers and digesting duplexes at restriction sites; FIG. 8G illustrates polymerization by cross-junction synthesis of cDNA conjugates and extended bridge strand conjugates to generate amphfiable single strand nucleic acids.

[0050] FIGURE 9 illustrates the cross-junction synthesis, generating a single strand of nucleic acid across hybridized strands.

[0051] FIGURE 10 illustrates a workflow for cross-junction synthesis across a concatemer of barcodes to generate a continuous nucleic acid.

[0052] FIGURE 11 describes exemplary workflows for biomolecular capture.

[0053] FIGURE 12 describes exemplary' workflows for proximity recording.

[0054] FIGURE 13 describes exemplary workflows for integrated biomolecule capture and proximity recording.

[0055] FIGURE 14A is a schematic illustrating the assessment of release of docking and cleaving oligonucleotide duplexes using cleaving oligonucleotides with different restriction site locations; a loss of signal indicates digestion and release of the duplex-imager probe complex.

[0056] FIGURE 14B is an image of a gel showing that all cleaving oligos tested mediate cleaving of a BC-dock strand when conjugated and cleaved in an in vitro reaction.

[0057] FIGURES 15A-15H are microscope images of cells treated with fluorescent-tagged docking strands hybridized to cleave oligo strands after reverse transcription using cleave oligos as primers without (FIGS. 15A-15D) and with (FIGS. 15E-15F) treatment with restriction endonuclease; cells contacted with Cleave Oligo 2 showed a loss of fluorescence following treatment with restriction enzy me, indicating digestion and release of duplexed oligonucleotides. Scale bars, 20 pm.

[0058] FIGURE 16 is an image of a gel showing ssDNA with increased migration and decreased intensity of DNA intercalating dye binding in samples treated with exonuclease.

[0059] FIGURE 17 is an image of a gel showing higher molecular weight in a CNVK cleaving bridge strand and docking strand sample exposed to UV illumination compared to sample not exposed to UV light, indicating retained ability to crosslink in duplexed oligonucleotides.

[0060] FIGURE 18A is a microscope image capture showing fluorescent imager probe detection of RCA products throughout a sample of cells containing barcode and docking sequences.

[0061] FIGURE 18B is a microscope image capture showing fluorescent imager probe binding in the cells after lightly cleaving RCA product and washing, removing background noise outside of cells and blurring concatemer strand visualization over the cell area. Scale bars, 15pm.

[0062] FIGURES 19A-19I are images and graphs illustrating cleaving oligo bridge strands are able to record proximity' between concatemers through polymerization followed by digestion, elution, and cross-junction synthesis. FIGs. 19A-19C are microscope images of cells showing BC- dock concatemer 1 (FIG. 19A) and concatemer 2 (FIG. 19B) and signal retention in UV- illuminated region (in white box) of a probe detecting the bridge strand, indicating crosslinking of cleaving bridge strands hybridized to concatemers. FIGs. 19D-G are microscope images of cells show ing reduced release of bridge strands without extension (FIGs. 19D-19E) than after extension to a second concatemer (FIGs. 19F-19G). FIG. 19H is a gel image showing the positions of PCRproducts amplified with primers that bind to sequences from Type 1 and Type 2 concatemers. FIG. 191 is a bar plot showing the qPCR Ct values of bridge complexes amplified by Type 1 and Type 2-sequence PCR primers in 3’ extension conditions as compared to no polymerase control conditions. Bridge strand length before extension was 438 bases. Scale bars, 20 pm.DETAILED DESCRIPTION

[0063] Photocrosslinked-oligo-mediated cleavage (POMC) represents a novel molecular mechanism for spatially controlled release of photo-crosslinked nucleic acid complexes. The complexes produced by POMC are capable of encoding both proximity and region of interest (ROI)-scale spatial information. In some embodiments described herein, complex components are flexibly functionalized with different molecular modifications. In some embodiments, components are utilized for different purposes including acting as primers, binders, or templates for further molecular reactions.

[0064] Provided herein are methods and compositions using photo-crosslinked oligo-mediated cleavage (POMC) in recording spatial proximity between biomolecules using DNA sequences. Such processes provide spatial measurement technologies that improve upon current limitations around resolution and tissue depth. Through molecular proximity recording followed by spatial inference on the resulting associations matrix, it is possible to map cellular and tissue spatial locations of biomolecules at high resolution without requiring spatial positions to be indexed. Provided here are systems where points in tissue space are represented by spatial barcode concatemers. In some embodiments, barcoded strands are released to label biomolecules or to conjugate, or handshake, to neighboring spatial barcodes. In some embodiments, release is effected by hybridizing a “cleaving oligo’7to the single stranded concatemer to generate a recognition sequence for an endonuclease. Endonuclease is added to the hybridized nucleic acids, strands are cleaved, liberating barcoded strands. In some embodiments, the digested fragments have extendable ends. Processes described herein are applicable to analysis of cellular states in many preparations such as tissue sample, synthetic tissue, isolated or cultured cells, or in solution. Further described herein are applications of such compositions and methods for information data storage purposes. In describing methods and compositions for POMC, provided are design and generation of docking oligonucleotide concatemers; design of cleaving oligonucleotides; methods for duplexing and processing oligonucleotides; and workflows for applying the described compositions and methods.Definitions

[0065] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” include theplural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "contains." “containing,’’ “including,” “includes,” “having,” “has,” "with." or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0066] The term “about” or “approximately” describes an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value, such as ±10% of the value modified by the term “about”.

[0067] The term “affinity binding” describes binding of an affinity molecule to a target. In some embodiments, an affinity molecule comprises a component of a ligand / receptor complex. In some embodiments, an affinity molecule comprises an antibody or an antibody component. In some embodiments, the antibody component is an antibody fragment, a nanobody, or other affinity reagent. In some embodiments, an antibody fragment, as described herein, is a F(ab’)2 fragment, an Fab’ fragment, an Fab fragment, an Fv fragment, an IgG fragment, an Fc fragment, or any combination thereof. In some embodiments, the antibody is an IgA, IgG, or IgM antibody or a functional fragment thereof. In some embodiments, an affinity molecule comprises an aptamer or a modified nucleic acid.

[0068] The term “homology,” as used herein, means calculations of “homology” or “percent homology" between two or more nucleotide or amino acid sequences that are determined by aligning the sequences for optimal comparison purposes e.g., gaps introduced in the sequence of a first sequence). The nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100). For example, a position in the first sequence may be occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent homology between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. In some embodiments, the length of a sequence aligned for comparison purposes is at least about: 30%, 40%, 50%. 60%, 65%, 70%, 75%. 80%. 85%. 90%. 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100%. of the length of the reference sequence. A BLAST® search may determine homology between two sequences. The homology can be between the entire lengths of two sequences or between fractions of the entire lengths of two sequences. The two sequences can be genes, nucleotides sequences, proteinsequences, peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90- 5873-5877 (1993). Such an algorithm may be incorporated into the NBLAST and XBLAST programs (version 2.0). as described in Altschul, S. et al.. Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, any relevant parameters of the respective programs (e.g., NBLAST) can be used. For example, parameters for sequence comparison can be set at score= 100, w ord length= 12, or can be varied (e.g. . W=5 or W=20). Other examples include the algorithm of Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA. In another embodiment, the percent identity between two amino acid sequences is accomplished using, for example, the GAP program in the GCG software package (Accelrys, Cambridge, UK).PHOTOCROSSLINKED OL1GO-MEDIATED CLEAVAGE (POMC)

[0069] In some embodiments, photocrosslinked oligo-mediated cleavage (POMC) involves photocrosslinking a “cleaving strand" to a “docking strand,'’ such that the photo-crosslinked strands form a double-stranded region. In some embodiments, the double-stranded region is cleaved enzymatically. In some embodiments, enzymatic cleavage is performed by restriction endonucleases. In some embodiments, the double-stranded region provides a recognition sequence for a restriction endonuclease. FIG. 1 illustrates how7enzymatic cleavage only affects duplexed oligonucleotides after light-directed crosslinking. A docking strand and cleaving strand are combined, allowing for hybridization of complementary bases. When light is applied, as shown in the upper line of the split, the photocrosslinker is activated, generating a crosslinked duplex of the cleaving and docking strands. Without light, as shown in the lower line in the illustration, the strands remain hybridized, a much w eaker association. The crosslinked oligonucleotides are still bound after w ashing. The weaker hybridized strands, as shown in the lower line, are disassociated with washing of the sample. In some embodiments, washing is done concurrently with, or subsequent to, the photo-crosslinking step. In some embodiments, washing is done concurrently with, or preceding, enzymatic cleavage. In some embodiments, w ashing is done with a stringent wash buffer. In some embodiments, w ashing is done with a less stringent buffer. The duplexed region in the crosslinked strands provides a recognition sequence for a endonuclease, as noted by the “restriction site” in the illustration. Application of the appropriate endonuclease cleaves the strands at the restriction site, yielding a photo-crosslinked nucleic acid complex and smaller fragments. On the other hand, disassociated strands do not provide a recognition sequence to allow7for cleavage of the oligonucleotides. In some embodiments, POMC is applied to non-concatemericdocking strands or concatemers that have been immobilized inside of fixed biological samples. Mapping samples using POMC allows for location mapping in intact sample tissues. Photomasking during POMC allows for mapping or sequencing only cells or regions of interest.

[0070] POMC has many advantages over other methods of spatial analysis. FIG. 2A illustrates binding and cleavage of associated products without performing targeted crosslinking and removal of non-crosslinked duplexes. Duplex fragments are released throughout the sample. Alternatively, as shown in FIG. 2B, light-directed crosslinking in regions of interest (ROIs), followed by removal of non-crosslinked duplexes, results in cleavage and generation of duplex fragments only in specified ROIs. As described in these figures, in some embodiments, one advantage is that because the cleavage is light-directed, the regions in which cleavage occurs are spatially controlled through the focused (spatial) delivery of light to ROIs. Light based techniques provide for precise control over which cells or regions are profiled in the case of spatial sequencing. In some embodiments, light-directed spatial barcoding using cleaving oligos functionalized with a photo-crosslinker is used to deliver or encode ROI-level spatial barcode information. In some embodiments, concatemeric barcodes are used to deliver or encode local proximity information.

[0071] A second advantage to POMC is that cleaving strands, in some embodiments, are readily functionalized with different modifications. Exemplary modifications for incorporation include, without limitation photo-crosslinkers, fluorophores. biotin, and photo-cleavable spacers. In some embodiments, a commercially ordered standard oligo is subjected to such modifications. This provides flexibility for what function the product photo-crosslinked nucleic acid complexes sen e as in downstream applications.

[0072] Also described herein are methods whereby light-directed oligo conjugation facilitates the transfer of spatial barcodes from concatemers to biomolecules for the purpose of proximity recording and spatial reconstruction. In some embodiments, concatemers provide a means to deliver spatial barcodes. However, conventional concatemers have limitations around engineering additional functionality such as random reverse transcription (RT) priming, unique molecular identification, diffusion control, and proximity recording to other concatemers. As a solution, POMC creates a way to release the barcodes from concatemers in stable association with other ty pes of strands that are more flexibly engineered and functionalized. Provided here are processes to allow' for strand functionalization and diffusion control in diffusionomics designs, non-diffusive designs, and spatial-proximity data collection in a focused and efficient way combined with coarse spatial indexing with light.Docking Oligonucleotides

[0073] Docking strands or docking oligonucleotides described herein are a template for generating concatemers as provided in methods and compositions described herein. In some embodiments, a docking oligonucleotide comprises ribonucleotides, deoxyribonucleotides, and / or any variation or combination thereof. In some embodiments, the ribonucleotides and deoxyribonucleotides described herein are non-naturally occurring ribonucleotides and deoxyribonucleotides. In some embodiments, the docking oligonucleotides comprises modified nucleobases. In some embodiments, docking oligonucleotides comprise at least one flanking region and at least one barcode sequence. A flanking region and barcode sequence are not exclusive; in some embodiments regions overlap, one encompasses the other, or they are sequentially distinct. In some embodiments, docking oligonucleotides comprise more than one flanking region, more than one barcode sequence, or both. In some embodiments, a docking oligonucleotide further comprises a restriction enzy me recognition sequence.

[0074] In some embodiments, a docking oligonucleotide described herein comprises from 1 to 100 flanking regions. In some embodiments, a docking oligonucleotide comprises 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 flanking regions. In some embodiments, the flanking regions comprise PCR primers, strand barcodes, affinity7sequences, hybridization sequences complementary' to downstream components, or other sequences to enable immobilization. In some embodiments, the affinity sequences comprise additional barcodes, aptamers, DNAzymes, RNAzymes, ISH probes, primers, or any combination thereof. In some embodiments, the flanking regions comprise binding sites for fluorophore-labeled oligos.

[0075] In some embodiments, the flanking regions comprise from 1 to about 500 nucleotides. In some embodiments, the flanking regions are about 1 to about 50 nucleotides, about 50 to 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300 nucleotides, about 300 to about 350 nucleotides, about 350 to about 400 nucleotides, about 400 to about 450 nucleotides, about 450 to about 500 nucleotides. In some embodiments, the flanking regions are about 10 to about 40 nucleotides. In some embodiments, the flanking regions comprise up to about 40, 50, 100, 200, 250, 300, 400. or 500 nucleotides.Barcodes

[0076] Provided here are barcodes for incorporation in nucleic acids described herein. In some embodiments, the barcodes provide an information tag in a sample. In some embodiments, a barcode is a short section of DNA or RNA wherein individual sequences are used to identify or differentiate samples, cells, ROIs, cell types, targets, target types, nodes, node ty pes, proximal molecules, spatial information, node information, or combinations thereof. Barcodes, in someinstances, are incorporated into a longer length of nucleic acid for further processing. In some embodiments, further processing of the nucleic acid comprises extraction, amplification, sequencing, analysis, or any combination thereof.

[0077] In some embodiments, a docking oligonucleotide comprises from 1 to 100 barcode sequences. In some embodiments, a docking oligonucleotide comprises 1-10, 10-20, 20-30, 30- 40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 barcode sequences.

[0078] In some embodiments, barcode sequences comprise canonical DNA bases, RNA bases, or any combination thereof. In some embodiments, bases are edited or modified in situ after production, using enzy matic editing or any other means. In some embodiments, bases further incorporate modified nucleotides such as dUTP, fluorophore-labeled dNTPs, fluorophore-labeled NTPs, 2’-O-Me NTPs, non-canonical bases, or otherwise modified dNTPs or NTPs.

[0079] In some embodiments, the barcode sequences comprise a series of random bases. In some embodiments, the barcode sequence comprises NNNNN, NNNNNNNNNNN, NNNNNNNNNNNNNNN, DDDDDD. DDDDDDDD, HHHHH, or HHHHHHHHH, wherein ■‘N’’ represents bases A, C, G, or T / U; “D” represents bases A, G, or T / U; and ‘"H” represents bases A, C, or T / U, or any combination of random and non-random bases. In some embodiments, barcode sequences are from 1 to 100 nucleotides in length or longer. In some embodiments, the barcode sequence is from about 1 to about 10 nucleotides, from about 10 to about 20 nucleotides, from about 20 to about 30 nucleotides, from about 30 to about 40 nucleotides, from about 40 to about 50 nucleotides, from about 20 to about 50 nucleotides, from about 50 to about 100 nucleotides, or more than 100 nucleotides in length. In some embodiments, the barcode sequence is about 3 to about 30 nucleotides in length. In some embodiments, the barcode sequence is 5 to 10 nucleotides in length. In some embodiments, the barcode sequence is 4. 5, 6. 7, 8, 9, or 10 nucleotides in length. In some embodiments, individual unique barcode domains are assigned a bit value of ‘0’ or ‘1’. In some embodiments a concatenated string of nucleic acid barcodes is equivalent to a string of 0’s and l ’s. In some embodiments, the barcode sequence comprises a three-letter code.

[0080] Nucleic acid barcodes optionally comprise additional detectable labels such as fluorophores, luminescent and bioluminescent markers (e.g, biotin, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, and aequorin), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g, galactosidases, glucorinidases, phosphatases (e.g, alkaline phosphatase), peroxidases (e.g, horseradish peroxidase), and cholinesterases), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g, polystyrene, polypropylene, and latex) beads. In some embodiments, barcodes described herein are modified to comprise the addition of a small molecule, antibody, enzyme (e g., Cas9), or metal isotope. In some embodiments, barcodedsamples are visualized. In some embodiments, visualization is by colorimetric, fluorescent, ultraviolet, or other means.Restriction site

[0081] In some embodiments, the docking template encodes a restriction site for a restriction enzyme or endonuclease. In some embodiments, the enzyme is a double-stranded endonuclease, wherein an endonuclease makes a double stranded cut at a restriction site. In some embodiments, the enzy me is a single-stranded endonuclease, wherein the endonuclease makes a single-stranded cut. In some embodiments, the single-stranded endonuclease, or "‘nickase,” makes a singlestranded cut in a double stranded region of a nucleic acid. In some embodiments the singlestranded endonuclease makes a single-stranded cut in a single-stranded region of a nucleic acid. In some embodiments, oligonucleotides comprising a complementary7sequence to the region encoding the recognition sequence are deposited on the concatemer and allowed to hybridize to the nucleic acid. In some embodiments, the endonuclease is then added to the concatemer to make a double-stranded cut at the restriction site. In some embodiments, an endonuclease is added concurrently with a polymerase. In some embodiments, amplification occurs at the same time as digestion. In some embodiments, the concatemer forms a secondary7hairpin structure comprising the region of the recognition sequence, thereby providing a double-stranded region for the endonuclease cleavage. In some embodiments, a double-strand specific endonuclease makes a single-stranded cut in a double stranded sequence. In some embodiments, a double-stranded restriction enzyme cuts across a single base pair, generating blunt ends. In some embodiments, a double-stranded restriction makes a staggered cut across different base pairs generating sticky ends. In some embodiments the endonuclease in naturally occurring modified. In some embodiments, the endonuclease is a DNAzyme, RNAzyme, or CRISPR endonuclease. In some embodiments, the restriction endonuclease is a Type I, Type II, Type IIS, Type IIG, Type III, Type IV, Type V endonuclease, or a Nickase. Exemplary7endonucleases and corresponding cleavage sites for incorporation are listed in Table 1. In some embodiments, the endonuclease comprises BtsI, Nb.BtsI, BbvCI, EcoRI, EcoRII, BamHI, HindTII, TaqI, Notl, HinFI, Sau3AI, PvuII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I, Kpnl , PstI, Sad, Sall, Seal , Spel, SphI, Stul, Xbal, Alwl, Nt. Alwl, Nb.BbvCI, Nt.BbvCI, BsmI, Nb.BsmI, BsmAI, Nt.BsmAI, BssSI, Nb.BssSI, Nt.BstNBI, BtsI, Nb.BtsI, or any combination thereof.Table 1. Restriction endonucleases with recognition sequences.A: cleavage site* : blunt endsN = C or G or T or AW = A or T

[0082] Type V enzymes utilize guide RNAs to target specific sequences. Type V enzy mes include Cas enzy mes. Within Cas enzymes, there are Type I, Type II, Type III, or Type IV enzy mes. Exemplary Cas enzymes include Cas9, Cas 12a, and Cas 13. In some embodiments, the endonuclease is a Type V enzyme. In some embodiments, the endonuclease is a CRISPR enzyme. In some embodiments, the endonuclease is a Cas enzyme. In some embodiments, the Case enzyme comprises Cas9, Cas 12a, Cas 13, or variants thereof.

[0083] In some embodiments, the enzyme is a single-stranded endonuclease. In some embodiments, the endonuclease makes a cut to a single-stranded nucleic acid. In some embodiments, the cleaving strand comprises a single-stranded restriction site. In some embodiments, the docking strand comprises a single-stranded restriction site.

[0084] In some embodiments, the enz me is a nicking endonuclease. In some embodiments, the endonuclease makes a cut to a single strand of a double-stranded nucleic acid. In some embodiments, the nickase makes a cut to the cleaving strand in a duplex. In some embodiments, the nickase makes a cut to the docking strand in a duplex.Photocaging

[0085] In some embodiments, docking oligonucleotides comprise one or more photolabile protecting groups. Such provision for light activation, or “photocaging,” provides for controlled initiation of concatemer production. In some embodiments, synthesis is time-delimited. In some embodiments, photocaging allows for spatial control of concatemer generation. In some embodiments, photocaging is controlled with targeted light microscopy. Without limitation, photolabile protecting groups include (2-nitrophenyl)ethyl groups, 2-nitrobenzyl-based groups, carbonyl-based groups, benzyl-based groups, coumarin-based groups, borondipyrromethene (BODIPY)-based groups, cyanine-based groups, ortho-nitrobenzyl (NB)-based groups, or any variation or combination thereof.ed

[0086] In some embodiments, a primer or promoter sequence comprises at least one photocaging protecting group. With light activation, the primer or promoter binds the template strand, allowingreplication to proceed. In some embodiments, the primer or promoter protected with photolabile protecting groups is added to the sample. In some embodiments, primer or promoter protected with photolabile protecting groups is integrated in the template strand. Concurrently or subsequent to addition of the primer or promoter, the sample is exposed to light, releasing the photolabile protecting groups, allowing hybridization of the primer or promoter to the complementary sequence to initial replication.Concatemers

[0087] Provided herein are compositions, methods and systems incorporating concatemers. Generally, concatemers are nucleic acids comprising repetitive sequences. In some embodiments, the concatemer is generated by isothermal amplification. In some embodiments, isothermal amplification is performed at ambient temperature and / or room temperature. In some embodiments, ambient temperature and / or room temperature is about 20 to 25 °C. In some embodiments, a concatemer is generated in situ. In some embodiments, a concatemer is generated outside of the sample in a test tube (in vitro). Exemplary generation methods comprise rolling circle amplification (RCA), strand-displacement amplification (SDA), Nicking Enzyme Amplification Reaction (NEAR), exponential amplification reaction (EXPAR), enzymatic oligonucleotide synthesis via internal inosine modification and exonuclease V, nick translation, Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Multiple Displacement Amplification (MDA), Ligase Chain Reaction (LCR), Recombinase Polymerase Amplification (RPA), Ramification amplification method (RAM), Nucleic Acid Sequence-Based Amplification (NASBA), signal amplification by exchange reaction (SABER), primer exchange reaction (PER), or any variation or combination thereof. In some embodiments, concatemers are generated by rolling circle amplification (RCA). In some embodiments, concatemers are generated from untethered pre-circularized template, hybridized pre-circularized template, splint-ligation of a padlock on specific targets, or gap-filling of a padlock.

[0088] In some embodiments, methods of generation of concatemers comprise use of primers that bind to template docking strands and are extended by a one or more polymerases to create copies of the template strand. In some embodiments, a primer is a short nucleotide sequence for amplification of target DNA. In some embodiments, a primer serves as a starting sequence for new strand synthesis. In some embodiments, generation is enabled by additional enzymes such as recombinases, helicases, single-stranded binding proteins, or nickases.

[0089] In some embodiments, generation of docking oligonucleotides is modulated over time using one or more methods, alone or in any combination, of time tracking or gating of docking oligonucleotide production. In some embodiments, NTPs and / or dNTPs that degrade are used inlieu of or in combination with standard NTPs / dNTPs. Incorporation of hot start NTPs and / or dNTPs allows for modulation or monitoring through temperature control. Addition of base editing enzymes or mutagens provides for mutation of barcode sequences and unique modifiers over time.

[0090] In some embodiments, sample or substrate temperature is modulated during production of the docking oligonucleotides. In some embodiments, the temperature is held constant, or it is cycled, e.g., between 12°C and 42°C, between 4°C and 95°C, or any temperature in between. Temperature modulation is by any means, such as PCR machines, flat top incubators, water baths, Piezo devices, heating chambers around a microscope, flowing of heated buffer across the sample or substrate, or any other means or combination thereof.

[0091] In some embodiments, template strands additionally comprise one or more unique modifiers. In some embodiments, a unique modifier comprises one or more universal bases, non- canonical bases, or other bases or modifications that a polymerase can pair with either random or incorrect bases to result in a random sequence that is specific or semi-specific to the particular docking oligonucleotide. In some embodiments, unique modifier domains comprise a mix of random sequences as described and normal nucleotides. In some embodiments, a docking oligonucleotide sequence or a cleaving oligonucleotide sequence comprises one or more unique modifiers. In some embodiments, concatemers produced from a docking oligonucleotide template comprise repeats of the same barcode sequence. In some embodiments, concatemers produced from a docking oligonucleotide template comprise different unique modifier sequences. FIG. 3 shows exemplary embodiments, wherein the unique modifier region is 3’ of the barcode sequence or 5’ of the barcode sequence. Flanking regions are indicated by L* and R*. In some embodiments, unique modifier information is used downstream, after next generation sequencing, to deduplicate reads and obtain more quantitative information about the number of proximity recording events. In some embodiments, an oligonucleotide described herein comprises a 5’ promoter or primer sequence.Rolling Circle Amplification

[0092] In some embodiments, concatemers are generated by Rolling Circle Amplification (RCA). RCA is a method of generating multiple copies of a nucleic acid in a continuous strand from a circular template. RCA amplifies a template nucleic acid into a long and repeated single-stranded oligonucleotide. In general, RCA comprises formation of a circular template from a length of nucleic acid. In some embodiments, a template nucleic acid, or padlock probe, is annealed with a ligation template, or nucleic acid splint. In some embodiments, the splint and the template nucleic acid comprise DNA or RNA. In some embodiments, the template nucleic acid is circularized using a ligase. In some embodiments, the ligase comprises T4 ligase, CircLigase®, Ampligase®,SplintR® Ligase, or any combination thereof. In some embodiments, a primer sequence, a polymerase, and deoxynucleotide triphosphates (dNTPs) are incubated with the template nucleic acid to generate a concatemer of single stranded DNA comprising repeat copies complementary to the circular template. In some embodiments, the polymerase comprises Phi29, Bst, Vent exo- DNA polymerase, or T7 RNA polymerase.

[0093] In some embodiments, the nucleic acid comprising the splint sequence is a region of a nucleic acid in a sample. In some embodiments, the splint sequence is a region of genomic DNA, mRNA, cDNA, or other nucleic acid in a sample. In some embodiments, the splint sequence is on mRNA encoding a gene. In some embodiments, the splint sequence is a regulatory RNA. In some embodiments, the splint sequence is circular RNA (circRNA), ribosomal RNA (rRNA), 16S ribosomal RNA (16S rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), a non-coding RNA, long non-coding RNA (IncRNA), or microRNA (miRNA).

[0094] In some embodiments, hybridizing to a nucleic acid in a sample allows for concatemer generation at a fixed location. In some embodiments, the padlock probe hybridizes to a splint sequence at a fixed location in a sample. In some embodiments, the splint sequence is located on a surface, in a cell, in a nucleus, or in a tissue. In some embodiments, the splint sequence acts as an anchor or tether for the RCA machinery. In some embodiments, the padlock probe hybridizes to a splint sequence on a nucleic acid encoding a gene in a sample. In some embodiments, RCA generates at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000. at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000 copies of the docking oligonucleotide.

[0095] In some embodiments, a ligation template or splint is conjugated to a surface, a protein, a small molecule, a nucleic acid, a lipid or any combination thereof. In some embodiments, a surface comprises glass, a bead, a microwell plate, a scaffold, or another substrate. In some embodiments, the protein comprises a receptor, a ligand, an antibody, or any functional fragment thereof.

[0096] In some embodiments, docking oligonucleotides are deposited onto a sample with a concentration from about 20 nM to about 500 nM. In some embodiments, docking oligonucleotides provided herein are deposited onto a sample with a concentration up to about 1 nM, 10 nM, 20 nM, 100 nM. 250 nM, 500 nM, 1000 nm, or more.

[0097] In some embodiments, oligonucleotides provided herein are non-specifically or specifically conjugated to another molecule in a sample. In some embodiments, an oligonucleotide is conjugated to a protein, an antibody, an antibody fragment, a nanobody, a small molecule, a nucleic acid therapeutic, an epitope, a lipid, nanoparticle, lipid nanoparticle, a liposome, a droplet,a bead, another nucleic acid LNP, AAV, exosome. a lentivirus, or an aptamer. In some embodiments, an antibody fragment, as described herein, is a F(ab’)2 fragment, a Fab’ fragment, an Fab fragment, an Fv fragment, an IgG fragment, an Fc fragment, or any combination thereof. In some embodiments, the lipid is an emulsion, a liposome, a particle, or any combination thereof. In some embodiments, the antibody is an IgA. IgG, or IgM antibody or a functional fragment thereof. In some embodiments, the tissue samples or population of cells provided herein are imaged after concatemer crosslinking.Cleaving oligonucleotides

[0098] Provided here are cleaving strands or cleaving oligonucleotides capable of hybridizing to a restriction site on a single-stranded nucleic acid, creating a double-stranded region around the restriction site. In some embodiments, functionality an endonuclease requires a double stranded recognition sequence. In some embodiments, functionality of an endonuclease requires a single stranded recognition sequence. In some embodiments, short cleaving oligonucleotides are crosslinked to concatemers to generate hybrid duplexes that cany' a barcode. In some embodiments, a cleaving oligonucleotide is attached to a single-stranded nucleic acid using light- directed photocrosslinking. In some embodiments, generation of a stable, covalently linked, structure with the in situ generated concatemers allows for use of additional functionalization available for synthesized oligonucleotides.

[0099] In some embodiments, the cleaving oligonucleotides comprise features for generating duplexes with at least one concatemer as described herein. In some embodiments, a cleaving oligonucleotide comprises at least one hybridization domain, wherein the hybridization domain is complementary to a region on a concatemer comprising docking oligonucleotides, and a restriction site. In some embodiments, a cleaving oligonucleotide further comprises a photocrosslinking agent as described herein.

[0100] In some embodiments, the length of a cleaving oligonucleotide imparts a functional characteristic upon the nucleic acid. Factor for functional manipulation include diffusion rate, diffusion of cleavage events and sequencing efficiency. In some embodiments, the cleaving oligonucleotide is from about 150 to about 200 nucleotides in length. In some embodiments, the cleaving oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 150, from about 150 to about 200, from about 200 to about 250, from about 250 to about 300, from about 300 to about 350, from about 350 to about 400, from about 400 to about 450, from about 450 to about 500, from about 500 to about 550, from about 550 to about 600, from about 600 to about 700, from about 700 to about 800, from about 800 to about 900, from about 900 to about 1000, from about 100 to about 1500, from or about 1500 to about 2000, from about 2000 toabout 2500, from about 2500 to about 3000, from about 3000 to about 3500. or from about 3500 to about 4000 nucleotides in length.

[0101] In some embodiments, cleaving oligonucleotides are deposited onto a sample with a concentration from about 20 nM to about 500 nM. In some embodiments, cleaving oligonucleotides provided herein are deposited onto a sample with a concentration up to about 1 nM, 10 nM, 20 nM, 100 nM, 250 nM, 500 nM, 1000 nm, or more.

[0102] In some embodiments, cleaving oligonucleotides comprise a crosslinking agent. Exemplary crosslinking agents include, aiw l azides, azido-methyl-coumarins, benzophenones, anthraquinones, certain diazo compounds, diazirines, psoralen derivatives, or any combination thereof. In some embodiments, the crosslinking agent is a photoreactive nucleobase. In some embodiments, the photo-reactive nucleobase comprises a vinyl, acrylate, N-hydroxy succinimide, amine, carboxylate or thiol chemical group. In some embodiments, the photo-reactive nucleobase comprises a bromo-deoxyuridine. In some embodiments, the photoreactive nucleobase is 3- cyanovinylcarbazole nucleoside (CNVK). In some embodiments, the photoreactive nucleobase is 5-phenylethynyl-2'-deoxyuridine (PdU).

[0103] In some embodiments, a plurality7of photo-crosslinker molecules are incorporated, such that different cleaving oligonucleotides comprise different photo-crosslinker molecules capable of crosslinking at different wavelengths. Such variance in the barcodes allows for varying crosslinking events at one or more regions of interest. In some embodiments, a reverse crosslinking reaction is performed. For example, irradiation at a particular wavelength for a sufficient period provides for crosslink reversal. In some embodiments, a series of crosslink-then-decrosslink reactions is performed. In some embodiments, molecules are deposited on a substrate provided herein to locally generate a light wavelength to support the light sensitive crosslinking reaction.Cleaving oligo modifications

[0104] In some embodiments, cleaving oligonucleotides comprise additional domains or modifications. In some embodiments, additional domains or modifications facilitate immobilization, detection, or visualization of an oligonucleotide duplex. In some embodiments, additional domains or modifications are used for spatial identification. In some embodiments, modifications comprise features that provide for tethering of an oligonucleotide, features to control diffusion, or features that direct localization. In some embodiments, cleaving oligonucleotides further comprise one or more of a unique modifier, polymerization stops to preserve singlestranded ends, a primer, biotin, an antibody, a photolinker, a fluorophore, a photo-cleavable spacer, a protein, a small molecule, a lipid, a nanoparticle, a lipid nanoparticle, a dye, an aptamer, a probe, a barcode, or any combination thereof.

[0105] Provided herein are methods and compositions for tethering of a nucleic acid or oligonucleotide. In some embodiments, a nucleic acid described herein comprises modification that prevent diffusion. In some embodiments, the nucleic acid is a docking oligonucleotide, a cleaving oligonucleotide, a cDNA generated as described herein, or another nucleic acid as described herein. In some embodiments, docking oligonucleotides described herein are tethered. In some embodiments, cleaving oligonucleotides described herein are tethered. In some embodiments, a cDNA as described herein is tethered. Exemplary tethering features include, without limitation, modified bases with amino groups (e.g. aminoallyl), acrylic phosphorami dite (e.g. Acrydite), alkynes, azidos, and other modifications such as chloroacetamide, vinylsulfonamide, or aliphatic aldehydes. In some embodiments, modifications provide for chemical crosslinking to fixed biomolecules or to gels polymerized in the cell or tissue. In some embodiments, the chemical crosslinking is to carbohydrates, lipid, nucleic acids, or proteins.

[0106] In some embodiments, features to control diffusion comprise modifications that constrain the diffusion of the cleaving oligonucleotide or the docking oligonucleotide. In some embodiments, features to control diffusion comprise complexes that include the oligonucleotide. In some embodiments, described features control diffusion before or after cleaving. In some embodiments, features comprise modifications to the sequence and base composition of the oligonucleotide to add bulk, secondary structure, or modify binding properties. In some embodiments, features comprise binding of additional strands to the cleaving oligonucleotide or the docking oligonucleotide. In some embodiments, features comprise conjugating peptides, dextran, PEG others short strand polymers such as poly(glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2 -hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl) methacrylamide) (HPMA), poly(vinylpyrrohdone) (PVP), poly(N.N- dimethyl acrylamide) (PDMA), and poly(N-acryloylmorpholine) (PacM). In some embodiments, a composition comprises individual features described herein. In some embodiments, a composition comprises a combination of described features.

[0107] Provided herein are compositions and methods providing features that direct localization of oligonucleotides. In some embodiments, features that direct localization comprise modifications to the oligonucleotide sequence. In some embodiments, features that direct localization comprise modification to the base composition to hybridize to other molecules. In some embodiments, the base composition hybridizes to endogenous RNA, DNA, or cDNA sequences, or to proteins. In some embodiments, hybridization is direct or via intermediate strands. In some embodiments, oligonucleotides described herein are conjugated to affinity binding molecules, such as ligands, receptors, aptamers, antibodies, nanobodies, or fragments, thereof that bind to target biomolecules.

[0108] A cleaving oligonucleotide is optionally designed to stably bind or transiently bind to a complementary strand. In some embodiments, a cleaving oligonucleotide is covalently or non- covalently bound to a complementary strand. In some embodiments, the hybridization region on a cleaving oligonucleotide is at least 20%. at least 30%. at least 40%. at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% complementary to the hybridization domain on a docking oligonucleotide. In some embodiments, the complementary region is a contiguous sequence. In some embodiments, the complementary region on one or both strands is not contiguous. In some embodiments, a cleaving oligonucleotide comprises hybridization regions complementary to more than one docking oligonucleotide.

[0109] In some embodiments, cleaving oligonucleotides further compnse a unique modifier. In some embodiments, a unique modifier comprises universal bases or other base modifications to create heterogeneity in the cleaving oligonucleotide. In some embodiments, a unique modifier comprises universal bases or other base modifications to create heterogeneity in the docking oligonucleotide. In some embodiments, each oligonucleotide comprises a unique modifier. In some embodiments, unique modifiers allow for more quantitative measurements. In some embodiments, unique modifiers further comprise targetable sequences for base editing. In some embodiments, oligonucleotides are edited using CRISPR techniques. In some embodiments, targetable sequences are modulated over time.

[0110] Provided herein are compositions and methods comprising primers. In some embodiments, a primer comprises a short nucleic acid sequence used to initiate nucleic acid synthesis. In some embodiments, the primer is DNA. In some embodiments, the primer is RNA. In some embodiments, the primer has a region complementary to a strand of DNA or RNA. In some embodiments, the primer provides a free 3 ’-OH at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain. In some embodiments, the primer comprises from about 5 to about 50 or more nucleotides. In some embodiments, the primer comprises about 10 to about 30 nucleotides. In some embodiments, a primer as described herein comprises the 3‘ sequence NNNNNNN (7N’s), NNNNNGGG (5N’s and 3G’s), NNNNNCCC (5N’s and 3C’s), NNNNNAAA (5N’s and 3A’s), or NNNNNTTT (5N’s and 3T’s). In some embodiments, a primer described herein comprises a photocrosslinking agent. In some embodiments, a primer comprises a CNVK group or a PdU group.[OHl] In some embodiments, oligonucleotides provided herein are non-specifically or specifically conjugated to another molecule in a sample. In some embodiments, an oligonucleotide is conjugated to a protein, an antibody, an antibody fragment, a nanobody, a small molecule, a nucleic acid therapeutic, an epitope, a lipid, nanoparticle, lipid nanoparticle, a liposome, a droplet, a bead, another nucleic acid LNP, AAV, exosome, a lentivirus, or an aptamer. In someembodiments, an antibody fragment, as described herein, is a F(ab’)2 fragment, an Fab’ fragment, an Fab fragment, an Fv fragment, an IgG fragment, an Fc fragment, or any combination thereof. In some embodiments, the lipid is an emulsion, a liposome, a particle, or any combination thereof. In some embodiments, the antibody is an IgA, IgG, or IgM antibody or a functional fragment thereof. In some embodiments, the tissue samples or population of cells provided herein are imaged after concatemer crosslinking.

[0112] In some embodiments, cleaving oligonucleotides are modified before, during, or after hybridization. In some embodiments, cleaving oligonucleotides are modified to incorporate one or more additional functionalities to the oligonucleotide.Cleaving oligo-cDNA generation

[0113] In some embodiments, the cleaving oligonucleotide functions as a primer on an RNA in the sample. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the target nucleic acid is a regulatory RNA. In some embodiments, the RNA is circular RNA (circRNA), ribosomal RNA (rRNA), 16S ribosomal RNA (16S rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), a non-coding RNA, long noncoding RNA (IncRNA), or microRNA (miRNA). For the purposes of this description, reference is to mRNA, however it is understood any other type of RNA is encompassed by this description. Transcription of the RNA generates a cDNA extension on the cleaving oligonucleotide. FIG. 4 shows an exemplary embodiment using a cleaving oligonucleotide as a primer for reverse transcription of a target mRNA. In the top view of FIG. 4, a cleaving oligonucleotide comprising a PCR primer and a 3’ polyT domain hybridizes to a polyA tail on an mRNA. Reverse transcription from the polyT domain generates a cDNA strand extension of the cleaving oligonucleotide, as shown in the middle view- of FIG. 4. In some embodiments, primer sequences are random or specific. In some embodiments, the primer sequence comprises TTTTTTTTTT (SEQ ID NO: 4),TTTTTTTTTTTTTTTTTTTTgnQID NQ TTTTTTTTTTTTTTTTTTTTVN NQ6), NNNNNNN, NNNNNNNN, NNNNNGGG, NNNNNCCC, NNNNNTTT, or any combination thereof. In some embodiments, the priming sequence is specific to an RNA of interest. In some embodiments, the primer sequence is a CRISPR guide RNA sequence, an mRNA therapeutic sequence, a T or B cell receptor sequence, or any combination thereof. In some embodiments, second strand synthesis is applied to the reverse transcription product. In some embodiments, template switching oligos (TSO) are used in the second strand synthesis. In some embodiments, second strand synthesis generates a strand comprising PCR primers flanking one or more barcode and cDNA sequences.

[0114] In some embodiments a 3’ overhang is added to the cDNA transcripts in a tailing step. In some embodiments, tailing is achieved through the use of a terminal transferase enzyme and dXTP (dATP, dCTP, dGTP, dTTP, or any combination thereof). Adding ddXTP (ddATP, ddCTP, ddGTP, ddTTP, or any combination thereof) or another terminating nucleotide at the 3‘ end prevents subsequent extension during the later cross-junction synthesis step. In some embodiments, ddXTP or another terminating nucleotide is included at a low concentration to randomly terminate the 3’ end. In some embodiments, other strategies are used to add a 3’ overhang. In some embodiments, an overhang is added by ligation.Cleaving oligo design

[0115] Provided herein are compositions and methods incorporating cleaving oligonucleotides comprising particular designs. In some embodiments, design of cleaving oligonucleotides comprises relative location of oligonucleotide features. In some embodiments, relative location of one or more photocrosslinking agents, enzyme restriction sites and proximity to the 5‘ or 3‘ end of the oligonucleotide. In some embodiments, the photocrosslinking agent is located from about 2 to about 12 bases from the restriction site. In some embodiments, the photocrosslinking agent is located from about 2 to about 5, from about 5 to about 8, from about 8 to about 12 bases from the restriction site. In some embodiments, the photocrosslinking agent is located about 4 bases from the restriction site. In some embodiments, the restriction site is located from about 5 to about 20 bases from the 5’ end of the cleaving oligonucleotide. In some embodiments, the restriction site is located from about 5 to about 10, from about 10 to about 15, from about 15 to about 20 bases from the 5’ end of the cleaving oligonucleotide. In some embodiments, the restriction site is located about 12 bases from the 5’ end of the cleaving oligonucleotide.

[0116] Provided herein are compositions and methods incorporating cleaving and docking oligonucleotides comprising one or more restriction sites located relative to a barcode sequence. FIG. 5A shows an exemplary duplexed concatemer, wherein a double-stranded restriction site is located 5?to a crosslinking site on the cleaving oligonucleotide and 3‘ to the barcode sequence on the concatemer. Resulting duplex fragments comprise the crosslinked cleaving oligonucleotide fragment comprising a 3’ primer and the docking oligonucleotide fragment comprising the barcode sequence 5’ of the crosslinking site. FIG. 5B shows an exemplary duplexed concatemer, wherein a single-stranded restriction site is located 5' to a crosslinking site and 3’ to a barcode sequence. Resulting duplex fragments comprise the crosslinked cleaving oligonucleotide fragment comprising a 5 ’ primer and the docking oligonucleotide fragment comprising the barcode sequence 5 ’ of the crosslinking site. FIG. 5C shows an exemplary duplexed concatemer, wherein a doublestranded restriction site is located 3’ to a crosslinking site and 5‘ to a barcode sequence and asingle-stranded restriction site is located 3‘ to the double-stranded restriction site and 5’ to a barcode sequence, wherein a barcode region is located within a docking site, resulting in some mismatches with randomization of the barcode sequence. In some embodiments, a similar duplexed concatemer only comprises a double-stranded restriction site.Hybridization and CrosslinkingHybridization

[0117] Some methods described herein comprise hybridization of nucleic acids or probes to oligonucleotides. In some embodiments, hybridization comprises in situ hybridization (ISH). ISH generally involves depositing nucleic acid probes on to a sample. In some embodiments, a nucleic acid probe recognizes a region or regions of a target nucleic acid. In some embodiments, the ISH nucleic acid probes recognize RNA. In the ISH method comprises smISH. In some embodiments, smlSH comprises deposing many small nucleic acids that each recognize an RNA. In some embodiments, where binding of a single probe results in a weak signal. In further embodiments, the signal from the ensemble of deposited probes is robust. In some embodiments, probes comprise a feature to generate a signal. In some embodiments, a detectable label is linked to probes described herein. In some embodiments, the probe comprises a radio-label, a hapten label, a fluorescent label, an antigen label, or any combination thereof. In some embodiments, an ISH. or modified ISH, reaction is applied to a sample described herein prior to or following an amplification reaction. In further embodiments, ISH probes target barcodes included in described concatemers.

[0118] In some embodiments, hybridization comprises fluorescent in situ hybridization (FISH). FISH generally involves depositing nucleic acid probes comprising a label. In some embodiments, the label is a fluorescent label. In some embodiments, a fluorescent nucleic acid probe recognizes a region or regions of genome. In some embodiments, the fluorescent nucleic acid probes recognize RNA. In the case of smFISH, many small nucleic acids together recognize an RNA, where binding of a single fluorescent probe results in weak signal, but the signal from the ensemble of all the probes is robust. In some embodiments, a FISH, or modified FISH, reaction is applied to a sample described herein prior to or following an amplification reaction. In further embodiments, FISH probes target barcodes included in described concatemers.Crosslinking

[0119] Light-directed crosslinking is, in some embodiments, selectively applied to a sample of cells. Crosslinking is selectively performed by shielding areas from radiation, allowing crosslinking only in unshielded areas. A photomask provides a template to shield areas of a sample that receive radiated light. In some embodiments, a photomask is generated to allow radiation of one or more ROIs. In some embodiments, the photomask is manually or machine generated. Insome embodiments, the photomask is generated according to sample imaging as previously described herein. In some embodiments a photomask is applied to a sample prior to radiation.

[0120] In some embodiments, in addition or in lieu of a photomask, as described herein, light is focused on one or more ROIs. In some embodiments, light is focused using a digital micromirror device (DMD). In some embodiments, light is focused using a confocal microscope. In some embodiments, the confocal microscope is a laser scanning confocal microscope. In some embodiments, light is focused using a DVD writer. In some embodiments, light is focused using a Blu-ray disc writer. In some embodiments, a light source is an independent entity from another device, such as a microscope. In some embodiments, the light source is a laser. In some embodiments, the laser has a primary wavelength from about 180 nm to about 1 mm. In some embodiments, the laser has a primary wavelength from about 180 nm to about 400 nm. In some embodiments, the laser has a primary wavelength from about 400 nm to about 700 nm. In some embodiments, the laser has a primary' wavelength from about 700 nm to about 1 mm.

[0121] In some embodiments, light is focused on a surface of the tissue samples or population of cells provided herein. In some embodiments, light is focused on a plane comprising at least one portion of the tissue samples or population of cells provided herein. In some embodiments, light is focused on one or more planar surfaces on or within the tissue samples or population of cells provided herein. In some embodiments, the light-directed crosslinking described herein is applied to a surface of the tissue samples or population of cells provided herein. In some embodiments, the light-directed crosslinking described herein is applied across multiple planes within the tissue samples or population of cells provided herein. In some embodiments, the light-directed crosslinking described herein is applied on multiple planes of a 3-dimensional tissue samples or population of cells provided herein.

[0122] In some embodiments, methods described herein comprise use of an optical system to provide for selective z-plane illumination. In some embodiments, the optical system provides light in a z-plane having from about 2 nm to about 10 pm resolution, from about 5 nm to about 9 pm resolution, from about 10 nm to about 8 pm resolution, from about 15 nm to about 7 pm resolution, from about 20 nm to about 6 pm resolution, from about 25 nm to about 5 pm resolution, from about 30 nm to about 4 pm resolution, from about 35 nm to about 3 pm resolution, from about 40 nm to about 2 pm resolution, from about 45 nm to about 1 pm resolution, from about 50 nm to about 800 nm resolution, from about 100 nm to about 600 nm resolution, from about 200 nm to about 400 nm resolution, or any range there between. In some embodiments, applied light in a z- plane has 10 pm or less resolution. In some embodiments, light is focused on a surface of the tissue samples or population of cells provided herein. In some embodiments, light is focused on a plane comprising at least one portion of the sample or population of cells provided herein. In someembodiments, light is focused on one or more planar surfaces on or within the sample or population of cells provided herein. In some embodiments, the light-directed crosslinking described herein is applied to a surface of a sample or population of cells provided herein. In some embodiments, the light-directed crosslinking described herein is applied across multiple planes within the tissue samples or population of cells. In some embodiments, the light-directed crosslinking described herein is applied on multiple planes of a 3-dimensional tissue samples or population of cells provided herein. Exemplary devices for illumination restricted in a z-plane comprise a 2-photon microscope and a light sheet microscope.

[0123] In some embodiments, an optical system used with methods described herein can provide for selective xy-plane illumination. In some embodiments, the optical system can provide light in a xy-plane that has from about 2 nm to about 10 gm resolution, from about 5 nm to about 9 pm resolution, from about 10 nm to about 8 pm resolution, from about 15 nm to about 7 pm resolution, from about 20 nm to about 6 pm resolution, from about 25 nm to about 5 pm resolution, from about 30 nm to about 4 pm resolution, from about 35 nm to about 3 pm resolution, from about 40 nm to about 2 pm resolution, from about 45 nm to about 1 pm resolution, from about 50 nm to about 800 nm resolution, from about 100 nm to about 600 nm resolution, from about 200 nm to about 400 nm resolution, or any range there between. In some embodiments, light in a xy-plane has 10 pm or less resolution.

[0124] In some embodiments, radiation is provided at a particular wavelength or range of wavelengths. In some embodiments, the radiation is ultraviolet (UV) radiation. In some embodiments, the radiation is provided at wavelengths in a range from about 300 nanometers (nm) to about 450 nm. In some embodiments, the radiation is provided at a wavelength of about 300 nm. about 305 nm, about 310 nm, about 315 nm, about 320 nm, about 325 nm, about 330 nm, about 335 nm, about 340 nm, about 345 nm, about 350 nm, about 355 nm, about 360 nm, about 365 nm, about 370 nm, about 375 nm, about 380 nm, about 385 nm, about 390 nm, about 395 nm, about 400 nm, about 405 nm, about 410 nm, about 415 nm, about 420 nm, about 425 nm, about 430 nm, about 435 nm, about 440 nm. about 445 nm, about 450 nm, or any combination thereof. In some embodiments, the radiation is provided at a wavelength of about 365 nm. In some embodiments, the radiation is provided at a wavelength of about 405 nm.

[0125] In some embodiments, radiation is provided for a particular amount of time. In some embodiments, the radiation is provided for about 1 second up to 10 seconds. In some embodiments, the radiation is provided for about 1 0 milliseconds (ms) up to 10 seconds. In some embodiments, the radiation is provided for at least about 10 ms or more, at least about 100 ms or more, at least about 500 ms or more, at least about 600 ms or more, at least about 700 ms or more, at least about 800 ms or more, at least about 900 ms or more, at least about 1 second or more, at least about 2seconds or more, at least about 3 seconds or more, at least about 4 seconds or more, at least about 5 seconds or more, at least about 6 seconds or more, at least about 7 seconds or more, at least about 8 seconds or more, at least about 9 seconds or more, at least about 10 seconds or more, at least about 11 seconds or more, at least about 12 seconds or more, at least about 13 seconds or more, at least about 14 seconds or more, at least about 15 seconds or more, at least about 16 seconds or more, at least about 17 seconds or more, at least about 18 seconds or more, at least about 19 seconds or more, at least about 20 seconds or more, or at least about 30 seconds or more.

[0126] The number of additional docking strands or cleavage strands added is not limited. In some embodiments, 1, 2. 3, 4, 5. 6, 7, 8. 9, 10, 11, 12, 13, 14. 15. 16. 17. 18. 19. 20, 21, 22, 23, 24, 25 or more additional strands are added. In some embodiments, 75, 100, 150, 200, 250 or more additional strands are added.

[0127] In some embodiments, cleaving strands are hybridized to docking strands by about 1 to about 10 bases, by about 10 to about 20 bases, by about 20 to about 30 bases, by about 30 to about 40 bases, by about 40 to about 50 bases, by about 50 to about 60 bases, by about 60 to about 70 bases, by about 70 to about 80 bases, by about 80 to about 90 bases, by about 90 to about 100 bases, or more than 100 bases. In some embodiments, salt concentrations, temperature, denaturant concentration, or strand concentration affects stability of strand binding. In some embodiments, stability of strand binding is adjusted such that cleaving strands bind for less than 1 second, between 1 and 10 seconds, or for less than 1 minute on average. In some embodiments, cleaving strands comprise increased homology domains to docking strands. In some embodiments, salt is increased, or temperature decreased, thereby adjusting strand binding on average for longer than one minute. In some embodiments, conditions are adjusted to adjust the average bound time of the cleaving strands to docking strands.

[0128] In some embodiments, a crosslinking reaction occurs concurrently with hybridization. In some embodiments, cycles of hybridization and crosslinking are repeated without a washing step to remove oligonucleotides that are not crosslinked.Concatemer-cleaving oligo crosslinking

[0129] Compositions and Methods described herein comprise concatemers of docking oligonucleotides comprise hybridization regions complementary' to a region on one or more cleaving oligonucleotides. In some embodiments, described concatemers are generated in a sample. In some embodiments, described concatemers are added to a sample. In some embodiments, one or more cleaving oligonucleotides are added to the sample after adding or generating the concatemers. In some embodiments, one or more cleaving oligonucleotides are added to the sample before. In some embodiments, one or more cleaving oligonucleotides areadded to the sample at the same time as adding or generating the concatemers. In an exemplary embodiment, the cleaving oligonucleotides comprise additional features, for example a crosslinking agent and a primer, as shown in FIG. 6A. The crosslinking agent is activated, covalently binding the cleaving oligonucleotide to the concatemer of docking oligonucleotides, as illustrated in FIG. 6B. The duplexed nucleic acids form a recognition sequence for an endonuclease. The duplex is contacted with the endonuclease to cleave the cross-linked oligonucleotides, generating duplexed fragments, as shown in FIG. 6C. In this embodiment, as shown in FIG. 6D, the primer on the cleaving oligonucleotide hybridized to an mRNA in the sample and contacted with a polymerase to generate a cDNA extension to the cleaving oligonucleotide. FIG. 6E illustrates polymerization of a continuous strand copy of the duplexed fragment / cDNA complex using crossjunction synthesis. The depicted method is exemplary. In some embodiments, steps are performed in an alternative order. In some embodiments, cDNA generation proceeds prior to light activation of the cross-linking agent or prior to endonuclease cleavage.

[0130] In some embodiments, the cleaving oligonucleotides are synthesized separately from the crosslinking environment. In some embodiments, the cleaving oligonucleotides are synthesized with randomers to enable unique molecular IDs and randomer priming. In some embodiments, cleaving oligonucleotides are modified with strand designs or chemical groups, allowing for improved diffusion control.Bridge crosslinking

[0131] In some embodiments, cleaving oligonucleotides hybridize with more than one concatemer comprising docking oligonucleotides, providing a source of relative spatial information, or handshake, between two points. Bridge strands are cleaving oligonucleotides of controlled length which crosslink to more than one type of concatemer. In some embodiments, without releasing from the first concatemer, the bridge strand hybridizes to, or copies, a barcode on a second concatemer. In some embodiments, the bridge strand comprises a light-activated crosslinking agent and crosslinks to a second concatemer. Endonuclease cleavage releases the complex comprising barcodes from two concatemers. In some embodiments, associated barcodes are resolved to a single strand by cross-junction synthesis for further analysis, including amplification and sequencing. In some embodiments, such proximity' recording without diffusion confers increased resolution in spatial mapping. An exemplary embodiment is illustrated in FIGs. 7A-7D. In this embodiment, a cleaving oligonucleotide hybridizes and is photocrosslinked to one of two ty pes of concatemers, as shown in FIG. 7A. The cleaving oligonucleotide comprises a primer that hybridizes and extends on the second concatemer to copy its barcode (See, FIG. 7B). In some embodiments, the second concatemer or the cleaving oligonucleotide carries a light-activatedcrosslinking agent. In some embodiments, the bridged cleaving oligonucleotide and second concatemer are crosslinked in ROIs by illumination of the ROIs. After extension, the bridged portion of the cleaving oligonucleotide and corresponding duplexed docking oligonucleotides (See, FIG. 7C) are released by restriction enzyme digestion. FIG. 7D illustrates cross-junction synthesis of a single strand nucleic acid associating the two barcodes.Combined crosslinking

[0132] In some embodiments, methods described herein add additional functionality to reverse transcription and handshake proximity recording. Compositions and methods described herein provide for opportunities for differentiated designs.

[0133] In some embodiments, the order of operations are varied by initiating the process with reverse transcription absent concatemers. FIG. 8A illustrates reverse transcription from a primer on a cleaving oligonucleotide with a photocrosslinking group, in this case CNVK. In some embodiments, the cDNA / cleaving oligonucleotide complex is fixed in place. In some embodiments, the cDNA / mRNA hybridization is maintained. In some embodiments, the cDNA is fixed. In some embodiments, template RNA is removed. In some embodiments, the tethered cDNA prevents diffusion and provides for improved spatial resolution. POMC allows the resulting scaffold to be disassembled and eluted, yielding crosslinked products that are again resolvable by cross-junction synthesis. FIG. 8C illustrates addition of multiple types of concatemers comprising barcoded docking oligonucleotides. In some embodiments, concatemers are generated in situ. In some embodiments, concatemers are generated ex situ. In some embodiments, concatemers are generated by RCA. In some embodiments, ROIs are irradiated to crosslink hybridized cDNA / cleaving oligonucleotides to a first type of concatemer (FIG. 8C). In further embodiments, bridge cleaving oligonucleotides comprising a photocrosslinking group are added to the sample (FIG. 8D). Bridge cleaving oligonucleotides are crosslinked to concatemers by irradiating (FIG. 8E). Bridge strands polymerize to copy a barcode on a second type of concatemer, as shown in FIG. 8F. After bridge strand extension, the networked oligonucleotides are cleaved using restriction enzymes. The resulting products, both cDNA / cleaving oligo complexes and bridge oligo complexes in this case, are resolved using cross-junction synthesis, as described in FIG. 8G. Data is only collected from illuminated regions of interest, as crosslinking is required for assembly of the cDNA. scaffold, and bridges as well as for digestion.Strand resolution

[0134] Crosslinked duplexes are further processed to generate a continuous strand nucleic acid.In some embodiments, the continuous nucleic acid is amplified and sequenced. In someembodiments, a continuous strand is generated from a branched or cross-linked strand by crossjunction synthesis.Cross-junction synthesis

[0135] Provided herein are methods for cross-junction synthesis. Cross-junction synthesis is performed directly in situ, or during / after displacement. The fundamental strategy for crossjunction synthesis is depicted in FIG. 9. Shown are two nucleic acids that have been hybridized together to form a junction. A primer is bound in front of that junction on the first nucleic acid (left). A strand-displacing polymerase is used to copy the x domain (also referred to as a junction domain herein) until it reaches a stopper (shown in black and also referred to as a blocking domain herein). The new and old x domains compete in a random walk branch migration process. Ultimately, the new x domain can bind to the exposed x* domain on the second template strand (right, also referred to as a synthesis region herein). Polymerization can then continue on the second template strand, copying along a new backbone.

[0136] In some embodiments, cross-junction synthesis produces a DNA, an RNA, a PNA, or an LNA. In some embodiments, the method of cross-junction synthesis comprises contacting a target nucleic acid or a barcode provided herein with a strand-displacing polymerase. In some embodiments, cross-junction synthesis is performed at room temperature (e.g., at least about 20 degrees C up to 25 degrees C). In some embodiments, cross-junction synthesis is performed at temperatures below room temperature (e.g., about 15 °C, about 10 °C, about 4 °C). In some embodiments, cross-junction synthesis is performed at temperatures above room temperature (e.g., about 30 °C, about 37 °C). In some embodiments, nucleic acid amplification or cross-junction synthesis is performed at a constant temperature. In some embodiments, nucleic acid amplification or cross-junction synthesis is performed at different or varied temperature.

[0137] Provided herein are methods, wherein a polymerase is prevented or stopped from continuing along the backbone of a template strand and / or copying the template strand by a blocking domain. In some embodiments, the blocking domain comprises one or more modified nucleotides known for increasing Tmof double-stranded nucleic acids. In some embodiments, modified nucleotides include, but are not limited to, locked nucleic acids (LNAs), 2’-0-methoxy- ethyl (2’-M0E) nucleotides, 2,6-diaminopurine, G-clamp (an analog of C having 4 hydrogen bonds) and guanidinium G-clamp nucleotides, and the like.

[0138] In some embodiments, the compositions and methods described herein are used for synthesis of arbitrary length prescribed sequences. FIG. 10 illustrates an exemplary embodiment, wherein multiple barcode strands are hybridized together and through cross-junction synthesis reactions, are replicated to form a continuous nucleic acid strand. Each junction betw een barcodestrands shows the same domain motifs, whereby the strand domains copied on the 3’ end of the growing strand before reaching a stopper (b, c, d, e) can reach across the junction and bind to the exposed complementary sequence (b*, c*, d*, e*) on the next barcode strand. Arbitrary7sequences can be added in the template regions between the motif domains (shown in gray) to enable longer sequences to be assembled.

[0139] In some embodiments, at least one of the blocking domain comprises a poly monomer stretch. For example, the blocking domain comprises a stretch of poly A, polyT, polyC, or polyG.

[0140] In some embodiments, the blocking domain comprises covalent cross-linking of two barcode strands. In some embodiments, the covalent cross-linking is a photo-crosslink or a chemical cross-link. In some embodiments, covalent cross-linking comprises cross-linking the nucleotide at the 5’-terminus of the first barcode strand to the nucleotide at 3’-end of the second template strand. In some embodiments, the cross-linking is at an oligonucleotide or a single nucleic acid backbone linkage. In some embodiments, the cross-linking is by a phosphodiester bond. In some embodiments, the cross-linking reaction is between a CNVK nucleotide and a complementary7nucleotide. In some embodiments, the crosslinked nucleotides act as a stopper to a polymerase.

[0141] In some embodiments, the blocking domain comprises any desired nucleotide sequence or number of nucleotides. Blocking domains are not restricted to a particular length. In some embodiments, each blocking domain is from one nucleotide to 100 nucleotides in length. In some embodiments, each blocking domain is independently one, two, three, four, five, six, seven, eight, nine or ten nucleotides in length. In some embodiments, a blocking domain is a single nucleotide.

[0142] In some embodiments, a polymerase described is an RNA or DNA polymerase. In some embodiments, a polymerase refers to an enzyme that performs template-directed synthesis of polynucleotides. In some embodiments, the DNA polymerase comprises Phi29, Pol I, Pol II, Pol III, Pol IV, Pol V, T4, Phusion®, Taq ligase, Klenow fragment, Bsm, Bst, reverse transcriptase, or any combination thereof. In some embodiments, the polymerase is an RNA polymerase. In some embodiments, the RNA polymerase comprises T7, T3, SP6, or any combination thereof. In some embodiments, a polymerase comprises exonuclease activity, degrading an encountered downstream strand via 5’ to 3’ exonuclease activity. In some embodiments, a DNA polymerase is isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. In some embodiments, polymerase enzymes include, but are not limited to: Klenow fragment (New England Biolabs Inc.), Taq DNA polymerase (QIAGEN). 9° N™ DNA polymerase (New England Biolabs Inc.), Deep Vent™ DNA polymerase (New England Biolabs Inc.), Manta DNA polymerase (Enzymatics), Bst DNA polymerase (New England Biolabs Inc.), or phi29 DNA polymerase (New England Biolabs Inc.). In some embodiments, a polymeraseis DNA-dependent. In some embodiments, a polymerase is RNA-dependent. In some embodiments, the polymerase is reverse transcriptase.SAMPLESSample Preparation

[0143] Provided herein are methods for preparing samples for downstream biological analysis steps. In some embodiments, samples as provided herein comprise a population of cells. In some embodiments, samples provided herein comprise a tissue sample. In some embodiments, samples provided herein comprise a laminar cell culture. Non-limiting examples of samples for use include: intact tissue, dissected tissue, dissociated cells, an organoid, engineered tissue, cultured cells, sub- cellular compositions, a suspension of cells, organs, organelles, tissue biopsies, frozen tissue, tissue section, tissue block, membrane-bound structures, cell-free systems or lysates, formalin- fixed paraffin-embedded (FFPE) samples, exosomes, nuclei, fixed nuclei, fixed exosomes, protein complexes, RNPs, or any combination thereof. In some embodiments, samples provided herein comprise a population of cultured cells. Types of cultured cells include, but are not limited to, primary cell cultures, dissociated cells, dissected cells, passaged cell lines, and / or transformed cells. In some embodiments, the population of cells provided herein comprise an engineered tissue. In some embodiments, the engineered tissue comprises a population of human in vitro- differentiated cells. In some embodiments, the human in vitro-di fferentiated cells are derived from human induced pluripotent stem cells (hiPSCs), embry onic stem cells, or adult stem cells. In some embodiments, the human in vitro-di fferentiated cells are in vitro-di fferentiated cardiomyocytes, in vitro-dd fferentiated neurons, in vitro-di fferentiated hepatocytes, in vitro-di fferentiated endothelial cells, in vitro-di fferentiated epithelial cells, in vitro-di fferentiated smooth muscle cells, in vitro- differentiated skeletal muscle cells, or in v / 7ro-differentiated kidney cells. In some embodiments, the population of cells provided herein comprise retinal cells. In some embodiments, the population of cells provided herein are obtained from a subject. In some embodiments, samples are unfixed or fixed. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a primate, a non-human primate, a human, a mouse, a rat, a goat, a rabbit, a dog, a camelid, or a cat.

[0144] In some embodiments, the population of cells comprises a homogeneous population of cells (e.g., cells of the same type or of the same species). In some embodiments, the population of cells comprise a heterogenous population of cells (<?.g., cells of different types, different species, or any combination thereof). In some embodiments, the population of cells comprises cells from one species. In some embodiments, the population of cells comprises cells from two or more species. In some embodiments, the population of cells comprises one cell type. In someembodiments, the population of cells comprises cells from two or more different cell types. In some embodiments the population of cells comprises cells from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 250, at least 500, at least 1000 or more species. In some embodiments the population of cells comprises cells from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 250, at least 500, at least 1000 or more different cell types.

[0145] In some embodiments, samples used in methods described herein are a tissue sample. In some embodiments, tissue samples provided herein comprise connective tissue, epithelial tissue, muscle tissue, or nervous tissue. In some embodiments, tissue samples provided herein comprise a combination of tissues. An organ comprises tissues of different types and has a specific function. In some embodiments, a combination of tissues is an organ. In some embodiments, a combination of tissues is an organoid. In some embodiments, the tissue sample is obtained from a subject. In some embodiments, tissues samples provided herein are obtained from a brain, an eye, a lung, a liver, a bladder, a kidney, a heart, a stomach, an intestine, a lymph node, a skeletal muscle, a smooth muscle, a pharynx, a larynx, an artery, a liver, a gallbladder, a bone, a spleen, a vein, a pancreas, a reproductive organ, a tumor, an infected tissue, or any combination thereof. In some embodiments, tissue samples provided herein are a whole organ or a biopsy tissue. In some embodiments, the biopsy tissue comprises a tissue that has or is suspected of having abnormal proliferation or growth. In some embodiments, the biopsy tissue comprises cancer cells.

[0146] In some embodiments, a population of cells described herein comprises one or more microorganisms. In some embodiments, the population of cells comprises prokaryotic or eukaryotic cells. In some embodiments, a population of cells provided herein comprise bacterial cells, fungal cells, archaeic cells, eukaryotic cells, or any combination thereof. In some embodiments, the population of cells comprises mammalian cells. In some embodiments, the mammalian cells are human, non-human primate, primate, pig, horse, sheep, cat, rat, mouse, dog, llama, rabbit, or goat cells. In some embodiments, the population of cells further comprises a virus. In some embodiments, the virus is a pathogenic virus. In some embodiments, the population of cells comprise a viral vector. In some embodiments, the population of cells have been genetically modified. Non-limiting examples of genetic modifications include base editing, gene silencing (e.g., by CRISPR / Cas9 systems), gene insertions, gene deletions.

[0147] In some embodiments, the population of cells are fixed and then frozen. In some embodiments, the population of cells are frozen, then fixed after thawing and / or sectioning. Insome embodiments, the fixed population of cells is sectioned and then fixed a second time. In some embodiments, the population of cells is stored at room temperature after fixing. In some embodiments, the population of cells is fixed with organic solvents, such as alcohols and acetones. In some embodiments, the population of cells (e.g., tissue) are embedded in paraffin prior to fixing. In some embodiments, the population of cells are fixed with cross-linking reagents. In some embodiments, the cross-linking reagent comprises formalin, formaldehyde, paraformaldehyde, dithio-bis(succinimidyl propionate) (DSP) or any combination thereof. In some embodiments, a permeabilization step is performed. In some embodiments, no permeabilization is performed. In some embodiments, permeabilization occurs concurrently with fixation. In some embodiments, permeabilization is performed following fixation. In some embodiments, a permeabilization reagent comprises acetone, alcohol, detergent, or any combination thereof.

[0148] In some embodiments, a population of cells for analysis as provided herein are live cells. In further embodiments, the live cells are contacted directly with a barcode sequence, or indirectly via a linking molecule. In some embodiments, the linking molecule binds to cell surface receptors. In some embodiments, the linking molecule binds a cell surface molecule. In some embodiments, the cell surface molecule is a protein or nucleic acid. In some embodiments, the nucleic acid is RNA or DNA. In some embodiments, the protein is a cell receptor. In some embodiments, the cell surface molecule is located at cell adhesion points. In some embodiments, barcode sequences are introduced intracellularly into live cells. In some embodiments, barcode sequences are attached to lipid nanoparticles (LNPs) as carriers for delivery into the cell. In some embodiments, AAV’s, lentiviruses, exosomes, or other encapsulations are carriers for delivery' of barcode sequences into the cell. In some embodiments, cells are transfected or transformed to receive barcode sequences. In some embodiments, cells are mechanically perturbed to enable cellular uptake of barcode sequences.

[0149] In some embodiments, a sample described herein comprises a nucleic acid. In some embodiments, a sample comprises nucleic acid bound or tethered directly or indirectly to a surface. In some embodiments, a sample comprises nucleic acid in solution. In some embodiments, a sample comprises nucleic acid in a hydrogel, expanded hydrogel, biological sample embedded in a hydrogel, biological sample embedded in an expanded hydrogel, with or without sample clearing. In some embodiments, a sample comprises nucleic acid in a scaffold. In some embodiments, the nucleic acid is attached directly or indirectly to a synthetic matrix. In some embodiments, the synthetic matrix comprises a bead or semi-solid gel.

[0150] In some embodiments, a sample described herein comprises a cell. In some embodiments, the cell is bound to a surface. In some embodiments, the cell is in solution. In some embodiments, the cell is connected to a hydrogel. In some embodiments, the cell is connected to a syntheticscaffold. In some embodiments, the cell is attached directly or indirectly to a synthetic scaffold. In some embodiments, the synthetic scaffold comprises a bead or semi-solid gel. In some embodiments, the synthetic scaffold comprises an extracellular matrix protein. In some embodiments, the extracellular matrix protein comprises collagen or fibronectin.

[0151] In some embodiments, samples provided herein are frozen at -5°C to -200°C. In some embodiments, samples provided herein are frozen at about -5°C, about -10°C, about -15°C, about -20°C, about -25°C, about -30°C, about -35°C, about -40°C, about -45°C, about -50°C, about - 55°C, about -60°C, about -65°C, about -70°C, about -75°C, about -80°C, about -85°C, about - 90°C, about -95°C, about -I00°C, or any combination thereof. In some embodiments, a tissue sample or a population of cells provided herein is flash frozen. In some embodiments, a tissue sample or a population of cells provided herein are frozen by slow, or equilibrium, freezing.

[0152] In some embodiments, fixation impacts protein biochemistry, masking an epitope. In some embodiments, a sample described herein is further treated with an antigen retrieval method. In some embodiments, the antigen retrieval method is protease-induced epitope retrieval (PIER). In some embodiments, the antigen retrieval method is heat-induced epitope retrieval (HIER). In some embodiments, the antigen retrieval method is any other method capable of reversing the masking of an epitope. In some embodiments, the antigen retrieval step is combined with a permeabilization step. In some embodiments, the antigen retrieval step is performed independently.

[0153] In some embodiments, samples provided herein are fixed on a microscope slide or coverslip. In some embodiments, the microscope slide is a chamber slide. In some embodiments, the chamber slide comprises one or more chambers. In some embodiments, the slide comprises polystyrene. In some embodiments, the slide comprises glass. In some embodiments, the coverslip comprises glass. In some embodiments, the coverslip comprises a UV transparent material. In some embodiments, the coverslip comprises quartz.

[0154] In some embodiments, the slide comprises a coating to promote adherence of a tissue sample or a population of cells provided herein to a surface of the slide. In some embodiments, the coating comprises poly-L-lysine or poly-D-lysine. In some embodiments, the coating comprises a gelatin. In some embodiments, the coating comprises a 3-aminopropyltriethoxysilane (APES). In some embodiments, samples are adhered to the slide using an adhesive tape. In some embodiments, the adhesive tape is poly vinylidene chloride, poly ester / silicone, polypropylene film, cellophane, synthetic rubber-resin hot melt, low density polyethylene film, polyester film, or any combination thereof.

[0155] In some embodiments, samples are embedded in a hydrogel. In some embodiments, the hydrogel comprises a synthetic gel or polyepoxide. In some embodiments, embedding a sample generates at tissue-hydrogel matrix. In some embodiments, embedding a sample provides forexpansion of a tissue sample or a population of cells provided herein. In some embodiments, the hydrogel is a natural hydrogel, a synthetic hydrogel, or a synthetic / natural hybrid hydrogel. In some embodiments, the natural hydrogel is a protein, a polysaccharide, a protein / polysaccharide, DNA, or any combination thereof. In some embodiments, the protein hydrogen comprises collagen, elastin, fibrin, silk, lysozyme, Matrigel® (Coming Inc., Coming. NY), genetically engineered proteins, or any combination thereof. In some embodiments, the polysaccharide comprises hyaluronic acid (HA), alginate, chitosan, dextran, or any combination thereof. In some embodiments, the protein / polysaccharide comprises collagen / HA, laminin / cellulose, fibrin / alginate, gelatin / agarose, chitosan, alginate, dextran, or any combination thereof. In some embodiments, the DNA hydrogel comprises X-DNA, Y-DNA, T-DNA, linear plasmid DNA, or any combination thereof. In some embodiments, the synthetic hydrogel is a nonbiodegradable hydrogel, a biodegradable hydrogel, a bioactive hydrogel, or any combination thereof. In some embodiments, the nonbiodegradable hydrogel comprises poly(2-hydroxyethyl methacrylate) (PHEMA), poly(2-hydroxypropyl methacrylate) (PHPMA), poly(N-isopropylacrylamide (PNIPAm), poloxamer (Pluronic®, ThermoFisher Scientific, Inc., Waltham, MA), poly(ethylene glycol) diacrylate (PEGDA), poly(vinyl alcohol (PVA), or any combination thereof. In some embodiments, the biodegradable hydrogel comprises degradable polyethylene glycol (PEG), polypropylene fumarate-PEG (PPF-PEG), poly(2-hydroxyethyl methacrylate -poly(e- caprolactone (PHEMA-PCL), synthetic peptides, or any combination thereof. In some embodiments, the bioactive hydrogel comprises a cell-adhesive hydrogel, an enzyme-sensitive hydrogel, a growth factor-bearing hydrogel, another bioactive hydrogel, or any combination thereof. In some embodiments, the synthetic / natural hybrid hydrogel comprises PEG / dextran, heparin, HA, chondroitin sulfate (CS), one or more proteins. PNIPAm / proteins. chitosan, alginate, synthetic peptides / proteins, polysaccharides, PVA / DNA, Pluronic / dextran, PHPMA / protein, or any combination thereof.

[0156] In some embodiments, a sample comprises cells. In some embodiments, a sample comprises cells from a suspension. In some embodiments, a sample comprises a whole organism. In some embodiments, the whole organism is a multicellular organism. In some embodiments, the whole organism is a single cell organism. Example multicellular organisms include, without limitation, roundworm (Caenorhabditis elegans), zebrafish (Danio rerio and a Drosophila. Example single cell organisms include, without limitation, bacteria and yeast. In some embodiments, a sample comprises tissue. In some embodiments, the sectioned tissue is sliced. In some embodiments, the slices are up to about 5 urn, about 10 um, about 15 um, about 20 um, about 25 um, about 30 um, about 35 um, about 40 um, about 45 um, about 50 um, or more in thickness.

[0157] In some embodiments, samples described herein are imaged using a microscope. In some embodiments, a microscope used in methods described herein includes a compound microscope, a fluorescent microscope, a confocal microscope, a light sheet microscope, a Raman microscope, or a digital microscope.Fixed nucleic acids

[0158] Analysis using methods described herein are applicable to an immobilized nucleic acid. In some embodiments, the immobilized nucleic acid is fixed to a substrate. In some embodiments, the fixation is via formalin / formaldehyde treatment. In some embodiments, the fixation is via ethanedial or oxalaldehyde treatment. Analysis using methods described herein are applicable to any fixed nucleic acid. In some embodiments, described methods are used to characterize nucleic acids, proteins, or any combination thereof. In some embodiments, fixed nucleic acids are immobilized through affinity binding, including hybridization, conjugation, binding, crosslinking, photo-crosslinking, priming and extension, or any combination thereof. In some embodiments, affinity binding is specific to particular subcellular, cellular, cell-type specific, or supercellular structures such as membranes, mitochondria, genomic DNA, proteins, nucleic acids, lipids, protein-protein complexes, protein-RNA complexes, or any combination thereof. Accordingly, in some embodiments, the fixed nucleic acids bind multiple targets in the sample. In some embodiments, the target or multiple targets further bind another set of multiple targets. In some examples, template strands bind to kinases, which in turn bind to ADP or ATP and a substrate. In such instances, the multiple kinases bind multiple substrates. In another example, template strands bind to ATP, which will bind to multiple kinases. In some embodiments, immobilization is randomly dispersed throughout a substrate through random binding, non-specific binding, or conjugation to a surface, defined or random incorporation into a hydrogel, or focused within a specific area. In some embodiments, focused deposition is through depositing reagents on a specific region, spatially confined crosslinking, or photo-crosslinking as described herein. In some embodiments, fixed nucleic acids are conjugated to an affinity molecule or immobilized to a scaffold, a substrate, a surface, a bead, or a column. In some embodiments, methods described herein are used to profile in situ hybridization (ISH) probes, DNA conjugated to antibodies, DNA encoded libraries, cDNA libraries, CRISPR libraries, viral libraries and / or genomes, nucleic acids covalently linked to cleared hydrogels, nucleic acid conjugated proteins, aptamers, guide RNAs, nucleic acids localized on a surface (e.g. , glass slide or micro-array), or in situ generated sequences (e.g., by in vitro transcription (IVT)). In some embodiments, methods described herein are used to profile peptide libraries, protein libraries, nanobody libraries, aptamer libraries, or antibody libraries. In some embodiments, proteins or their variants are attached directly to their respectivecoding mRNA transcripts, such as through ribosomal stalling during translation. In some embodiments, attached mRNA transcripts are treated as a nucleic acid '‘tag” in identifying the associated protein using methods as described herein. In some embodiments, samples are fixed prior to concatemer generation and / or subsequent to concatemer generation.

[0159] In some embodiments, the fixed nucleic acid is conjugated to an affinity molecule. In some embodiments, an affinity molecule comprises a component of a ligand / receptor complex. In some embodiments, an affinity molecule comprises an antibody or component thereof. In some embodiments, the antibody component is an antibody fragment, a nanobody, or other affinity reagent. In some embodiments, an antibody fragment, as described herein, is a F(ab’)2 fragment, an Fab’ fragment, an Fab fragment, an Fv fragment, an IgG fragment, an Fc fragment, or any combination thereof. In some embodiments, the antibody is an IgA, IgG, or IgM antibody or a functional fragment thereof. In some embodiments, the fixed nucleic acid is conjugated to an aptamer.

[0160] In some embodiments, the fixed nucleic acid is conjugated to a scaffold. In some embodiments, a scaffold is biodegradable. In some embodiments, a scaffold is non-biodegradable. In some embodiments, a scaffold is biocompatible. In some embodiments, a scaffold comprises synthetic materials. In some embodiments, the synthetic materials comprise silicone, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL). poly-lactic-co-glycolic acid (PLGA), or any combination thereof. In some embodiments, the scaffold comprises natural materials. In some embodiments, the natural materials comprise derivatives of the extracellular matrix, collagen, fibrin, polysaccharides including chitosan or glycosaminoglycans (GAGs), or any combination thereof. In some embodiments, glycosaminoglycans (GAGs) comprise hyaluronic acid.

[0161] In some embodiments, the fixed nucleic acid is conjugated to a surface comprising a flexible material. In some embodiments, the flexible material comprises, without limitation, modified nylon, unmodified nylon, nitrocellulose, polypropylene or any combination thereof. In some embodiments, the surface comprises a rigid material. In some embodiments, the rigid material comprises, without limitation, glass, fuse silica, silicon, silicon dioxide, silicon nitride, plastics (for example, polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, metals (for example, gold, platinum), or any combination thereof. In some embodiments, surfaces described herein are fabricated from a material comprising silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combination thereof. In some embodiments, the fixed nucleic acid is conjugated to a bead. In come embodiments, the bead is a magnetic bead. In some embodiments, the bead is an immunoaffinity bead.Sample conditions

[0162] Described herein are compositions and methods comprising modulation of a diffusion factor. In some embodiments, diffusion in a sample is modulated by adjusting the sample environment, nucleic acid strand length, or any other factor affecting diffusion, immobilization, or mobility within the sample. In some embodiments, diffusion in a solution is controlled through modulation of parameters affecting the rate that molecules move through a solution. Exemplary parameters affecting diffusion comprise viscosity, time, temperature, crowding agents, pH, electric field, physical features, polymerization of reagents in solution, fixation, concentration of reagents within solution, attachment of molecules to scaffolds endogenous to the sample, attachment of molecules to externally introduced scaffolds, or any combination thereof. In some embodiments, diffusion of nucleic acids are modulated by addition of molecular crowding agents. In some embodiments, crowding agents comprise dextran sulfate, glycerol, polymers, crystals. In some embodiments, diffusion of nucleic acids are enhanced by heating the tissue samples or population of cells provided herein. In some embodiments, diffusion is enhanced by heating to more than 30°C, more than 35°C, more than 40°C, more than 45°C, more than 50°C. . In some embodiments, diffusion is enhanced by heating to about 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C.

[0163] In some embodiments, viscosity of a sample is modulated with a hydrogel or any excipient that increases the poise measure of the environment. In some embodiments, the sample medium has a viscosity^ greater than 1 centipoise (cP). In some embodiments, the sample medium has a viscosity of from about 1 to about 10 cP, from about 10 to about 20 cP, from about 20 to about 30 cP, from about 30 to about 40 cP, from about 40 to about 50 cP, from about 50 to about 60 cP, from about 60 to about 70 cP, from about 70 to about 80 cP, from about 80 to about 90 cP, from about 100 to about 150 cP, from about 150 to about 300 cP, from about 300 to about 500 cP, from about 500 to about 1000 cP, from about 1000 to about 2000 cP, from about 2000 to about 3000 cP. Exemplary crowding agents comprise dextran sulfate, glycerol, polymers, crystals, or any combination thereof.

[0164] Sample fixation is achieved through various methods including, without limitation, heating, microwaving, cryopreservation, or chemical means. Chemical fixation generally comprises crosslinking fixatives, such as aldehydes, including formaldehyde or glutaraldehyde; precipitating fixatives, such as alcohols, including ethanol, methanol, or acetone; oxidizing agents, such as osmium tetroxide, potassium dichromate, chromic acid, and potassium permanganate; mercurials, such as B-5 and Zenker’s fixative; picrates; or hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) fixative. In some embodiments, the sample iscrystallized, for example by silver fixation. In some embodiments, a sample is attached to a sample substrate using any of the methods described herein.

[0165] Methods as described herein comprise application of reagents, including oligonucleotide strands, wash sequences, and the like, to a sample. In some embodiments, application methods comprise traditional microfluidics. passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handlers (e.g., Echo Liquid Handler”. Beckman Coulter, Indianapolis, IN) for depositing new solutions, inkjet printers with modified contents, automatic liquid handlers, optionally incorporating pipettors, robotic arms for processing slides between solution baths, or any combination thereof. In some embodiments, application methods are performed on-stage. In some embodiments, application methods are performed off-stage.

[0166] In some embodiments, samples are treated to prevent or reduce nonspecific binding of oligonucleotide strands. In some embodiments, a blocking agent is added to the tissue samples or population of cells provided herein prior to application of oligonucleotide strands. In some embodiments, a blocking agent is added to the tissue samples or population of cells provided herein concurrently to application of oligonucleotide strands. In some embodiments, the blocking agent comprises non-specific nucleic acid sequences, sheared salmon sperm DNA, tRNA, yeast tRNA, single-stranded oligonucleotides, double-stranded oligonucleotides, polysaccharides, charged polysaccharides, negatively charged polysaccharides, negatively charged molecules, charged molecules bovine serum albumin (BSA), dried milk, detergent, a nonionic polymeric surfactant (e.g., Poly (ethyleneoxide) / poly (propyleneoxide) triblock copolymers - PLURONIC™ F-127, Gibco® Pluronic® F-68), crowding agents, or any combination thereof. In some embodiments, ribosomal depletion or targeted depletion of high abundance sequences occurs prior to any of: reverse transcription, barcoding, sequence extraction, cross-junction synthesis, or PCR amplification. In some embodiments, ribosomal depletion or targeted depletion of high abundance sequences occurs after PCR amplification. In some embodiments, in situ hybridization (ISH) probes targeting high abundance sequences are included before or during reverse transcription to block specific the high abundance sequences from acting as binding sites for reverse transcription primers. In some embodiments, ISH probe binding is combined with endonuclease activity (e.g. RnaseH) to specifically digest high abundance sequences in situ prior to reverse transcription. In some embodiments, a double-strand specific endonuclease (e.g. dsDNAse) is applied after PCR to specifically de-enrich high abundance sequences.

[0167] Following irradiation and crosslinking reactions, in some embodiments, oligonucleotides that are not crosslinked are removed. In some embodiments, oligonucleotides that are not crosslinked are removed by washing. In some embodiments, oligonucleotides that are not crosslinked are not removed prior to additional cycles of crosslinking. In some embodiments, asample is washed with a chemical denaturant solution. In some embodiments, the chemical denaturant solution comprises one or more salts. In some embodiments, the salt is NaCl. In some embodiments, the salt concentration is from 0.1 M to 2 M. In some embodiments, the salt concentration is about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M. about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2.0 M. In some embodiments, the chemical denaturant is formamide. In some embodiments, the chemical denaturant is ethylene carbonate. In some embodiments, the formamide solution is 50-60% formamide. In some embodiments, the denaturant solution is in a PBS buffer. In some embodiments, the denaturant solution comprises a detergent. In some embodiments, the concentration of detergent in the denaturant solution is about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1.0%, about 2%, about 3%, about 4%, or about 5% by volume. In some embodiments, the detergent is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 (TWEEN® 20, Merck KgaA. Darmstadt, Germany) or polysorbate 80 (TWEEN® 80, Merck KgaA, Darmstadt, Germany). In some embodiments, the detergent is a nonionic surfactant. In some embodiments, the non-ionic surfactant is a non-ionic polyoxyethylene detergent. In some embodiments, the detergent is polyethylene glycol (PEG), 2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy] ethanol (CAS number: 9002-93-1), N.N dimethyltetradecylamine N-oxide (TDAO, CAS number: 3332-27-2). In some embodiments, the detergent is Triton X-100, Nereid, TDAO, or a combination thereof. In some embodiments, the washing is performed at room temperature. Hadn some embodiments, the washing is performed at a temperature warmer or cooler than room temperature. In some embodiments, the washing is performed at 4 degrees C. 16 degrees C, 25 degrees C, 37 degrees C. 40 degrees C, 45 degrees C. 50 degrees C, 55 degrees C. 60 degrees C, or higher temperature.Surface Materials

[0168] In some embodiments, surfaces of nucleic acid storage provided herein surface comprise a flexible or a rigid material. Exemplary rigids materials include, without limitation: glass; fused silica; silicon such as silicon dioxide or silicon nitride; metals such as gold or platinum; plastics such as polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and a combination thereof. In some embodiments, a rigid surface is fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass, or any combination thereof. Flexible materials are generally capable of being bent, folded or similarly manipulated without breakage. In some embodiments, a flexible material is bent at least 30 degrees around a roller. In some cases, aflexible material is bent at least 180 degrees around a roller. In some cases, a flexible surface is bent at least 270 degrees around a roller. In some instances, a flexible material is bent about 360 degrees around a roller. In some cases, the roller is less than about 10 cm, 5 cm, 3 cm, 2 cm or 1 cm in radius. Exemplary flexible materials include, without limitation: nylon (unmodified nylon, modified nylon, clear nylon), nitrocellulose, polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene, acetal, acrylic, acrylonitrile, butadiene styrene (ABS), polyester films such as polyethylene terephthalate, polymethyl methacrylate or other acrylics, polyvinyl chloride or other vinyl resin, transparent PVC foil, transparent foil for printers, Poly (methyl methacrylate) (PMMA), methacrylate copolymers, styrenic polymers, high refractive index polymers, fluorine- containing polymers, polyethersulfone, polyimides containing an alicyclic structure, rubber, fabric, metal foils, and any combination thereof. In some embodiments, nylon and PMAA surfaces are provided as a sheet or alternatively provided as a layer that is coated over another material. In some embodiments, the coating is over silicon. In some embodiments, plasticizers or modifiers are used with polymeric substrate materials to achieve selected flexibility characteristics. In some instances, the flexible material is a hydrogel such as agarose or polyacrylamide.

[0169] In some embodiments, nucleic acid strands are covalently or non-covalently attached to the surface. In some embodiments, a surface is first coated with biotin, then streptavidin. In further embodiments, biotinylated nucleic acids are attached to the coated surface. In some embodiments, a surface is coated with copolymers of N, N-dimethylacrylamide. In some embodiments, nucleic acid are baked onto a glass surface using desiccation and UV light. In some embodiments, nucleic acid strands contain an acrydite modification and are covalently linked to a hydrogel. In some embodiments, nucleic acid strands are attached to beads that are immobilized on the surface. In further embodiments immobilized strands are UV-cleavable from the beads. In some embodiments, beads are hydrogel beads, metallic beads, streptavidin beads, magnetic beads, magnetic streptavidin beads, gold nanoparticles, metallic nanoparticles, or any combination thereof. In some embodiments, nucleic acid strands are encapsulated by a bead, LNP, AAV, virus, exosome in the biological sample. In some embodiments, the biological sample is a fixed tissue or cells. In some embodiments, the biological sample is live cells.

[0170] In some embodiments, the surface is silanized. In further embodiments nucleic acid strands comprise bases modified by amination or other reactive groups, and are covalently bound to the silanized surface. In some embodiments, attachment to the silanized surface is direct by reaction with an epoxy group. In some embodiments, attachment to the silanized surface is indirect, using crosslinkers such as 1,4-phenylenediisothiocyanate (PDC). In some embodiments, nucleic acid strands are covalently bound to the surface using click chemistry'. In some embodiments nucleic acid strands are modified with azide or alkyne reactive groups, which react with alkyne or azidegroups, respectively, on the surface. In some embodiments, the reactions are in the presence of copper.Visualization

[0171] Provided herein are methods for image capture and enhancement thereof which incorporate application of imaging reagents. Tn some embodiments, imaging reagents are used for distinguishing cells, cell types, and / or subcellular structures. In some embodiments, a tissue sample or a population of cells provided herein is contacted with imaging reagents. In some embodiments the imaging reagent is a dye or stain. In some embodiments, the dye or stain comprises 7-AAD, acridine orange, Bismarck brown, calcein, CFSE, carmine, Coomassie blue, cresyl violet, crystal violet, DAPI, eosin, ethidium bromide, acid fuchsin, haematoxylin, Hoechst stains, iodine, malachite green, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide (formal name: osmium tetraoxide), propidium iodide, resazurin. rhodamine, safranin, Trypan Blue, tetrazolium salts, or any combination thereof.

[0172] In some embodiments, oligonucleotides, concatemers, templates, generated copies, or any combination thereof visualized by incorporating one or more dyes or stains. In some embodiments, the dye or stain comprises fluorophore-labelled NTPs, fluorescent-labelled probes, dyes or stains as described elsewhere herein. In some embodiments, a dye or stain intercalates into nucleic acid. In some embodiments, a modified nucleotide, dye or stain is added during synthesis of the node barcode strands. In some embodiments, an oligonucleotide is hybridized to a complementary fluorophore-labelled oligonucleotide. In some embodiments, the modified nucleotide is selected from a fluorophore-labeled, DIG-labeled, or biotin-labeled dNTP / NTP. In some embodiments, the modified nucleotide is selected from an EdU, BrdU, or PdU.

[0173] In some embodiments, samples are visualized with immunofluorescent (IF) staining. In some embodiments, the IF staining reagent comprises a fluorophore conjugated streptavidin. In some embodiments, the imaging reagent is a fluorophore. Exemplary' fluorophores for inclusion in workflows described herein include, but are not limited to, 1.5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5- Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5- Carboxyfluorescein): 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 6- Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7- Amino-4-methylcoumarin; 7 -Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2- methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen S1TSA; Aequorin (Photoprotein); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; AlexaFluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA- S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO- TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y 66H); BG-647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™ -3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650- X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy FI; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1 ; BO-PRO™ -3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green- 1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green- 5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X- rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CFDA; CFP - Cyan Fluorescent Protein; Chlorophyll; Chromomycin A; CMFDA; Coelenterazine ; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidin; CPM Methylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Diehl orodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8- ANEPPS (non-ratio); DiA (4-D1-16-ASP); DIDS; Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DilCl 8(7)); Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer- 1 (EthD- 1); Euchrysin; Europium (III) chloride; Europium; EYFP; Eva Green; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4; Fluorescein Diacetate; FluoroEmerald; Fluoro-Gold (Hydroxy stilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura-2, high calcium; Fura-2, low calcium; Genacr l Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1;JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; Lucifer Yellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green 488-X; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoery thrin B [PE]; Phycoery thrin R [PE]; PKH26 ; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium lodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufm; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200 ; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L; S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ (6-methoxy-N- (3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; SYBR Gold; SYBR Green I; SYBR Green II; SYBR Safe; Tetracycline; Tetramethylrhodamine ; Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO- PRO- 1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC (TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. Many suitable forms of these fluorescent compounds are available and can be used.

[0174] In some embodiments, sample imaging provides a basis for identification of cellular regions of interest (ROI). An ROI, in some embodiments, comprises an area of a sample targeted for spatial analysis. In some embodiments, the ROI is based on morphology7of a sample, for example cells, cellular structures, tissue layers, or sub-cellular structures. In some embodiments,the ROI has a regular or irregular boundary. In some embodiments, the ROI describes a boundary for spatial analysis. In some embodiments, the ROI is grid-based. In some embodiments, the ROI is in two dimensions (2D). In some embodiments, the ROI is in three dimensions (3D).

[0175] A photomask provides a template to control areas of a sample that receive radiated light. In some embodiments, a photomask is generated to allow radiation of one or more ROIs. In some embodiments, one or more photomasks are generated. In some embodiments, a photomask is generated relevant to each ROI. In some embodiments, each crosslinking step comprises a unique photomask. In some embodiments, the photomask is manually or machine generated. In some embodiments, the photomask is generated according to sample imaging as previously described herein.Record extraction and sequencing

[0176] In some embodiments, generated cross-junction synthesis products are displaced from the tissue samples or population of cells as provided herein. In some embodiments, these steps occur sequentially or simultaneously. In some embodiments, continuous nucleic acid strands are pooled. In some embodiments nucleic acid strands are then amplified and sequenced. In some embodiments, sequencing data is incorporated with earlier imaging data to provide spatially defined sequencing information.

[0177] Provided herein are methods for nucleic acid strand displacement. In some embodiments, cDNA bound to RNA is displaced from the tissue samples or population of cells provided herein. In some embodiments, nucleic acid strands are displaced chemically. In some embodiments, nucleic acid strands are displaced enzymatically. In some embodiments, nucleic acid strands are displaced using an enzyme that specifically cleaves RNA (e.g., RnaseH). In some embodiments, nucleic acid strands are displaced using an enzyme that specifically cleaves DNA, a DNAse (e.g., DNAse I). In some embodiments, nucleic acid strands are displaced physically. In some embodiments, displacement and cross-junction synthesis occur concurrently.

[0178] Provided herein are methods of amplification of nucleic acids. In some embodiments, amplification comprises strand-displacement amplification (SDA), Nicking Enzyme Amplification Reaction (NEAR), exponential amplification reaction (EXPAR), enzymatic oligonucleotide synthesis via internal inosine modification and exonuclease V, nick translation, Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HD A), Multiple Displacement Amplification (MDA), Ligase Chain Reaction (LCR), Recombinase Polymerase Amplification (RPA), Ramification amplification method (RAM), Nucleic Acid Sequence-Based Amplification (NASBA), signal amplification by exchange reaction (SABER), primer exchange reaction (PER), or any variation or combination thereof. In some embodiments,the PER is a Terminal deoxynucleotidyl transferase (Tdt)-based PER assay. In some embodiments, the amplification is performed at multiple temperatures, for example in a cycle. In some embodiments, the amplification is performed using non-isothermal, linear, or exponential amplification methods. In some embodiments, the amplification is PCR. In some embodiments, the PCR is asymmetric PCR. In some embodiments, PCR amplification uses a DNA polymerase to synthesize DNA from deoxynucleotide substrates on a single-stranded template. In some embodiments described herein, the PCR method comprises Real-time PCR, Quantitative real time PCR (Q-RT PCR), Reverse Transcriptase PCR (RT-PCR), Multiplex PCR, Nested PCR, Long- range PCR. Single-cell PCR. Fast-cycling PCR, Methylation-specific PCR (MSP), Hot start PCR, High-fidelity PCR, In situ PCR, Variable Number of Tandem Repeats (VNTR) PCR, Asymmetric PCR, Repetitive sequence-based PCR, Overlap extension PCR, Assemble PCR, Intersequencespecific PCR(ISSR), Ligation-mediated PCR, Methylation -specifm PCR, Miniprimer PCR, Solid phase PCR, Touch down PCR, or any combination thereof. In some embodiments, a reverse transcriptase is applied for an amplification described herein. In some embodiments, the reverse transcriptase is a MuLV reverse transcriptase, or Avian myeloblastosis virus (AMV) reverse transcriptase. In some embodiments, a DNA polymerase is applied for an amplification described herein. In some embodiments, the DNA polymerase is a T7 DNA polymerase, thermophilic eubacterial microorganism Thermus aquaticus DNA polymerase, Pfu DNA polymerase, or Bst DNA Polymerase. In some embodiments, a DNA ligase is applied for joining strands. In some embodiments, the DNA ligase is EC 6.5. 1.1. In some embodiments, the DNA ligase is a T4 DNA ligase.

[0179] In some embodiments, barcode sequences, or primers complementary to such sequences, comprise a polymerase promoter sequence. In some embodiments, the promoter is an RNA polymerase promoter. In some embodiments, the polymerase promoter sequence comprises, without limitation, a T7 RNA polymerase promoter region (SEQ ID NO: 1: TAATACGACTCACTATAG), a T3 RNA polymerase promoter region (SEQ ID NO: 2: AATTAACCCTCACTAAAG), or a SP6 RNA polymerase promoter region (SEQ ID NO: 3: ATTTAGGTGACACTATAG).

[0180] Provided herein are methods for sequencing. In some embodiments, sequencing comprises next-generation sequencing (NGS) technologies. In some embodiments, sequencing comprises massively parallel sequencing. In some embodiments, sequencing comprises chain termination sequencing, nanopore sequencing, sequencing by hgation, combinatorial probe anchor synthesis, sequencing by synthesis, pyrosequencing, ion semiconductor, or single-molecule real-time sequencing.Data analysis

[0181] In some embodiments, a density of barcoded nucleic acids is measured by the number of unique sequences detected. In some embodiments, the density of barcoded nucleic acids is the number of barcodes generated within one square micrometer (pm2) of a tissue sample, a population of cells, or a population of cells. In some embodiments, the density of barcoded nucleic acids is the number of barcodes generated within a cubic micrometer (pm3) of a tissue sample, a population of cells, an organoid, or an organ. In some embodiments, the methods provided herein generate about 55 tags or barcodes per square micrometer (pm2). In some embodiments, the methods provided herein generate about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about , about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000, about 2250, about 2500, about 2750, about 3000, about 3500. about 4000, about 4500. about 5000 tags per pm2.

[0182] In some embodiments, the methods provided herein generate up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, up to about 50, up to about 55, up to about 60, up to about 65, up to about 70, up to about 80, up to about 90. up to about 100, up to about 150, up to about 200, up to about 250, up to about 300, up to about , up to about 350, up to about 400, up to about 450, up to about 500, up to about 550, up to about 600, up to about 650, up to about 700, up to about 800, up to about 900, up to about 1000, up to about 1200, up to about 1400, up to about 1 00, up to about 1800, up to about 2000. up to about 2250, up to about 2500, up to about 2750, up to about 3000, up to about 3500, up to about 4000, up to about 4500, up to about 5000 tags per pm2.

[0183] In some embodiments, the methods provided herein generate about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 80, about 90, about 100, about 150, about 200. about 250, about 300, about , about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000, about 2250, about 2500, about 2750, about 3000, about 3500, about 4000, about 4500, about 5000 , about 6000, about 70000, about 8000, about 9000, about 10,000 tags per cubic micrometer (pm3).

[0184] In some embodiments, the methods provided herein generate up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, up to about 50, up to about 55, up to about 60, up to about 65, up to about 70, up to about 80, up to about 90, up to about 100, up to about 150, up to about 200, up to about 250, up to about 300, up to about , up to about 350, up to about 400, up to about 450, up to about 500, up toabout 550, up to about 600, up to about 650. up to about 700, up to about 800, up to about 900. up to about 1000, up to about 1200, up to about 1400, up to about 1600, up to about 1800, up to about 2000, up to about 2250, up to about 2500, up to about 2750, up to about 3000, up to about 3500, up to about 4000, up to about 4500, up to about 5000, up to about 6000, up to about 70000, up to about 8000, up to about 9000, up to about 10,000 tags per cubic micrometer (pm3).

[0185] Provided herein are methods for spatial analysis of transcribed nucleic acids in selected regions of interest (ROIs) in cells and tissues. In some embodiments, the transcribed nucleic acid is a reverse transcribed DNA or a transcribed RNA. In some embodiments, barcode sequences from sequencing reads provides spatial analysis data. In some embodiments, spatial analysis data combined with imaging methods described earlier herein provides a link between cellular phenotype information and transcriptome information.WORKFLOWS

[0186] Methods described herein are not temporally constrained in the order or even if all steps must be performed. Described herein are workflows comprising non-limiting combinations of methods described herein comprising POMC. Representative workflows describe processes of biomolecular capture (FIG. 11), proximity recording (FIG. 12), or integrated biomolecule capture and proximity recording (FIG. 13) comprising methods as described herein. Modular processes provide flexibility in methodology.

[0187] Non-limiting methods for biomolecule capture in a sample are described in FIG. 11. In some embodiments: first, a concatemer of barcoded docking oligonucleotides is generated in situ second, a cleaving oligonucleotide is hybridized and photocrosslinked to the concatemer of docking oligonucleotides to form a duplex; third, the duplex is cleaved to release a crosslinked barcode complex; fourth, mRNA is reverse transcribed to generate cDNA using the cross-linked barcode complex as transcription primer; and fifth, the cDNA / cross-linked barcode complex is read using cross-junction synthesis and NGS. In some embodiments: first, a cleaving oligonucleotide is used as a primer to generate a cDNA extension from an mRNA; second, a concatemer of barcoded docking oligonucleotides is generated in situ; third, the cleaving oligonucleotide / cDNA is hybridized and photocrosslinked to the concatemer of docking oligonucleotides to form a cDNA / cross-linked barcode complex; fourth, the complex is cleaved to release a cDNA / cross-linked barcode complex; fifth, the cDNA / cross-linked barcode complex is read using cross-junction synthesis and NGS. In some embodiments: first, a cleaving oligonucleotide is used as a primer to generate a cDNA extension from an mRNA; second, a concatemer of barcoded docking oligonucleotides generated ex situ is introduced to the sample; third, the cleaving oligonucleotide / cDNA is hybridized and photocrosslinked to the concatemer ofdocking oligonucleotides to form a cDNA / cross-linked barcode complex; fourth, the complex is cleaved to release a cDNA / cross-linked barcode complex; fifth, the cDNA / cross-linked barcode complex is read using cross-junction synthesis and NGS. In some embodiments: first, a concatemer of barcoded docking oligonucleotides is generated in situ second, a cleaving oligonucleotide is used as a primer to generate a cDNA extension from an mRNA; third, the cleaving oligonucleotide / cDNA is hybridized and photocrosslinked to the concatemer of docking oligonucleotides to form a cDNA / cross-linked barcode complex; fourth, the complex is cleaved to release a cDNA / cross-linked barcode complex; fifth, the cDNA / cross-linked barcode complex is read using cross-junction synthesis and NGS.

[0188] Nonlimiting methods of proximity recording in a sample are described in FIG. 12. In some embodiments: first, a concatemer of barcoded docking oligonucleotides is generated in situ; second, a cleaving oligonucleotide is hybridized and photocrosslinked to the concatemer of docking oligonucleotides to form a duplex; third, the cleaving oligonucleotide is hybridized to a nearby barcoded concatemer and extended to copy the barcode; fourth, the duplex is cleaved to release a cross-linked multi-barcode complex; fifth, the cross-linked multi-barcode complex is read using cross-junction synthesis and NGS. In some embodiments: first, a concatemer of barcoded docking oligonucleotides generated ex situ is introduced to the sample; second, a cleaving oligonucleotide is hybridized and photocrosslinked to the concatemer of docking oligonucleotides to form a duplex; third, the cleaving oligonucleotide is hybridized to a nearby barcoded concatemer and extended to copy the barcode; fourth, the duplex is cleaved to release a cross-linked multi-barcode complex; fifth, the cross-linked multi-barcode complex is read using cross-junction synthesis and NGS. In some embodiments: first, concatemers of barcoded docking oligonucleotides are generated in situ; second, a cleaving oligonucleotide is hybridized and photocrosslinked to a first concatemer of docking oligonucleotides to form a duplex; third, the cleaving oligonucleotide is hybridized and photocrosslinked to a second concatemer of docking oligonucleotides to form a bridged cross-linked complex; fourth, the cross-linked complex is cleaved to release a cross-linked multi-barcode complex; fifth, the cross-linked multi-barcode complex is read using cross-junction synthesis and NGS.

[0189] Nonlimiting methods of integrated biomolecule capture and proximity recording in a sample are described in FIG. 13. In some embodiments: first, a cleaving oligonucleotide is used as a primer to generate a cDNA extension from an mRNA; second, concatemers of barcoded docking oligonucleotides generated ex situ are introduced to the sample; third, the cleaving oligonucleotide / cDNA is hybridized and photocrosslinked to a first concatemer of docking oligonucleotides to form a duplex; fourth, a bridge cleaving oligonucleotide is hybridized and photocrosslinked to the first concatemer of docking oligonucleotides; fifth, the bridge cleavingoligonucleotide is hybridized to a nearby barcoded concatemer and extended to copy the barcode; sixth; duplexes and complexes are cleaved to release cross-linked barcoded products; seventh, the cross-linked barcoded products are read using cross-junction synthesis and NGS. In some embodiments: first, concatemers of barcoded docking oligonucleotides are generated in situ second a bridge cleaving oligonucleotide is hybridized and photocrosslinked to a first concatemer of docking oligonucleotides; third, the bridge cleaving oligonucleotide is hybridized to a nearby barcoded concatemer and extended to copy the barcode; fourth, a primer / cleaving oligonucleotide is hybridized and photocrosslinked to the first or second concatemer of docking oligonucleotides to form a duplex: fifth, duplexes are cleaved to release cross-linked barcoded products; sixth, the primer / cleaving oligonucleotide is used as a primer to generate a cDNA extension from an mRNA; seventh, the cross-linked barcoded products are read using cross-junction synthesis and NGS.

[0190] In some embodiments, steps of described workflows are temporally distinct. In some embodiments, steps of described workflows occur without temporal distinction. In some embodiments, components for each step of described workflows are provided separately. In some embodiments, components for each step of described workflows are provided simultaneously, or in a “one pot” reaction.Screening

[0191] Application of methods described herein include and are not limited to identification of drug targets, identification of biomarkers, profiling, characterization of cells and disease models, characterization of differentiation status and cell state, tissue mapping, and multi-dimensional analysis.Kits

[0192] Provided herein are kits comprising compositions described herein. In some embodiments, kits comprise one or more containers comprising compositions described herein. In some embodiments, containers are selected from boxes, ampules, bottles, vials, tubes, bags, pouches, blister- packs, or other suitable container forms known in the art in any combination. In some embodiments, kits are for detection or treatment of a disease or condition. In some embodiments, a kit comprises instructions for performing methods as described herein.Oligonucleotide library generation

[0193] In some embodiments, populations of oligonucleotides described herein are provided in a library of nucleic acids. In some embodiments, a library' of nucleic acids comprises a population of docking oligonucleotides as described herein. In some embodiments, a library of nucleic acidscomprises a population of cleaving oligonucleotides as described herein. In some embodiments, a library of nucleic acids comprises a concatemer of docking oligonucleotides as described herein.

[0194] In some embodiments, methods of POMC as described herein are used to generate a library of duplexed oligonucleotide fragments. In some embodiments, methods of POMC and crossjunction synthesis as described herein are used to generate a library of barcoded nucleic acids.EXAMPLES

[0195] The examples below further illustrate the described embodiments without limiting the scope of this disclosure.EXAMPLE 1: IN VITRO DIGESTION ASSAY

[0196] The following assay demonstrates crosslinked cleaving oligonucleotides are able to direct cleaving of cross-linked strands. Cleavage and docking oligonucleotides as depicted in FIG. 14A were combined 1: 1 in 1DTE buffer (10 mM Tris. 0.1 mM EDTA) at a 50 mM final concentration. The oligo pairs were annealed by heating to 90°C for 3 minutes, then gradually cooling to 20°C over the course of 30 min. The annealing reactions were crosslinked by placing the tubes under a 365 nm LED (Thorlabs, Inc., Newton, NJ) for 30 seconds. To remove any uncrosslinked oligos, reactions were denatured by adding formamide (25% final concentration) and heating to 98°C for 3 min before loading onto a 4% e-gel. The highest molecular weight band (crosslinked product) was excised from the gel and purified using a Monarch DNA gel purification kit (New England BioLabs, Ipswich, MA (NEB)). Next, paired digestion reactions were set up that each contained 4 pL of purified product as input, 2 pL of 10X rCutsmart buffer (NEB). 12 pL of ultrapure water (Invitrogen, Waltham, MA), and either 2 pL of restriction enzyme (digest conditions) or 2 pL of ultrapure water (no digest conditions). Digestion reactions were incubated at 37°C for 1 hour and subsequently run on a gel to assess cutting. A photo of the gel comparing the size of digested and intact crosslinked oligonucleotides is shown in FIG. 14B. Cleaving oligonucleotide design is described in Table 2.Table 2. Cleaving oligonucleotideEXAMPLE 2: IN SITU DIGESTION ASSAY

[0197] Cleavage of crosslinked oligonucleotides in cells was demonstrated in an in situ digestion assay.Cell seeding and fixation

[0198] 3T3 cells were seeded and grown overnight in chambered coverslips (Ibidi USA, Inc., Fitchburg, WI). The next day, media was removed and the cells were washed with pre-warmed DPBS (no calcium, no magnesium) before fixation with 4% paraformaldehyde solution in IX PBS (4% PF A). Cells were incubated in fixative for 5 minutes at room temperature before washing 2 x 2 minutes in IX PBS with 0.1% Tween-20 (PBST). Cells were then permeabilized with IX PBS with 0.25% Triton-X-100 (PBSTx) for 10 minutes, then washed with 2 x 1 min with IX PBS. Finally, cells were washed with PBST 2 x 2 minutes.Reverse transcription

[0199] Reverse transcription (RT) was conducted using the candidate cleave oligos from Example 1 as primers. Cells were incubated in reverse transcription reaction mix that contained 300 pM dNTPs (NEB), 0.5% Triton X-100, 6 mM RNaseOUT (Invitrogen), 1 pM cleave oligo (one of Cleave Oligos 1-4), and 8 U / pL Maxima RT H Minus enzyme in IX RT buffer (Thermo Fisher Scientific, Waltham, MA). Samples were incubated on a flat-top thermocycler using the following program: 30 min at 22 °C, followed by a 12-cycle ramp program of 8 °C for 30 s, 15 °C for 30 s, 25 °C for 30 s, 30 °C for 1 min, 37 °C for 1 min and 42 °C for 2 min, followed by 42 °C for 30 min. After RT, samples were washed in PBST containing 60% formamide 3 times, then in PBST supplemented with 1 M NaCl (Invitrogen) twice, and finally PBST twice.Docking strand hybridization

[0200] Following RT, docking strands were introduced by incubating the cells in IX PBS. 500 mM NaCl, 10% Dextran Sulfate, 0.1% Tween-20, 2 mg / mL salmon sperm DNA, and 0.25 pM oligo for 30 minutes at room temperature. Excess docking oligo was removed by washing three times in high-salt wash buffer (IX PBS, 1 M NaCl, 0.1% Tween- 20). Docking strands were crosslinked to the RT oligos by placing them under a 365 nm LED for 30s.Digestion

[0201] Digestion was performed in situ by adding 50pL reaction mixture (IX rCutsmart buffer and 5 pL of restriction enzyme, either Bbvcl or BtsI as appropriate for the cleave oligo) to each well, and then incubating for 1 h at 37 °C. For control wells, water was added in place of the restriction enzyme. Following digestion, samples were rinsed twice with stringent wash buffer (0.1% PBST, 60% formamide), and then 3X1 min in 6X SSC (Invitrogen).Probe hybridization and imaging

[0202] To visualize cleavage, an imaging probe was hybridized to the docking oligo by adding 50 pL of the following mixture to the wells: IX PBS, 0.2% Tween-20 and 0.1 pM imaging probe. Samples were incubated for 30 min at 37 °C, then washed 2X5 min in PBST at 37 °C. Samples were imaged on a confocal microscope to compare the fluorescence intensity of the imaging probe between paired samples without and without enzyme treatment. A loss of fluorescence indicates increased cleavage of the duplexed oligonucleotides. Images are shown in FIGs 15A-15H. FIGs. 15A-15D are microscope images of fluorescent-tagged docking strands hybridized to Cleave Oligo 1 (FIG. 15A), Cleave Oligo 2 (FIG. 15B), Cleave Oligo 3 (FIG. 15C), or Cleave Oligo 4 (FIG. 15D) after reverse transcription using cleave oligos as primers on cells without treatment with restriction enzyme. All images show fluorescent binding, indicating docking strands are fixed on the cells. FIGs. 15E-15H are microscope images of fluorescent-tagged docking strands hybridized to Cleave Oligo 1 (FIG. 15E), Cleave Oligo 2 (FIG. 15F), Cleave Oligo 3 (FIG. 15G), or Cleave Oligo 4 (FIG. 15H) after reverse transcription using cleave oligos as primers on cells with treatment with restriction enzyme. FIG. 15E, FIG. 15G, and FIG. 15H show similar levels of fluorescence as in samples not treated with restriction enzyme, indicating docking strands are fixed on the cells after treatment with restriction enzyme. FIG. 15F showed loss of fluorescence, indicating digestion and release of docking strands after treatment with restriction enzyme.

[0203] Results indicate that when cleaving oligos are used to generate cDNA in cells, efficiency of cleavage is dependent on dock sequences. Samples contacted with Cleave Oligo 2, having spacing of restriction enzyme (RE) site 4 bases from crosslink and 12 bases from the 5’ end mediated efficient cleaving in situ of cDNA / concatemer conjugates. Scale bars, 20um.EXAMPLE 3: BRIDGE STRAND PRODUCTION

[0204] Variable length photocrosslinkable cleaving oligo bridge strands were generated through PCR and exonuclease treatment. Initial bridge amplicons were produced by amplifying a gBlock with a CNVK-modified forward primer and one of three 5 '-phosphorylated reverse primers designed to produce fragments of various lengths (255 bases, 438 bases, and 655 bases). The PCR reaction mixture consisted of IX Q5 Reaction Buffer, 0.2mM dNTPs, 20U / mL Q5 High-Fidelity DNA Polymerase, 10 pM gBlock, and 0.5 pM each primer. The reaction program was 98°C 30 s, 35 cycles of [98°C 10 s, 68°C 20 s. 72°C 20 s], 72°C 2 min. The reactions were then purified with the Monarch PCR & DNA Cleanup Kit (NEB) following the manufacturer’s directions. The expected lengths of the products were confirmed by gel electrophoresis. The resulting fragments are showing in the gel image in FIG. 16. As shown, conversion to single stranded DNA was confirmed on gel as evidenced by faster electrophoretic migration and diminished intensity of DNA intercalating dye binding. To preferentially digest the 5 ’-phosphorylated strands to releasesingle-stranded, CNVK-modified bridge product, the purified amplicons were then treated with an exonuclease in a reaction containing 5ng / pl amplicon, 0.5 U / pl Lambda Exonuclease, and IX Lambda Exonuclease Reaction Buffer. Reactions were incubated at 37°C for 30 min, then heat inactivated at 75°C for 10 min and used directly in downstream reactions.EXAMPLE 4: BRIDGE STRAND CROSSLINKING IN VITRO.

[0205] CNVK cleaving bridge strands generated through PCR + exonuclease retain ability to crosslink to complementary strands as evidenced by higher MW on gel under denaturing conditions following UV illumination in presence of a duplexed oligo. The 255-base bridge strand generated in Example 3 was annealed to a docking oligo in a solution containing 2pL of 10 pM docking oligo (in IDTE) and 8 pL of unpurified bridge strand by heating to 90°C for 3 min, then cooling to 20°C over 30 min. The reaction was split, and one half of the reaction was crosslinked by placing the sample under a 365 nM LED for 30 s. To denature any non-crosslinked products, all reactions incubated in 25% formamide at 98°C for 3 min before being loaded and run on a gel. FIG. 17 shows an image of the gel, in columns from left to right. Molecular Weight ladder, docking and cleaving oligo without UV light exposure, and duplexed sample exposed to UV light to crosslink and stabilize the duplexed strands. As shown, the crosslink strands have increased MW, indicating crosslinking between cleaving and docking strands.EXAMPLE 5: CONCATEMER STRAND GENERATION AND DISTRIBUTION.

[0206] This example described generation and release of concatemeric strands comprising repeats of barcode domain and docking domain in situ.Cell seeding and fixation

[0207] 3T3 cells were seeded and grown overnight in chambered coverslips. The next day, media was removed and the cells were washed with pre-warmed DPBS (no calcium, no magnesium) before fixation with 4% paraformaldehyde solution in IX PBS (4% PF A). Cells were incubated in fixative for 5 minutes at room temperature before washing 2 x 2 minutes in IX PBS with 0.1% Tween-20 (PBST). Cells were then permeabilized with IX PBS with 0.25% Triton-X-100 (PBSTx) for 10 minutes, then washed with 2 x 1 min with IX PBS. Finally, cells were washed with PBST 2 x 2 minutes.Reverse transcription

[0208] Reverse transcription was performed using the candidate cleave oligos as primers. Cells were incubated in reverse transcription reaction mix that contained 300 pM dNTPs (NEB), 0.5% Triton X-100, 6 mM RNaseOUT (Invitrogen), 1 pM cleave oligo, and 8 U / pL Maxima RT H Minus enzyme in IX RT buffer (Thermo Fisher). Samples were incubated on a flat-top thermocycler using the following program: 30 min at 22°C, followed by a 12-cycle ramp programof 8 °C for 30 s, 15 °C for 30 s. 25 °C for 30 s, 30°C for 1 min, 37 °C for 1 min and 42 °C for 2 min, followed by 42 °C for 30 min. After RT, samples were washed in PBST containing 60% formamide three times, then in PBST supplemented with 1 M NaCl (Invitrogen) two times, and finally PBST two times.Concatemer template preparation

[0209] Templates for concatemer generation were produced by circularizing phosphorylated oligos (obtained from IDT) in vitro. The template oligos were stabilized in a circular configuration using a splinted ligation. First, a mixture containing 4.5 pM template oligo, 4.5 pM splint oligo, and 50 mM NaCl) was heated to 90 °C for 3 min and gradually cooled to 20 °C over 30 minutes. The splinted templates were then ligated using the following reaction mixture: IX T4 DNA ligase buffer (NEB), 13.3 U / pL T4 DNA ligase (NEB) and 0.9 pM annealed template. The ligation reactions were incubated at 23°C for 16 hrs and then heat inactivated 65°C for 10 min. The product was used directly in downstream steps without further purification.Template introduction and Rolling Circle Ampli fication

[0210] Circular templates were introduced into wells cells by adding 50 uL of a solution containing IX Ampligase buffer (VWR), 50 mM KC1, 20% formamide, 0.2 mg / mL BSA and 0.3 pM circular template. The samples were incubated at 55°C for 15 min and 45°C for 120 min to allow the templates to diffuse into the cells. Excess template was removed by washing 3X5 min in washing buffer (10% formamide, 2X SSC) and then 3X1 min in PBST. Templates were amplified using rolling circle amplification (RCA) by adding a reaction mixture consisting of IX Phi29 buffer (Thermo Fisher), 5% Glycerol, 0.2 mg / mL BSA, 0.25 pM dNTPs, 0.1 pM RCA initiator oligo, and 0.5 U / pL Phi29 polymerase (Thermo Fisher). Samples were incubated at 30°C overnight, then washed 3X1 min in PBST. 3X5 min in 65% formamide, and 2X1 min in PBST.Imaging RCA Product

[0211] To confirm successful template amplification, a fluorescent imager probe was hybridized to the concatemeric product by adding a solution containing IX SSC, 5% formamide, 0.06 pM fluorescent probe and 0.1 mg / mL BSA. The samples were incubated at 33 °C for 30 min, washed 3X5 min in washing buffer (10% formamide in 2X SSC), and 3X1 min in 6X SSC. Images such as FIG. ISA were acquired using a confocal microscope and the presence of fluorescent puncta confirmed successful amplification.Concatemer distribution

[0212] To distribute the concatemer more uniformly over the cell, the RCA product was lightly digested. To do this, a low concentration of a cleaving oligo was hybridized to the concatemer by applying a mixture containing IX SSC, 5% formamide, 0.1 mg / mL BSA, and 6 pM cleave oligo. The samples were incubated at 33 °C for 30 min, washed 3X5 min in washing buffer (10%61formamide. 2X SSC), and 3X1 min in 6X SSC. Digestion was performed by adding 50 pL of solution containing IX rCutsmart and 0.4 U / pL Btsl-v2 (NEB) and incubating for 1 hr at 37 °C. Samples were washed 3X1 min in 6X SSC.Imaging distributed concatemer

[0213] Images of the hybridized probe after multiple wash steps were acquired using a confocal microscope, shown in FIG. 18B. Successful concatemer distribution was visualized as blurring of the previously punctate fluorescence signal. Image shows cleaving with restriction enzyme and a low concentration of a duplex oligo as detected by imager oligo. This reaction removes background amplification products adhered to the coverslip, while abundant concatemeric strands are retained in cells after multiple wash steps. Scale bars, 15um.EXAMPLE 6: CLEAVING OLIGO BRIDGE STRANDS CROSSLINKING, POLYMERIZATION, DIGESTION, ELUTION, AND CROSS-JUNCTION SYNTHESIS

[0214] This example describes crosslinking of cleaving badge strands hybridized to BC-dock concatemers, 3' end extension of cleaving bridge strands followed by digestion, elution, and crossjunction synthesis.Selective crosslinking of bridge strand

[0215] Cell seeding and fixation. 3T3 cells were seeded and grown overnight in chambered coverslips. The next day, media was removed, and the cells were washed with pre-warmed DPBS (no calcium, no magnesium) before fixation with 4% paraformaldehyde solution in IX PBS (4% PF A). Cells were incubated in fixative for 5 minutes at room temperature before washing 2 x 2 minutes in IX PBS with 0.1% Tween-20 (PBST). Cells were then permeabilized with IX PBS with 0.25% Triton-X-100 (PBSTx) for 10 minutes, then washed with 2 x 1 min with IX PBS. Finally, cells were washed with PBST 2 x 2 minutes.

[0216] Reverse transcription. Reverse transcription was performed using the candidate cleave oligos as primers. Cells were incubated in reverse transcription reaction mix that contained 300 pM dNTPs (NEB). 0.5% Triton X-100, 6 mM RNaseOUT (Invitrogen), 1 pM cleave oligo, and 8 U / pL Maxima RT H Minus enzyme in IX RT buffer (Thermo Fisher). Samples were incubated on a flat-top thermocycler using the following program: 30 min at 22 degrees Celsius, followed by a 12-cycle ramp program of 8 degrees Celsius for 30 s, 15 degrees Celsius for 30 s, 25 degrees Celsius for 30 s, 30 degrees Celsius for 1 min, 37 degrees Celsius for 1 min and 42 degrees Celsius for 2 min, followed by 42 °C for 30 min. After RT, samples were washed in PBST containing 60% formamide three times, then in PBST supplemented with 1 M NaCl (Invitrogen) twice, and finally PBST twice.

[0217] Concatemer template preparation. Templates for concatemer generation were produced by circularizing phosphorylated oligos (obtained from IDT) in vitro. The template oligos were stabilized in a circular configuration using a splinted ligation. First, a mixture containing 4.5 pM template oligo, 4.5 pM splint oligo, and 50 mM NaCl) was heated to 90 degrees Celsius for 3 min and gradually cooled to 20 degrees Celsius over 30 minutes. The splinted templates were then ligated using the following reaction mixture: IX T4 DNA ligase buffer (NEB), 13.3 U / pL T4 DNA ligase (NEB) and 0.9 pM annealed template. The ligation reactions were incubated at 23°C for 16 hrs and then heat inactivated 65 degrees Celsius for 10 min. The product was used directly in downstream steps without further purification.

[0218] Template introduction and rolling cycle amplification. Circular templates were introduced into w ells cells by adding 50 uL of a solution containing IX Ampligase buffer (VWR), 50 mM KC1, 20% formamide, 0.2 mg / mL BSA and 0.3 pM each of the two circular template Wpes. The samples were incubated at 55 degrees Celsius for 15 min and 45 degrees Celsius for 120 min to allow the templates to diffuse into the cells. Excess template was removed by washing 3X5 min in washing buffer (10% formamide, 2X SSC) and then 3X1 min in PBST. Templates were amplified using rolling circle amplification (RCA) by adding a reaction mixture consisting of IX Phi29 buffer (Thermo Fisher), 5% Glycerol, 0.2 mg / mL BSA, 0.25 pM dNTPs, 0.1 pM RCA initiator oligo, and 0.5 U / pL Phi29 polymerase (Thermo Fisher). Samples were incubated at 30°C overnight, then washed 3X1 min in PBST, 3X5 min in 65% formamide, and 2X1 min in PBST.

[0219] Concatemer distribution. To distribute the concatemer more uniformly over the cell, the RCA product was lightly digested. To do this a low' concentration of a cleaving oligo was hybridized to the concatemer by applying a mixture containing IX SSC, 5% formamide, 0.1 mg / mL BSA, and 6 pM cleave oligo. The samples were incubated at 33 degrees Celsius for 30 min, washed 3X5 min in washing buffer (10% formamide, 2X SSC), and 3X1 min in 6X SSC. Digestion w as performed by adding 50 pL of solution containing IX rCutsmart and 0.4 U / pL Btsl- v2 (NEB) and incubating for 1 hr at 37 degrees Celsius. Samples were washed 3X1 min in 6X SSC.

[0220] cDNA hybridization and crosslinking. To ensure hybridization of cDNAs to the distributed concatemers, the samples w ere heated to 55 degrees Celsius for 15 min, 45 degrees Celsius for 60 min and 37 degrees Celsius for 60 min, The hybridized cDNAs were subsequently crosslinked to the type 1 concatemer by placing the samples under a 365 nM LED for 30s.

[0221] Bridge strand production. Bridge strands were produced in vitro by PCR amplification of a gBlock template using a photocrosslinkable forward primer and phosphorylated reverse primer. PCR reactions contained IX Q5 reaction buffer (NEB), 0.2 pM dNTPs, 0.01 nM template, 0.02 U / uL Q5 HiFi Polymerase (NEB), and 0.5 pM each of forward and reverse prime. Thefollowing cycling program was used for amplification: 1 cycle of 98 degrees Celsius for 30 sec, 25 cycles of 98 degrees Celsius for 10 sec, 68 degrees Celsius for 20 sec, 72 degrees Celsius for 20 sec, and 1 cycle of 72 degrees Celsius for 2 min. Reactions were cleaned using a Monarch PCR clean up kit (NEB) according to the manufacturer’s directions. The expected product length (438 bases) was verified by gel electrophoresis. A single stranded fragment was produced by degradation of the botom strand using an exonuclease reaction consisting of IX lambda exonuclease buffer (NEB), 0.5 U / pL lambda exonuclease (NEB), and 12 ng / uL of PCR product. The reaction was incubated at 37 degrees Celsius for 30 min, heat inactivated at 75 degrees Celsius for 10 min, and then purified using a Monarch PCR clean-up kit following the manufacturer’s directions. Successful degradation was confirmed by faster migration as visualized by gel electrophoresis (data not shown).

[0222] Bridge Strand Hybridization. Bridge strands were introduced into the samples by adding 50 uL of solution containing 2X SSC, 20 % formamide, 0.2 mg / rnL BSA, 0.2 mg / rnL yeast tRNA (Invitrogen), and 10 nM bridge strand to each well. Samples were incubated at 55°C for 15 min, 45°C for 60 min and 37°C for 60 min, then washed 3X1 min in 6X SSC.

[0223] Bridge Strand Crosslinking. Bridge strands were crosslinked to the type 1 concatemer strands within a specific ROI by targeted illumination on a confocal microscope equipped with a digital micromirror device (DMD). A rectangular region of interest within each well was exposed to 365 nm light for 10s. The samples were removed from the microscope, and the non-crosslinked bridge strand was removed by washing 2X5 min at 40°C with 70% formamide in 2X SSC and 2X1 min in PBST.

[0224] Probe hybridization and imaging. For visualization, spectrally distinct probes were hybridized to each of the two concatemer types and the bridge strand using a mixture containing I X SSC, 5% formamide, 0.06 pM each probe and 0.1 mg / mL BSA. The samples were incubated at 33 °C for 30 min, washed 3X5 min in washing buffer (10% formamide in 2X SSC), and 3X1 min in 6X SSC. Images stacks were acquired in fluorescence channels corresponding to each probe using a 20X objective. The image stacks were max projected for visualization. Detection of the first barcoded concatemer is show in in FIG. 19A. Detection of the second barcoded concatemer is shown in FIG. 19B. The UV -illuminated region of interest is indicated by the white box in FIGs. 19A-19C. Detection of bridge strands is shown in FIG. 19C. Fluorescence only in the illumination ROI indicates cross-linking of bridge strands to barcoded concatemers.Elution of extended bridge strand

[0225] Bridge strand extension. Bridge strands were extended onto the type 2 concatemers by adding a 50 pL of a mixture containing 0.3 mM dATP (NEB), 0.3 mM dTTP (NEB), 0.3 mM dCTP (NEB), IX RT buffer (Thermo Fisher), and 8 U / pL Maxima RT H Minus to each well. Forno extension controls, water was added in place of the enzyme. Samples were incubated on a flat- top thermal cycler with the following program: 50°C for 5 min, followed by 5 cycles of 65 degrees Celsius for 30 sec, 50 degrees Celsius for 1 min.

[0226] Bridge strand release. Once extended, bridge strands were released by adding a 50 uL of digestion mixture (IX rCutsmart, 0.4 U / pL Pad, and either 0.4 U / pL BtsI or 0.4 U / pL Nb.BtsI. Samples were incubated for Ih at 37 degrees Celsius, and then the supernatant was collected and replaced with 6X SSC. The sample was again heated to 37 degrees Celsius and the 6X SSC was removed and pooled with the previously collected supernatant and stored at -80 degrees Celsius until use. The sample chambers were washed 3X5 min with PBST.

[0227] Post displacement imaging. For visualization, spectrally distinct probes were hybridized to each of the two concatemer types and any remaining bridge strand using a mixture containing IX SSC, 5% formamide, 0.06 pM each probe and 0.1 mg / mL BSA. The samples were incubated at 33 degrees Celsius for 30 min, washed 3X5 min in washing buffer (10% formamide in 2X SSC), and 3X1 min in 6X SSC. Images stacks were acquired in fluorescence channels corresponding to each probe using a 20X objective in regions where the bridge strand was previously crosslinked. The image stacks were max projected for visualization.

[0228] FIG. 19D and FIG. 19E are images of remaining concatemers and bridge strands after digestion and washing without extension of the bridge strands to the second template. Only one duplex restriction recognition sequence is available for digestion. FIG. 19F and FIG. 19G are images of remaining concatemers and bridge strands after digestion and washing with extension of the bridge strands to the second template. A second restriction site is generated by extension of the bridge strand to hybridize with a second concatemer. Reduced fluorescence in FIGS. 19F and 19G indicate more complete digestion and release in samples with bridge strand extension. This shows bridge strand 3’ end extension onto Type 2 concatemers facilitates bridge-concatemer conjugate release, the extension mediates release by converting a second restriction site (Pad) on type 2 concatemers to double stranded form, exposing it to restriction enzy me mediated digestion. qPCR amplification of barcoded and extended bridge strand

[0229] Returning to the displaced bridge strand, the eluate was cleaned using AmpureXP beads (Beckman Coulter) at a 0.8X ratio according to the manufacturer’s directions. A cross-junction synthesis (CJS) step was performed to stitch the photocrosslinked bridge strand into a continuous product. Reactions containing 10 pL of crosslinked bridge strand, 1.29 pL of 10X ThermoPol Reaction buffer (NEB), 0.13 pL of 10 mM dNTPs (NEB), 0.26 pL of 1 pM CJS primer, and 1.24 pL of 8 U / pL of BST LF Polymerase were incubated at 60 °C for 30 min and heat inactivated at 80°C for 20 min. PCR reactions consisting of 0.5X SYBR green, IX Q5 Buffer, 0.3 pM forward primer, 0.5 pM reverse primer, 0.2 mM dNTPs and 0.02 U / pL Q5 HotStart HiFi DNA polymerasewere amplified on a QuantStudio Real Time PCR System (Thermo Fisher) using the following program: 1 cycle of 98°C for 30s, 30 cycles of 98°C for 10s, 66°C for 20s, 72°C for 2 min (plate read). Samples were removed when the fluorescence readings were approximately halfway to saturation. The product was visualized on a 2% EX gel (invitrogen).

[0230] As shown in FIG. 19H, photocrosslinked, extended, and eluted bridge complexes are conjugated to sequences from both Type 1 and Type 2 concatemers. PCR amplification with primers that bind to sequences from Type 1 and Type 2 concatemers conjugated to Bridge strands generate bands of expected size. Samples were amplified to differing numbers of cycles to prevent overamplification, as indicated, and therefore gel band intensities are not representative of eluted product abundance. qPCR quantification of bridge strand abundance

[0231] Data were exported from the QuantStudio system, and the Ct values were extracted and plotted, averaged over two biological replicates. Error bars represent the standard deviation. Bridge strand length before extension was 438 bases. Scale bars was 20 pm. As shown in FIG. 191, qPCR Ct Values demonstrate that bridge complexes amplifiable by Type 1 and Type 2-sequence PCR primers are significantly more abundant in 3’ extension conditions than in no polymerase control conditions.

[0232] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of biological information generation, the method comprising: a) immobilizing a concatemer onto a biological sample, wherein the concatemer is a nucleic acid and comprises two or more copies of a first docking oligonucleotide comprising: a barcode sequence; and a flanking region, b) contacting the biological sample with at least one cleaving oligonucleotide, wherein the cleaving oligonucleotide comprises a region complementary to a region on the first docking oligonucleotide, and wherein the first docking oligonucleotide or the cleaving oligonucleotide comprise at least one of each independently: a photocrosslinking agent, and at least one restriction site; c) illuminating predefined regions of the sample to activate the photocrosslinking agent, thereby binding the first docking oligonucleotide and the cleaving oligonucleotide to generate a photocrosslinked oligonucleotide complex; d) contacting the photocrosslinked oligonucleotide complex with an endonuclease, wherein the endonuclease cleaves the photocrosslinked oligonucleotide complex at one of the at least one restriction site, thereby generating a duplexed fragment of the photocrosslinked oligonucleotide complex, and wherein the duplexed fragment comprises the barcode sequence; e) polymerizing the duplexed fragment using cross-junction synthesis to generate a contiguous nucleic acid comprising copy of a region from each of the first docking oligonucleotide comprising the barcode sequence and the cleaving oligonucleotide; f) sequencing the contiguous nucleic acid, wherein an association of barcode sequence and cleaving oligonucleotide provides biological information.

2. The method of claim 1. wherein the biological sample comprises a population of cells, a tissue sample, or a protein.

3. The method of claim 2, wherein the biological sample is fixed on a surface.

4. The method of claim 1, wherein the docking oligonucleotide and the cleaving oligonucleotide each independently comprise ribonucleotides, deoxyribonucleotides, or any variation or combination thereof.

5. The method of claim 1. further comprising generating the concatemer by rolling circle amplification (RCA), strand-displacement amplification (SDA), Nicking Enzyme Amplification Reaction (NEAR), exponential amplification reaction (EXPAR), enzymatic oligonucleotide synthesis via internal inosine modification and exonuclease V, nick translation, Loop-Mediated Isothermal Amplification (LAMP). Helicase-Dependent Amplification (HD A), Multiple Displacement Amplification (MDA), Ligase Chain Reaction (LCR), Recombinase Polymerase Amplification (RPA), Ramification amplification method (RAM), Nucleic Acid Sequence-Based Amplification (NASBA), signal amplification by exchange reaction (SABER), or primer exchange reaction (PER).

6. The method of claim 1, further comprising generating the concatemer by rolling circle amplification (RCA).

7. The method of claim 1, wherein the concatemer comprises at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000. at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000 copies of the first docking oligonucleotide.

8. The method of claim 1, wherein the barcode sequence is from about 3 to about 30 nucleotides.

9. The method of claim 1, wherein the first docking oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, or more than 1000 nucleotides.

10. The method of claim 1. wherein the cleaving oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, from about 1000 to about 1500, from about 1500 to about 2000, from about 2000 to about 2500, from about 2500 to about 3000, from about 3500 to about 4000, or more than 4000 nucleotides.

11. The method of claim 1. wherein the photocrosslinking agent is located from about 2 to about 12 bases from the restriction site.

12. The method of claim 11, wherein the photocrosslinking agent is located about 4 bases from the restriction site.

13. The method of claim 1. wherein the restriction site is from about 5 to about 20 bases from the 5’ end of the cleaving oligonucleotide.

14. The method of claim 13, wherein the restriction site is about 12 bases from the 5’ end of the cleaving oligonucleotide.

15. The method of claim 1. wherein the immobilizing is by affinity binding, conjugation, incorporation into a hydrogel, crosslinking, or photo-crosslinking.

16. The method of claim 15, wherein the immobilizing is at random locations throughout the biological sample.

17. The method of claim 15, wherein the immobilizing is at specific targets in the biological sample.

18. The method of claim 1, wherein the cleaving oligonucleotide further comprises a modification.

19. The method of claim 18, wherein the modification comprises a photolinker. a fluorophore, a photo-cleavable spacer, a protein, a small molecule, a lipid, a nanoparticle, a lipid nanoparticle, or any combination thereof.

20. The method of claim 18, wherein the modification comprises a modification to affect tethering, diffusion, or localization of an oligonucleotide.

21. The method of claim 20, wherein the modification to affect tethering of an oligonucleotide comprises a base modified with an amino group, acrylic phosphoramidite, an alky ne, an azido, chloroacetamide, vinylsulfonamide, an aliphatic aldehyde, or any combination thereof.

22. The method of claim 20, wherein the modification to affect diffusion comprises conjugation with additional nucleic acid strands, peptide, dextran, a polyethylene glycol (PEG), a short strand polymer, or any combination thereof.

23. The method of claim 22, wherein the short strand polymer comprises a poly (glycerol) (PG), poly(oxazoline) (POX), poly(hydroxypropyl methacrylate) (PGPMA), poly(2- hydroxyethyl methacrylate) (PHEMA), poly(A-(2- hydroxy propyl) methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), or any combination thereof.

24. The method of claim 20, wherein the modification to affect localization comprises a hybridization sequence directed to endogenous RNA, DNA. or cDNA.

25. The method of claim 20, wherein the modification to affect localization comprises a conjugation to a biotin, a protein, an antibody or fragment thereof, a nanobody, a lipid, a nanoparticle, a lipid nanoparticle, other affinity' reagent, or any combination thereof.

26. The method of any one of claims 1 to 25, wherein the cleaving oligonucleotide further comprises a primer at the 3’ end of the cleaving oligonucleotide.

27. The method of claim 26, further comprising hybridizing the primer to a complementary' domain on an mRNA in the sample.

28. The method of claim 27, further comprising contacting the primer with a reverse transcriptase, thereby extending the primer to generate a complementary copy of a region of the mRNA.

29. The method of claim 27, wherein the hybridizing step occurs prior to the illuminating step.

30. The method of claim 27, wherein the hybridizing step occurs after the illuminating step.

31. The method of claim 26, further comprising hybridizing the primer to a second docking oligonucleotide, wherein the second docking oligonucleotide comprises a barcode sequence.

32. The method of claim 31, further comprising contacting the primer with a polymerase, thereby extending the primer to generate a duplexed region comprising a region of the cleaving oligonucleotide and the second docking oligonucleotide.

33. The method of claim 32, wherein the duplexed region further comprises a crosslinking agent.

34. The method of claim 33, further comprising activating the crosslinking agent to generate a covalently bound duplex.

35. The method of claim 34, wherein the activating is in the illuminating step.

36. The method of claim 34, wherein the activating is in a second illuminating step.

37. The method of any one of claims 1 to 36, further comprising controlling a diffusion factor of the sample, wherein the diffusion factor comprises a viscosity, a time, a temperature, a presence of crow ding agents, a pH, an electric field, physical features, or any combination thereof.

38. The method of claim 37, wherein the viscosity is greater than 1 cP.

39. The method of claim 38, wherein the viscosity is from about 1 to about 10 cP, from about10 to about 20 cP, from about 20 to about 30 cP, from about 30 to about 40 cP, from about40 to about 50 cP, from about 50 to about 60 cP, from about 60 to about 70 cP, from about70 to about 80 cP, from about 80 to about 90 cP, from about 100 to about 150 cP, from about 150 to about 300 cP, from about 300 to about 500 cP, from about 500 to about 1000 cP, from about 1000 to about 2000 cP, from about 2000 to about 3000 cP.

40. The method of any one of claims 1 to 39, wherein the cleaving oligonucleotide further comprises one or more unique molecular modifiers (UMI).

41. The method of claim 40, wherein the UMI comprises one or more noncanonical bases, universal bases, other bases which a polymerase will pair randomly or incorrectly, or any combination thereof.

42. The method of any one of claims 1 to 41, wherein the endonuclease is a Type I. Type II, Type IIS, Type IIG, Type III, Type IV, or Type V endonuclease, or a Nickase.

43. The method of claim 42, wherein the endonuclease comprises BtsI, Nb.BtsI, BbvCI, EcoRI, EcoRII, BamHI, Hindlll, TaqI, Notl, HinFI, Sau3AI, PvuII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I. Kpnl , PstI, SacI, Sall, Seal , Spel, SphI, StuE Xbal, Alwl, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, BsmI, Nb.BsmI, BsmAI, Nt.BsmAI, BssSI, Nb.BssSI, Nt.BstNBI, BtsI, Nb.BtsI, or any combination thereof.

44. The method of any one of claims 1 to 41, wherein the endonuclease is a Cas enzyme.

45. The method of claim 44, wherein the Cas enzyme comprises a Type I. Type II, Type III, or Type IV Cas endonuclease.

46. The method of claim 44, wherein the Cas enzyme is a Cas9 enzyme.

47. The method of claim 44, further providing a guide RNA specific for a sequence on the docking oligonucleotide or the cleaving oligonucleotide.

48. The method of any one of claims 1 to 47, wherein the contacting the biological sample with an endonuclease step is before the illuminating step.

49. The method of any one of claims 1 to 47, wherein the contacting the biological sample with at least one cleaving oligonucleotide comprises contacting the biological sample with more than one cleaving oligonucleotide, wherein each cleaving oligonucleotide comprises a region complementary to a different region on the docking oligonucleotide.

50. The method of any one of claims 1 to 47, wherein the photocrosslinking agent comprises a photoreactive nucleobase.

51. The method of claim 50, wherein the photoreactive nucleobase comprises 3- cyanovinylcarbazole phosphoramidite (CNVK) or 5-phenylethynyl-2'-deoxyuridine (phedU).

52. The method of any one of claims 1 to 51, wherein the polymerizing comprises depositing a DNA polymerase or an RNA polymerase on the sample.

53. The method of claim 52, wherein the DNA polymerase comprises a Phi29 DNA Pol, an E. coli Pol I, an E. coli Pol II, an E. coli Pol III, an E. coli Pol IV, an E. coli Pol V, a T4 DNA Pol, a Bsm DNA Pol I, a Bst DNA Pol, a Reverse Transcriptase, or any combination thereof.

54. The method of claim 52, wherein the RNA polymerase comprises T7, T3. SP6, or any combination thereof.

55. The method of any one of claims 1 to 54, further comprising analyzing a frequency of association of barcode sequence and cleaving nucleic acid to generate spatial mapping of the biological sample.

56. A method of biological information generation, the method comprising: a) immobilizing a first docking oligonucleotide template onto a biological sample, wherein the first docking oligonucleotide template comprises: a barcode sequence; and a flanking region. b) amplifying the first docking oligonucleotide template to generate a concatemer, wherein the concatemer comprises two or more copies of a first docking oligonucleotide; c) contacting the biological sample with at least one cleaving oligonucleotide, wherein the cleaving oligonucleotide comprises a region complementary to a region on the first docking oligonucleotide, wherein the first docking oligonucleotide and the cleaving oligonucleotide comprise at least one of each independently: a photocrosslinking agent, and at least one restriction site; d) illuminating predefined regions of the sample to activate the photocrosslinking agent, thereby binding the first docking oligonucleotide and the cleaving oligonucleotide to generate a photocrosslinked oligonucleotide complex; e) contacting the photocrosslinked oligonucleotide complex with an endonuclease, wherein the endonuclease cleaves the photocrosslinked oligonucleotide complex at one of the at least one restriction site, thereby generating a duplexed fragment of the photocrosslinked oligonucleotide complex, and wherein the duplexed fragment comprises the barcode sequence; f) polymerizing the duplexed fragment using cross-junction synthesis to generate a contiguous nucleic acid comprising copy of a region from each of the first docking oligonucleotide comprising the barcode sequence and the cleaving oligonucleotide; g) sequencing the contiguous nucleic acid, wherein an association of barcode sequence and cleaving oligonucleotide provides biological information.

57. The method of claim 56, wherein the biological sample comprises a population of cells, a tissue sample, or a protein.

58. The method of claim 57, wherein the biological sample is fixed on a surface.

59. The method of claim 56, wherein the docking oligonucleotide and the cleaving oligonucleotide each independently comprise ribonucleotides, deoxyribonucleotides, or any variation or combination thereof.

60. The method of claim 56, further comprising generating the concatemer by rolling circle amplification (RCA), strand-displacement amplification (SDA). Nicking EnzymeAmplification Reaction (NEAR), exponential amplification reaction (EXPAR), enzymatic oligonucleotide synthesis via internal inosine modification and exonuclease V, nick translation, Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HD A), Multiple Displacement Amplification (MDA), Ligase Chain Reaction (LCR), Recombinase Polymerase Amplification (RPA), Ramification amplification method (RAM), Nucleic Acid Sequence-Based Amplification (NASBA), signal amplification by exchange reaction (SABER), or primer exchange reaction (PER).

61. The method of claim 56, further comprising generating the concatemer by rolling circle amplification (RCA).

62. The method of claim , wherein the concatemer comprises at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000. at least 10,000 copies of the first docking oligonucleotide.

63. The method of claim 56, wherein the first docking oligonucleotide is immobilized to a splint sequence in a sample.

64. The method of claim 63, wherein the splint sequence is a region of genomic DNA, mRNA. cDNA. or other nucleic acid in a sample.

65. The method of claim 56, wherein the barcode sequence is from about 3 to about 30 nucleotides.

66. The method of claim 56, wherein the first docking oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, or more than 1000 nucleotides.

67. The method of claim 56, wherein the cleaving oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, from about 1000 to about 1500, from about 1500 to about 2000. from about 2000 to about 2500, from about 2500 to about 3000, from about 3500 to about 4000, or more than 4000 nucleotides.

68. The method of claim 56, wherein the photocrosslinking agent is located from about 2 to about 12 bases from the restriction site.

69. The method of claim 68, wherein the photocrosslinking agent is located about 4 bases from the restriction site.

70. The method of claim 56, wherein the restriction site is from about 5 to about 20 bases from the 5’ end of the cleaving oligonucleotide.

71. The method of claim 70, wherein the restriction site is about 12 bases from the 5’ end of the cleaving oligonucleotide.

72. The method of any one of claims 56 to 71, wherein the immobilizing is by an affinity binding, conjugation, incorporation into a hydrogel, crosslinking, or photocrosslinking.

73. The method of claim 72, wherein the immobilizing is at random locations throughout the biological sample.

74. The method of claim 72, wherein the immobilizing is at specific targets in the biological sample.

75. The method of any one of claims 56. wherein the cleaving oligonucleotide further comprises a modification.

76. The method of claim 75, wherein the modification comprises a photolinker, a fluorophore, a photo-cleavable spacer, a protein, a small molecule, a lipid, a nanoparticle, a lipid nanoparticle, or any combination thereof.

77. The method of claim 75, wherein the modification comprises a modification to affect tethering, diffusion, or localization of an oligonucleotide.

78. The method of claim 77, wherein the modification to affect tethering of an oligonucleotide comprises a base modified with an amino group, acrylic phosphoramidite. an alkyne, an azido, chloroacetamide, vinylsulfonamide, an aliphatic aldehyde, or any combination thereof.

79. The method of claim 77, wherein the modification to affect diffusion comprises conjugation with additional nucleic acid strands, peptide, dextran, a polyethylene glycol (PEG), a short strand polymer, or any combination thereof.

80. The method of claim 79, wherein the short strand polymer comprises a poly(glycerol) (PG), poly(oxazoline) (POX), poly(hydroxypropyl methacrylate) (PGPMA), poly (2- hydroxyethyl methacrylate) (PHEMA), poly(JV-(2- hydroxy propyl) methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poty(N-acryloylmorpholine) (PAcM), or any combination thereof.

81. The method of claim 77, wherein the modification to affect localization comprises a hybridization sequence directed to endogenous RNA, DNA, or cDNA.

82. The method of claim 77, wherein the modification to affect localization comprises a conjugation to a biotin, a protein, an antibody or fragment thereof, a nanobody, a lipid, a nanoparticle, a lipid nanoparticle, other affinity reagent, or any combination thereof.

83. The method of any one of claims 56 to 82, wherein the cleaving oligonucleotide further comprises a primer at the 3’ end of the cleaving oligonucleotide.

84. The method of claim 83, further comprising hybridizing the primer to a complementary domain on an mRNA in the sample.

85. The method of claim 84, further comprising contacting the primer with a reverse transcriptase, thereby extending the primer to generate a complementary' copy of a region of the mRNA.

86. The method of claim 84, wherein the hybridizing step occurs prior to the illuminating step.

87. The method of claim 84, wherein the hybridizing step occurs after the illuminating step.

88. The method of claim 83, further comprising hybridizing the primer to a second docking oligonucleotide, wherein the second docking oligonucleotide comprises a barcode sequence.

89. The method of claim 88, further comprising contacting the primer with a polymerase, thereby extending the primer to generate a duplexed region comprising a region of the cleaving oligonucleotide and the second docking oligonucleotide.

90. The method of claim 89, wherein the duplexed region further comprises a crosslinking agent.

91. The method of claim 90, further comprising activating the crosslinking agent to generate a covalently’ bound duplex.

92. The method of claim 91, wherein the activating is in the illuminating step.

93. The method of claim 91, wherein the activating is in a second illuminating step.

94. The method of any one of claims 56 to 93, further comprising controlling a diffusion factor of the sample, wherein the diffusion factor comprises a viscosity, a time, a temperature, a presence of crowding agents, a pH. an electric field, physical features, or any combination thereof.

95. The method of claim , wherein the viscosity is greater than 1 cP.

96. The method of claim 95, wherein the viscosity is from about 1 to about 10 cP. from about 10 to about 20 cP, from about 20 to about 30 cP, from about 30 to about 40 cP, from about 40 to about 50 cP, from about 50 to about 60 cP, from about 60 to about 70 cP, from about 70 to about 80 cP, from about 80 to about 90 cP, from about 100 to about 150 cP, from about 150 to about 300 cP, from about 300 to about 500 cP, from about 500 to about 1000 cP, from about 1000 to about 2000 cP, from about 2000 to about 3000 cP.

97. The method of any one of claims 56 to 96. wherein the cleaving oligonucleotide further comprises one or more unique molecular modifiers (UMI).

98. The method of claim 97, wherein the UMI comprises one or more noncanonical bases, universal bases, other bases which a polymerase will pair randomly or incorrectly, or any combination thereof.

99. The method of any one of claims 56 to 98, wherein the endonuclease is a Type I, Type II, Type IIS, Type IIG. Type III, Type IV, or Type V endonuclease, or a Nickase.

100. The method of claim 99, wherein the endonuclease comprises BtsI, Nb.BtsI, BbvCI, EcoRI, EcoRII, BamHI, Hindlll, TaqI, Notl, HinFI, Sau3AI, PvuII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I, Kpnl , PstI, SacI, Sall, Seal , Spel, SphI, Stul, Xbal, Alwl, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, BsmI, Nb.BsmI. BsmAI, Nt.BsmAI, BssSE Nb.BssSI, Nt.BstNBI, BtsI, Nb.BtsI, or any combination thereof.

101. The method of any one of claims 56 to 98, wherein the endonuclease is a Cas enzyme.

102. The method of claim 101, wherein the Cas enzyme comprises a Type I, Type II, Type III, or Type IV Cas endonuclease.

103. The method of claim 101, wherein the Cas enzyme is a Cas9 enzyme.

104. The method of any one of claims 101 to 103, further providing a guide RNA specific for a sequence on the docking oligonucleotide or the cleaving oligonucleotide.

105. The method of any one of claims 56 to 104, wherein the contacting the biological sample with an endonuclease step is before the illuminating step.

106. The method of any one of claims 56 to 105, wherein the contacting the biological sample with at least one cleaving oligonucleotide comprises contacting the biological sample with more than one cleaving oligonucleotide, wherein each cleaving oligonucleotide comprises a region complementary to a different region on the docking oligonucleotide.

107. The method of any one of claims 56 to 106, wherein the photocrosslinking agent comprises a photoreactive nucleobase.

108. The method of claim 107, wherein the photoreactive nucleobase comprises 3- cyanovinylcarbazole phosphoramidite (CNVK) or 5-phenylethynyl-2'-deoxyuridine (phedU).

109. The method of any one of claims 56 to 108, wherein the polymerizing comprises adding a DNA polymerase or an RNA polymerase to the sample.

110. The method of claim 109, wherein the DNA polymerase comprises a Phi29 DNA Pol, an E. coli Pol I, an E. coli Pol II, an E. coll Pol III, an E. coli Pol IV, an E. coli Pol V, a T4 DNA Pol. a Bsm DNA Pol I. a Bst DNA Pol, a Reverse Transcriptase, or any combination thereof.

111. The method of claim 109, wherein the RNA polymerase comprises T7, T3, SP6, or any combination thereof.

112. The method of any one of claims 56 to 111, further comprising analyzing a frequency of association of barcode sequence and cleaving nucleic acid to generate spatial mapping of the biological sample.

113. A composition, the composition comprising: a) a concatemer. wherein the concatemer is a nucleic acid and comprises two or more copies of a first docking oligonucleotide comprising: a barcode sequence; and a flanking region, b) at least one cleaving oligonucleotide, wherein the cleaving oligonucleotide comprises a region complementary to a region on the first docking oligonucleotide, wherein the first docking oligonucleotide and the cleaving oligonucleotide comprise at least one of each independently: a photocrosslinking agent, and at least one restriction site; and c) an endonuclease, wherein the endonuclease recognizes the at least one restriction site.

114. The composition of claim 113, wherein the docking oligonucleotide and the cleaving oligonucleotide each independently comprise ribonucleotides, deoxyribonucleotides, or any variation or combination thereof.

115. The composition of claim 113, wherein the concatemer comprises at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000. at least 3000, at least 4000, at least 5000. at least 6000. at least 7000, at least 8000, at least 9000, at least 10,000 copies of the first docking oligonucleotide.

116. The composition of claim 113, wherein the barcode sequence is from about 3 to about 30 nucleotides.

117. The composition of claim 113, wherein the first docking oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, or more than 1000 nucleotides.

118. The composition of claim 113, wherein the cleaving oligonucleotide is from about 20 to about 50, from about 50 to about 100, from about 100 to about 200, from about 200 to about 500, from about 500 to about 1000, from about 1000 to about 1500, from about 1500 to about 2000, from about 2000 to about 2500, from about 2500 to about 3000, from about 3500 to about 4000, or more than 4000 nucleotides.

119. The composition of claim 113. wherein the photocrosslinking agent is located from about 2 to about 12 bases from the restriction site.

120. The composition of claim 119, wherein the photocrosslinking agent is located about 4 bases from the restriction site.

121. The composition of claim 113, wherein the restriction site is from about 5 to about 20 bases from the 5’ end of the cleaving oligonucleotide.

122. The composition of claim 121, wherein the restriction site is about 12 bases from the 5' end of the cleaving oligonucleotide.

123. The composition of claim 113, wherein the cleaving oligonucleotide further comprises a modification.

124. The composition of claim 123, wherein the modification comprises a photolinker, a fluorophore, a photo-cleavable spacer, a protein, a small molecule, a lipid, a nanoparticle, a lipid nanoparticle, or any combination thereof.

125. The composition of claim 123. wherein the modification comprises a modification to affect tethering, diffusion, or localization of an oligonucleotide.

126. The composition of claim 125, wherein the modification to affect tethering of an oligonucleotide comprises a base modified with an amino group, acrylic phosphoramidite. an alkyne, an azido, chloroacetamide, vinylsulfonamide, an aliphatic aldehyde, or any combination thereof.

127. The composition of claim 125, wherein the modification to affect diffusion comprises conjugation with additional nucleic acid strands, peptide, dextran, a polyethylene glycol (PEG), a short strand polymer, or any combination thereof.

128. The composition of claim 127. wherein the short strand polymer comprises a poly(glycerol) (PG), poly(oxazoline) (POX), poly(hydroxypropyl methacrylate) (PGP MA), poly(2-hydroxy ethyl methacrylate) (PHEMA), poly(A-(2- hydroxypropyl) methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), or any combination thereof.

129. The composition of claim 125, wherein the modification to affect localization comprises a hybridization sequence directed to endogenous RNA, DNA, or cDNA.

130. The composition of claim 125, wherein the modification to affect localization comprises a conjugation to a biotin, a protein, an antibody or fragment thereof, a nanobody, a lipid, a nanoparticle, a lipid nanoparticle, other affinity reagent, or any combination thereof.

131. The composition of any one of claims 113 to 130, wherein the cleaving oligonucleotide further comprises a primer at the 3’ end of the cleaving oligonucleotide.

132. The composition of claim 131. wherein the primer further comprises a photocrosslinking agent.

133. The composition of any one of claims 113 to 132, wherein the cleaving oligonucleotide further comprises one or more unique molecular modifiers (UMI).

134. The composition of claim 133, wherein the UMI comprises one or more noncanonical bases, universal bases, other bases which a polymerase will pair randomly or incorrectly, or any combination thereof.

135. The composition of any one of claims 113 to 134, wherein the endonuclease is a Type I, Type II. Type IIS, Type IIG, Type III, or Type IV endonuclease, Type V. or a Nickase.

136. The composition of claim 135, wherein the endonuclease comprises BtsL Nb.BtsI, BbvCI, EcoRI, EcoRII, BamHI, Hindlll, TaqI, Notl, HinFI, Sau3AI, PvuII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I, Kpnl , PstI, SacI, Sall, Seal , Spel, SphI, Stul, Xbal, Alwl, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, BsmI, Nb.BsmI. BsmAI, Nt.BsmAI, BssSI. Nb.BssSI, Nt.BstNBI. BtsI, Nb.BtsI, or any combination thereof.

137. The composition of any one of claims 113 to 134, wherein the endonuclease is a Cas enzyme.

138. The composition of claim 137, wherein the Cas enzyme comprises a Type I, Type II, Type III, or Type IV Cas endonuclease.

139. The composition of claim 137, wherein the Cas enzyme is a Cas9 enzyme.

140. The composition of any one of claims 137 to 139, further providing a guide RNA specific for a sequence on the docking oligonucleotide or the cleaving oligonucleotide.

141. The composition of any one of claims 113 to 140, wherein the photocrosslinking agent comprises a photoreactive nucleobase.

142. The composition of claim 141 , wherein the photoreactive nucleobase comprises 3- cyanovinylcarbazole phosphoramidite (CNVK) or 5-phenylethynyl-2'-deoxyuridine (phedU).

143. The composition of any of claims 113 to 142, further comprising a DNA polymerase or an RNA polymerase.

144. The composition of claim 143, wherein the DNA polymerase comprises a Phi29 DNA Pol, an E. coli Pol I, an E. coli Pol II, an E. coll Pol III, an E. coll Pol IV, an E. coll Pol V, a T4 DNA Pol, a Bsm DNA Pol I, a Bst DNA Pol, a Reverse Transcriptase, or any combination thereof.

145. The composition of claim 143, wherein the RNA polymerase comprises T7, T3, SP6, or any combination thereof.

146. A kit comprising the composition of any one of claims 113 to 145.

147. A library of nucleic acids comprising: a. a population of docking oligonucleotides, wherein the population of docking oligonucleotides comprises: i. a barcode sequence: and ii. a flanking region; and b. a population of cleaving oligonucleotides, wherein the population of cleaving oligonucleotides comprises at least one region complementary' to a region on a docking oligonucleotide in the population of docking oligonucleotides, wherein the population of docking oligonucleotides and the population of cleaving oligonucleotides comprise at least one independently: a photocrosslinking agent, and at least one restriction site.

148. The library of nucleic acids of claim 147, wherein the population of cleaving oligonucleotides further comprises a plurality of regions complementary to more than one docking oligonucleotide.

149. The library of nucleic acids of claims 147 or 148, wherein the population of cleaving oligonucleotides further comprises a 3’ primer.

150. The library of nucleic acids of claim 147, further comprising a population of concatemers, wherein each concatemer comprises at least two copies of a docking oligonucleotide of the population of docking oligonucleotides.

151. A library' of nucleic acids, wherein the library' of nucleic acids comprises duplexed fragments generated according to the method of any one of claims 1 to 112.

152. A library of nucleic acids, wherein the library of nucleic acids comprises contiguous nucleic acids generated according to the method of any one of claims 1 to 1 12.

Images