WO2022006042A1

WO2022006042A1 - Methods, compositions, and kits for nucleic acid barcoding of biomolecules

Info

Publication number: WO2022006042A1
Application number: PCT/US2021/039506
Authority: WO
Inventors: Sinem K. SAKA; Jocelyn Y. KISHI; Peng Yin
Original assignee: President And Fellows Of Harvard College
Priority date: 2020-06-29
Filing date: 2021-06-29
Publication date: 2022-01-06

Abstract

Provided herein are methods, compositions, and kits related to barcoding target binding molecules for diagnostics, treatments, and research applications. In some aspects, provided herein is a method for biomolecule tagging and barcoding.

Description

METHODS, COMPOSITIONS, AND KITS FOR NUCUEIC ACID BARCODING OF

BIOMOUECUUES

CROSS-REFERENCE TO REUATED APPUICATIONS [0001] This application claims benefit under 35 U.S.C. § 119(d) of the US Provisional Application No. 63/045,343 filed June 29, 2020, the contents of which are incorporated herein by reference in their entirety.

GOVERNTMENT SUPPORT

[0002] This invention was made with government support under N00014-18-1-2549 awarded by U.S. Department of Defense Office of Naval Research, and under HL145600, GM133052, and GM124401 awarded National Institutes of Health. The government has certain rights in the invention.

TECHNICAU FIEUD

[0003] The present disclosure relates to methods, compositions, and kits for nucleic acid barcoding.

BACKGROUND

[0004] Nucleic acid barcoding of molecular probes enables simultaneous multiplexed detection of biomolecules. The generation of the biomolecules or probes tagged with different sequences to create barcoded libraries constitutes a bottleneck step in barcoding biomolecules for research applications. Thus, there is an unmet need for methods, compositions, and kits to generate a new barcode from an existing nucleic acid moiety on a biomolecule.

SUMMARY

[0005] Provided herein are methods, compositions, and kits that can generate barcodes from existing nucleic acid moieties attached to target binding molecules for use in diagnostic and research applications.

[0006] In one aspect, provided herein is a method for extending a nucleic acid strand attached to a target binding molecule. Generally, the method comprises: (i) annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, extending the primer strand with a polymerase to produce a synthesized nucleic acid strand and removing the template strand; or (ii) ligating or crosslinking an extension nucleic acid strand to a primer strand, optionally using a template nucleic acid. [0007] In some embodiments of any one of the aspects, the method comprises: annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand; extending the primer nucleic acid strand from its 3 ’-end by a polymerase to synthesize a nucleic acid strand; and removing the template nucleic acid strand to produce a synthesized single-strand nucleic acid. Generally, the primer nucleic acid stand can be attached to a target binding molecule. The primer strand comprises a hybridization domain (a). The template nucleic acid strand comprises a hybridization domain (a*) and a second domain (b*), wherein the hybridization domain (a*) of the template strand is substantially complementary to the hybridization domain (a) of the primer strand. Optionally, the template nucleic acid strand comprises a linker domain between the hybridization domain and the second domain.

[0008] In some embodiments of any one of the aspects, the method comprises: annealing or hybridizing together a primer nucleic acid strand, an extension nucleic acid strand and a template nucleic acid strand; ligating an end of the primer nucleic acid strand with an end of the extension nucleic acid strand; and removing the template nucleic acid strand to produce a synthesized nucleic acid strand. The primer nucleic acid strand comprises a hybridization domain (a) and optionally a second domain. The primer strand can be linked to a target binding molecule. The extension nucleic acid strand comprises a hybridization domain (b), a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c). The template nucleic acid strand comprises a first hybridization domain (a*); and a second hybridization domain (b*), where the first hybridization domain (a*) of the template nucleic acid strand is substantially complementary to the hybridization domain (a) of the primer nucleic acid strand, and the is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand, and the second hybridization domain (b*) of the template nucleic acid strand is substantially complementary to the hybridization domain (b) of the extension nucleic acid strand.

[0009] In some embodiments of any of the aspects, the method comprises: linking a primer nucleic acid strand with an extension nucleic acid strand to produce a synthesized strand, and optionally, removing any excess extension strands. The primer stand is attached to a target binding molecule. The extension nucleic acid strand comprises a barcode sequence/domain and, optionally, a hybridization domain. At least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety, and/or one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[0010] In some embodiments of any one of the aspects, the method comprises: annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, extending the target nucleic acid strand from its 3 ’-end by a polymerase having strand displacing activity to produce a synthesized strand; and optionally, removing any excess template nucleic acid strand. The primer strand is attached to a target binding molecule. The primer strand comprises a hybridization domain (a). The template strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker domain between the first stem domain (b*) and the second stem domain (b). The first and second stem domains of the template strand are substantially complementary to each other and capable of forming a double-stranded structure and the hybridization domain (a*) of the template nucleic acid strand is substantially complementary to the hybridization domain (a) of the primer strand. At least one of the first stem domain, the second stem domain and the linker domain of the template strand comprises a modification capable of terminating nucleic acid polymerization. Optionally, the template strand is in form of a hairpin. [0011] In some embodiments of any of the aspects, the method comprises a step of isolating/purifying the target binding molecule bound to the template strand prior to removing the template strand. Such an isolating/purifying step can comprise affinity purification using a ligand attached to the template strand. Further, the isolating/purifying step can be done prior to the step of removing the template strand.

[0012] In another aspect, provided herein is a composition. Generally, the composition comprises a primer nucleic acid strand, a template nucleic acid strand, and optionally an extension nucleic acid strand. The composition can be useful in the methods described herein.

[0013] In some embodiments of any of the aspects, the composition comprises: a primer nucleic acid strand attached to a target binding molecule and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain is at the 3 ’-end of the primer strand. The template strand comprises a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain. The hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand. Optionally, neither the primer strand nor the template strand comprises a hairpin structure.

[0014] In some embodiments of any one of the aspects, the composition comprises a primer nucleic acid stand attached to a target binding molecule, an extension nucleic acid strand, and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain is at a terminus of the primer strand. The extension strand comprises a hybridization domain (b), a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c). The template strand comprises a first hybridization domain (a*); and a second hybridization domain (b*). The hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand. The hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the second hybridization domain (b*) of the template nucleic acid strand.

[0015] In some embodiments of any one of the aspects, the composition comprises: a primer nucleic acid stand is attached to a target binding molecule and an extension nucleic acid strand comprising a barcode sequence/domain and, optionally, a hybridization domain. At least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety, and/or one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[0016] In some embodiments of any one of the aspects, the composition comprises: a primer nucleic acid strand attached to a target binding molecule, and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain (a) is at 3 ’-terminus of the primer strand. The template strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker domain between the first stem domain and the second stem domain, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure. At least one of the first stem domain (b*), the second stem domain (b) and the linker domain of the template strand comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure. The hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand. Optionally, the template strand is in form of a hairpin.

[0017] In yet another aspect, provided herein is a kit. Generally, the kit comprises a primer nucleic acid strand, a template nucleic acid strand, and optionally an extension nucleic acid strand. The kit can be useful in the methods described herein.

[0018] In some embodiments of any of the aspects, the kit comprises: a primer nucleic acid strand attached to a target binding molecule and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain is at the 3 ’-end of the primer strand. The template strand comprises a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain. The hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand. Optionally, neither the primer strand nor the template strand comprises a hairpin structure. [0019] In some embodiments of any one of the aspects, the kit comprises a primer nucleic acid stand attached to a target binding molecule, an extension nucleic acid strand, and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain is at a terminus of the primer strand. The extension strand comprises a hybridization domain (b), a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c). The template strand comprises a first hybridization domain (a*); and a second hybridization domain (b*). The hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand. The hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the second hybridization domain (b*) of the template nucleic acid strand.

[0020] In some embodiments of any one of the aspects, the kit comprises: a primer nucleic acid stand is attached to a target binding molecule and an extension nucleic acid strand comprising a barcode sequence/domain and, optionally, a hybridization domain. At least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety, and/or one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[0021] In some embodiments of any one of the aspects, the kit comprises: a primer nucleic acid strand attached to a target binding molecule, and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain (a) is at 3’- terminus of the primer strand. The template strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker domain between the first stem domain and the second stem domain, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure. At least one of the first stem domain (b*), the second stem domain (b) and the linker domain of the template strand comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure. The hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand. Optionally, the template strand is in form of a hairpin.

BRIEF DESCRIPTION OF THE DRAWINGS [0022] FIG. 1 is a schematic representation of swapping the sequence X on biomolecules with an X, Y sequence. An antibody, a protein and a lipid are shown here as example of biomolecules that can be barcoded with nucleic acids. [0023] FIGS. 2A-2E are schematic representatins of barcode remapping according to some exemplary embodiments. Employing the methods detailed here (FIG. 2A) the original barcode, X, on the biomolecule, such as an antibody, can be remapped to a (FIG. 2B) single-stranded XY sequence, (FIG. 2C) a double-stranded XY sequence, (FIG. 2D) a partially double stranded XY sequence with a 5’ overhang, (FIG. 2E) a partially double stranded sequence where a Y domain on a new strand via hybridization, crosslinking or photocrosslinking to the original X strand on the biomolecule.

[0024] FIGS. 3A-3H are schematic representations of the reactions according to exemplary embodiments for creating new DNA barcodes on biomolecules. Here shown for an antibody that has a universal DNA barcode a, which can be directly conjugated to the biomolecule to a functional group (such as azide, alkyne, tetrazine, DBCO, maleimide, next generation maleimide, carbomide, NHS-ester, sulfo-NHS, thiol, amine, carbonyl, carboxyl and others) or indirectly, for example via a linker sequence (as shown in light grey), a hapten, another biomolecule, an affinity binder or a DNA-labeled tag. The original barcode a (also referred as a primer) on the biomolecule (here depicted as an antibody) can be converted into a new DNA barcode via several exemplary approaches:

[0025] a Primer exchange reaction ((FIG. 3A), extension of the original DNA barcode via polymerase and exonuclease activity (FIG. 3B), extension of the original DNA barcode via polymerase using a DNA template with modified bases (FIG. 3C), extension of the original DNA barcode using an RNA template via polymerase and RNAse H (FIG. 3D), barcode swap by photocrosslinking using a modified residue marked by the grey circle (FIG. 3E, the modified base can be incorporated in the template strand or in both the primer and template strand), barcode swap by crosslinking (FIG. 3F, functional groups can be placed internally or as end modifications), barcode swap by binding of paired binders on the original and new barcode sequence (FIG. 3G) and new barcode generation by ligation (FIG. 3H, the ligation can be performed in a non-templated or templated manner).

[0026] FIGs. 4A and 4B are schematic representation of shows single hairpin (FIG. 4A) and multiple hairpin/ repeated extension (FIG. 4B) of the original barcode for barcode swapping according to some exemplary embodiments.

[0027] FIG. 4C shows the barcode swap on an Antibody (IgG) based on band shift on a polyacrylamide gel (PAGE) by electrophoresis. The 9-nt original barcode on the antibody was swapped either to a 19 nucleotide barcode by hairpin-templated extension via Primer Exchange reaction, as shown in panel a, or is extended to a long concatemer of several b units by use of a 2 hairpin system as shown in panel b. Reaction (rxn) conditions are noted above the lanes. The rightmost 4 lanes show the antibody run under reducing conditions to see the band shift more clearly via the smaller size due to separation of the heavy chain of the antibody. Red boxes indicate the shifted antibody band that carries the new barcode.

[0028] FIGS. 5A and 5B are schematic representation of DNA-templated (FIG. 5A) or RNA- templated (FIG. 5B) barcode swap according to some exemplary embodiments.

[0029] FIG. 5C shows the barcode swap on an Antibody (IgG) based on band shift on a polyacrylamide gel (PAGE) by electrophoresis. The 9-nt original barcode on the antibody is swapped either to a new 30 nucleotide barcode by DNA-templated or RNA templated extension. Reaction (rxn) conditions are noted above the lanes. The rightmost 3 lanes show the antibody run under reducing conditions to see the band shift more clearly via the smaller size due to separation of the heavy chain of the antibody. Red boxes indicate the shifted antibody band that carries the new barcode.

[0030] FIG. 6 shows testing of different reaction conditions (buffer type, polymerase concentration, incubation time, sequential vs. simultaneous clean-up) for DNA or RNA templated barcode swapping and nuclease based clean-up for single-stranded barcode generation. Reaction (rxn) conditions were as follows: Lane 1: Protein ladder, Lane 2: Antibody in PBS, Lane 3, Just antibody in PBS, Lane 4: DNA-templatated barcodes synthesis (3 min at 37°C, 2400 units polymerase in buffer with bovine serum albumin (BSA)), Lane 5: DNA-templatated barcodes synthesis (3 min at 37°C, 1200 units polymerase in buffer with BSA), Lane 6: DNA-templatated barcodes synthesis (5 min at 37°C, 1200 units polymerase in buffer with BSA), Lane 7: DNA- templated barcode synthesis and simultaneous Lambda exonuclease clean-up (10 min at 37°C, 1200 units polymerase in buffer with BSA), Lane 8: DNA-templated barcode synthesis and simultaneous Lambda exonuclease clean-up (10 min at 37°C, 1200 units polymerase in buffer without BSA), Lane 9: DNA templated synthesis (10 min 37°C, 1200 units polymerase) + sequential Lambda exonuclease cleanup (30 min at 37°C) in buffer with BSA, and Lane 10: RNA templated synthesis (10 min 37°C, 1200 units polymerase) + sequential RNase H cleanup (30 min at 37°C) in buffer with BSA. The boxes indicate the shifted antibody band that carries the new barcode.

[0031] FIGS. 7A and 7B are schematic representation of barcode swapping integrated with one step purification of ssDNA barcode swapped antibodies according to some exemplary embodiments.

[0032] FIG. 7C shows the barcode swap in FIGS. 7A and 7B and band shifts on a polyacrylamide gel (PAGE) by electrophoresis. Boxes indicate the shifted antibody band that carries the new barcode. Fraction A: collected after polymerase step; Fraction B: collected after exonuclease step; Fraction C: total elution from the beads; New bar code: 30 nucleotide added based oin DNA template; Co-incubation: lstep polymerase + exonuclease at 37°C. DETAILED DESCRIPTION

[0001] The fundamental strategy for nucleic acid barcoding of a target binding molecule provided herein is depicted in FIGS. 1-3H.

[0033] Generally, the methods, compositions, and kits provided herein relate, in part, to the discovery that a single-stranded nucleic acid sequence can be appended to a target binding molecule by enzymatic synthesis using a template nucleic acid. Accordingly, the methods, compositions, and kits provided herein allow for high-throughput detection of a target molecule and the production of sequence and spatial information. Specifically, the template nucleic acid strand does not become a part of the barcoded biomolecular complex as it either dissociates from the newly synthesized barcode via branch migration and toehold mediated displacement or is cleaved via the action of a nuclease that can be added after the synthesis or can be included in the synthesis reaction. The methods and compositions provided herein are useful in many applications, such diagnostics, pathology, and basic research.

[0034] In some embodiments of any one of the aspects, the method comprises: (a) annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, wherein the primer nucleic acid stand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises a hybridization domain (a), wherein the template nucleic acid strand comprises a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain, wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand; (b) extending the primer nucleic acid strand from its 3 ’ -end by a polymerase to synthesize a nucleic acid strand; and (c) removing the template nucleic acid strand to produce a synthesized single-strand nucleic acid, and wherein the synthesized strand is attached to the target binding molecule.

[0035] In some embodiments of any one of the aspects, the method comprises: (a) annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, wherein the primer nucleic acid stand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises a hybridization domain (a), wherein the template nucleic acid strand comprises a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain, wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand; (b) extending the primer nucleic acid strand from its 3 ’ -end by a polymerase to synthesize a nucleic acid strand; (c) optionally, removing the template strand; (d) annealing or hybridizing the synthesized strand from with a new template nucleic acid strand, wherein the new template strand comprises a hybridization domain (al*), a second domain (bl*), and optionally a linker domain between the hybridization domain and the second domain, wherein the hybridization domain (al*), of the new template strand comprises a nucleotide sequence having substantial identity to the second domain (b*) of the previous template strand; (e) extending the synthesized nucleic acid strand from its 3 ’-end by a polymerase to synthesize a new nucleic acid strand; (f) optionally, removing the template nucleic acid strand to produce a synthesized single-strand nucleic acid; (g) repeating steps (d)-(f); and (h) removing the template nucleic acid strand to produce a synthesized single-strand nucleic acid, and wherein the synthesized strand is attached to the target binding molecule.

[0036] In some embodiments of any one of the aspects, the method comprises: (a) annealing or hybridizing together a primer nucleic acid strand, an extension nucleic acid strand and a template nucleic acid strand, wherein the primer nucleic acid stand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises at one end a hybridization domain (a), wherein the extension nucleic acid strand comprises a hybridization domain (b), wherein the template nucleic acid strand comprises a first hybridization domain (a*), and a second hybridization domain (b*), optionally linked together via a linker domain, wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand, and wherein the hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the second hybridization domain (b*) of the template nucleic acid strand; (b) ligating an end of the primer nucleic acid strand with an end of the extension nucleic acid strand; and (c) removing the template nucleic acid strand to produce a synthesized single-strand nucleic acid, and wherein the synthesized strand is attached to the target binding molecule.

[0037] In some embodiments of any one of the aspects, the method comprises: (a) annealing or hybridizing together a primer nucleic acid strand, an extension nucleic acid strand and a template nucleic acid strand, wherein the primer nucleic acid stand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises at one end a hybridization domain (a), wherein the extension nucleic acid strand comprises a hybridization domain (b), wherein the template nucleic acid strand comprises a first hybridization domain (a*), and a second hybridization domain (b*), optionally linked together via a linker domain, wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand, and wherein the hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the second hybridization domain (b*) of the template nucleic acid strand; (b) ligating an end of the primer nucleic acid strand with an end of the extension nucleic acid strand; (c) optionally, removing the template strand; (d) annealing or hybridizing together the synthesized strand, a new extension nucleic acid strand and a new template nucleic acid strand, wherein the new extension nucleic acid strand comprises a hybridization domain (bl), a second domain (cl), and optionally a linker domain linking the hybridization domain (bl) and the second domain (cl), wherein the new template nucleic acid strand comprises a first hybridization domain (al*) and a second hybridization domain (bl*), optionally linked together via a linker domain, wherein the first hybridization domain (al*) of the template new nucleic acid strand is substantially complementary to the second domain of the previous extension nucleic acid strand, and wherein the hybridization domain (bl) of the new extension nucleic acid strand is substantially complementary to the second hybridization domain (bl *) of the new template nucleic acid strand; (e) ligating an end of the primer nucleic acid strand with an end of the extension nucleic acid strand; (f) optionally, removing the template nucleic acid strand; (g) optionally repeating steps (d) - (f); and (h) removing the template nucleic acid strands to produce a synthesized single-strand nucleic acid, and wherein the synthesized strand is attached to the target binding molecule.

[0038] In some embodiments of any one of the aspects, the method comprises: (a) linking a primer nucleic acid strand with an extension nucleic acid strand to produce a synthesized strand, wherein the primer stand is attached to a target binding molecule, wherein the extension nucleic acid strand comprises a barcode sequence/domain and, optionally, a hybridization domain, and wherein: (i) at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety; and/or (ii) one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair; and (b) optionally, removing any excess extension strands, and wherein the synthesized strand is attached to the target binding molecule.

[0039] In some embodiments of any one of the aspects, the method comprises: (a) linking a primer nucleic acid strand with an extension nucleic acid strand to produce a synthesized strand, wherein the primer stand is attached to a target binding molecule, wherein the extension nucleic acid strand comprises a barcode sequence/domain and, optionally, a hybridization domain, and wherein: (i) at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety; and/or (ii) one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair; (b) optionally, removing any excess extension strands, and wherein the synthesized strand is attached to the target binding molecule; (d) linking the synthesized strand with a new extension nucleic acid strand to produce a new synthesized strand, wherein the new extension nucleic acid strand comprises a barcode sequence/domain and, optionally, a hybridization domain; (e) optionally, removing any excess extension strands; and (f) optionally, repeating steps (d) and (e). [0040] In some embodiments of any one of the aspects, the method comprises: (a) annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, wherein the primer strand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises a hybridization domain (a), wherein the template nucleic acid strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker domain between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker domain comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, wherein the hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand; (b) extending the target nucleic acid strand from its 3 ’-end by a polymerase having strand displacing activity to produce a synthesized strand; and (c) optionally, removing any excess template nucleic acid strand, and wherein the synthesized strand is attached to the target binding molecule.

[0041] In some embodiments of any one of the aspects, the method comprises: (a) annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, wherein the primer strand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises a hybridization domain (a), wherein the template nucleic acid strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker domain between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker domain comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, wherein the hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand; (b) extending the target nucleic acid strand from its 3 ’-end by a polymerase having strand displacing activity to produce a synthesized strand; and (c) optionally, removing any excess template nucleic acid strand, and wherein the synthesized strand is attached to the target binding molecule.

[0042] In some embodiments of any one of the aspects, the method comprises: (a) annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, wherein the primer strand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises a hybridization domain (a), wherein the template nucleic acid strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker domain between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker domain comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, wherein the hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand; (b) extending the target nucleic acid strand from its 3 ’-end by a polymerase having strand displacing activity to produce a synthesized strand; (c) optionally, removing any excess template nucleic acid strand; (d) annealing or hybridizing synthesized strand with a new template nucleic acid strands, wherein the new template nucleic acid strand comprises in 3’ to 5’ direction: a hybridization domain (al*), a first stem domain (bl*), a second stem domain (bl), and a linker domain between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker domain comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, wherein the hybridization domain (al*) of the new template nucleic acid strand comprises a sequence having substantial identity to first stem domain of the previous template strand; (e) extending the target nucleic acid strand from its 3 ’-end by a polymerase having strand displacing activity to produce a synthesized strand; (f) optionally, removing any excess template strands; and (g) optionally, repeating steps (d) - (f).

Primer Strand

[0043] The methods, compositions, and kits provided herein include a primer nucleic acid strand, also referred to as a primer strand herein. Generally, the primer strand is attached to target binding molecule. It is noted that the primer strand can attached to the target binding molecule at any position of the primer strand. For example, the primer strand can be attached to the target binding molecule by its 5 ’-end, 3 ’-end or at an internal position. In some embodiments of any one of the aspects, the primer strand is attached to the target binding molecule by its 5 ’-end.

[0044] Further, the primer strand can be attached to the target binding molecule covalently or non-covalently. For example, the primer strand can be attached to the target binding molecule covalently by a direct bond or linker. Alternatively, the primer strand can be attached to the target binding molecule non-covalently, e.g. , by hybridizing to a nucleic acid strand attached to the target binding molecule. In some embodiments of any one of the aspects, the primer strand is attached to the target binding molecule covalently.

[0045] In some embodiments of any one of the aspects, the primer strand comprises a hybridization domain. The hybridization domain can be present in anywhere in the primer strand. For example, the hybridization domain can be at the 5 ’-end, the 3 ’-end or at an internal position. In some embodiments of any one of the aspects, the hybridization domain is at the 3 ’-end of the primer strand. Generally, the hybridization domain of the primer nucleic acid strand is substantially complementary to a hybridization domain of a template nucleic acid strand provided herein.

[0046] Without limitations, the hybridization domain of the primer strand can be of any desired length. For example, the hybridization domain of the primer strand can be of length sufficient to allow hybridization with a template strand. Thus, in some embodiments of any one of the aspects, the hybridization domain of the primer strand has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5- 35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the hybridization domain of the primer strand has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[0047] In some embodiments of any one of the aspects, the primer strand comprises a second domain. Optionally, a linker can be present between the hybridization domain and the second domain. The second domain can be positioned 5’ or 3’ of the hybridization domain of the primer strand. Preferably, the second domain is 5’ of the hybridization domain.

[0048] Without limitations, the second domain of the primer strand can be of any desired length. For example, the second domain of the primer strand can be 1 or more nucleotides. Accordingly, in some embodiments of any one of the aspects, the second domain of the primer strand has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the second domain of the primer strand has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[0049] In some embodiments of any of the aspects, primer strand comprises a barcode sequence/domain. It is noted that the barcode sequence/domain can be a discrete domain or overlap, partially or fully with the hybridization domain and/or, if present, the second domain of the primer strand.

[0050] The barcode sequence/domain of the primer strand can be of any desired length. For example, the barcode sequence/domain of the primer strand can be 1 or more nucleotides. Accordingly, in some embodiments of any one of the aspects, the barcode sequence/domain of the primer strand has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the second domain of the primer strand has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides. [0051] In some embodiments of any one of the aspects, the primer strand can comprise a nucleic acid modification, e.g., a nucleic acid modification selected from the group consisting of modified intemucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof. Exemplary nucleic acid modifications described herein. The nucleic acid modification can be located anywhere in the primer strand. For example, the nucleic acid modification can be at the 5 ’ -end, the 3 ’ -end or at an internal position of the primer strand. Generally, the primer strand comprises a 3 ’-OH group at the 3 ’-end.

[0052] In some embodiments of any one of the aspects, the primer strand comprises a nucleic acid modification, wherein said modification is in the hybridization domain of the primer strand. [0053] In some embodiments of any one of the aspects, the primer strand comprises a nucleic acid modification, wherein said modification is in the second domain of the primer strand.

[0054] In some embodiments of any one of the aspects, the primer strand comprises a nucleic acid modification, wherein said modification is present at position between the hybridization domain and the second domain.

[0055] In some embodiments of any one of the aspects, the primer strand comprises a cross- linking moiety. Cross-linking moieties include, but are not limited to, photo-cross linking moieties, chemical cross-linking moieties, binding pair members, and any combinations thereof. When present, the cross-linking moiety can be present at any position of the primer strand. For example, the cross-linking moiety can be or at a terminus of the primer nucleic acid strand. In other words, the cross-linking moiety can be at the 5 ’-end, 3 ’-end or at an internal position of the primer strand. [0056] In some embodiments of any of the aspects, the cross-linking moiety can be at the 3’- end of the primer strand.

[0057] In some embodiments of any of the aspects, the cross-linking moiety is present at an internal position of the primer strand. For example, the cross-linking moiety is present in the hybridization domain of the primer strand.

[0058] In some embodiments of any one of the aspects, the primer strand comprises a photo cross linking moiety. For example, a photo-cross linking moiety selected from the group consisting of 3-Cyanovinylcarbazole (CNVK) nucleotide; 5-bromo deoxycytosine; 5-iodo deoxycytosine; 5- bromo deoxyuridine (Bromo dU); 5-iodo deoxyuridine; and nucleotides comprising an aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluorinated aryl azide (FAB-dUMP) or diazirine (DB- dUMP), psoralen, 4-thio-dT (S4dT), and the like.

[0059] In some embodiments of any one of the aspects, the primer strand comprises a chemical- cross linking moiety comprising a chemical functional group for linking. Exemplary chemical functional group for cross-linking include, but are not limited to, azide, alkyne, tetrazine, DBCO, thiol, amine, carbonyl, carboxyl group, maleimide, next generation maleimide, carboiimide, NHS- ester, sulfo-NHS, and the like.

[0060] In some embodiments of any one of the aspects, the cross-linking moiety comprises a binding pair. For example, the primer strand comprises a one member of a binding pair. The other member of the binding pair can be present in an extension strand described herein.

[0061] As used herein, the term “binding pair” refers to a pair of moieties that specifically bind each other with high affinity, generally in the low micromolar to picomolar range. When one member of a binding pair is conjugated to a first element and the other member of the pair is conjugated to a second element, the first and second elements will be brought together by the interaction of the members of the binding pair. Non-limiting examples of binding pairs include biotin: avidin, biotin: streptavidin, biotin: neutravidin (or other variants of avidin that bind biotin), a receptor ligand pair, an antibody antigen pair, antigen binding fragment of an antibody and antigen pair, and the like. Additional molecule for binding pair can include, neutravidin, strep-tag, strep- tactin and derivatives, and other peptide, hapten, dye-based tags-anti-Tag combinations such as SpyCatcher-SpyTag, His-Tag, Fc Tag, Digitonin, GFP, FAM, haptens, SNAP-TAG. HRP, FLAG, HA, myc, glutathione S-transferase (GST), maltose binding protein (MBP), small molecules, and the like.

[0062] In some embodiments of any of the aspects, a domain, e.g., the hybridization domain, the second domain, and/or the barcode sequence/domain of the primer strand comprises a sequence of nucleotides that lacks one of A, T, C or G.

[0063] In some embodiments, provided herein is a use of a nucleic acid primer strand for creating combinatorial barcodes to assign unique identities to a target binding molecule provided herein.

[0064] The primer strand provided herein can be single or double stranded. In embodiments of any of the aspects, the primer strand is single-stranded.

[0065] The primer strand can be any length that permits attachment to the target binding molecule and hybridization with a template stand. For example, the primer strand is generally 25- 300 nucleotides in length. In some embodiments of any of the aspects, the primer strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50-250, 50-200, 50-150 or 50- 100 nucleotides. In some embodiments of any of the aspects, the primer strand has a length of 25- 100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100-150 or 100-200 nucleotides. For example, the primer strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,

40, 41, 42, 43, 44, 45, 46, 47, 48, 49. 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,

92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides. Template strand

[0066] The methods, compositions, and kits provided herein include a template nucleic acid strand, also referred to as a template strand herein. Generally, the template strand comprises a hybridization domain and a second domain. Optionally, the hybridization domain and the second domain of the template strand can be linked together by a linker domain. The hybridization domain of the template strand is also referred to as a toehold domain herein.

[0067] The hybridization domain of the template strand can be present in anywhere in the template strand. For example, the hybridization domain can be at the 5 ’-end, the 3 ’-end or at an internal position. In some embodiments of any one of the aspects, the hybridization domain is at the 3 ’-end of the template strand. Generally, the hybridization domain of the template strand is substantially complementary to the hybridization domain of a primer strand provided herein. [0068] Without limitations, the hybridization domain of the template strand can be of any desired length. For example, the hybridization domain of the template strand can be of length sufficient to allow hybridization with a primer strand. Thus, in some embodiments of any one of the aspects, the hybridization domain of the template strand has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35- 40 nucleotides. For example, the hybridization domain of the template strand has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[0069] The second domain of the template strand can be positioned 5 ’ or 3 ’ of the hybridization domain of the template strand. Preferably, the second domain is 5’ of the hybridization domain. [0070] Without limitations, the second domain of the template strand can be of any desired length. In some embodiments of any one of the aspects, the second domain of the template strand has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the second domain of the template strand has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[0071] In some embodiments of any one of the aspects, template strand comprises a second hybridization domain. For example, the second domain can be the second hybridization domain. Generally, the second hybridization domain of the template strand is substantially complementary with a hybridization domain of an extension nucleic acid strand provided herein.

[0072] In some embodiments of any of the aspects, the second domain of the template strand comprises a double stranded structure. For example, the second domain comprises a first stem domain, a second stem domain, and a linker domain between the first stem domain and the second stem domain, and wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure. Accordingly, in some embodiments of any of the aspects, the template strand comprises in 3’ to 5’ direction: a hybridization domain, a first stem domain, a second stem domain, and a linker domain between the first stem domain and the second stem domain, and wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure.

[0073] In some embodiments of any of the aspects, the template strand comprises in 3’ to 5’ direction: a hybridization domain, a first stem domain, a second stem domain, and a linker domain between the first stem domain and the second stem domain, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, and wherein the hybridization domain is substantially complementary to the second stem domain of another template strand.

[0074] In some embodiments of any of the aspects, the template strand comprises in 3’ to 5’ direction: a hybridization domain, a first stem domain, a second stem domain, and a linker domain between the first stem domain and the second stem domain, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, and wherein the hybridization domain comprises a nucleotide sequence substantially identical to a nucleotide sequence of the first stem domain. In other words, the hybridization domain is substantially complementary to the second stem domain of same template strand.

[0075] Without limitations, the first and second stem domain of the template strand can be of any desired length. In some embodiments of any one of the aspects, the first and second stem domain of the template strand independently have a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5- 35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the first and second stem domain of the template strand independently have a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides. [0076] In some embodiments of any of the aspects, the first stem domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the first stem domain is of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides in length.

[0077] In some embodiments of any of the aspects, the second stem domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10- 30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25- 35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the second stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides in length.

[0078] In some embodiments of any of the aspects, the first stem domain and the second stem domain hybridize to form a double-stranded structure, also referred to as a stem domain herein. The stem domain can have a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. In some embodiments of any of the aspects, the stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[0079] In some embodiments of any one of the aspects, the template strand can comprise a nucleic acid modification, e.g., a nucleic acid modification described herein. The nucleic acid modification can be located anywhere in the template strand. For example, the nucleic acid modification can be at the 5 ’-end, the 3 ’-end or at an internal position of the template strand. [0080] In some embodiments of any one of the aspects, the template strand comprises a nucleic acid modification, wherein said modification is in the hybridization domain of the template strand. [0081] In some embodiments of any one of the aspects, the template strand comprises a nucleic acid modification, wherein said modification is in the second domain of the template strand. [0082] In some embodiments of any one of the aspects, the primer strand comprises a nucleic acid modification, wherein said modification is present at position between the hybridization domain and the second domain.

[0083] In some embodiments of any one of the aspects, the template strand comprises a nucleic acid modification, wherein said modification is in the first stem domain of the template strand. [0084] In some embodiments of any one of the aspects, the template strand comprises a nucleic acid modification, wherein said modification is in the second stem domain of the template strand. [0085] In some embodiments of any one of the aspects, the template strand comprises a nucleic acid modification, wherein said modification is in the linker domain linking the first and second stem domains of the template strand.

[0086] In some embodiments of any one of the aspects, the template strand comprises a nucleic acid modification, wherein said modification is present between the linker domain linking the first and second stem domains of the template strand and either first stem domain or the second stem domain.

[0087] In some embodiments of any of the aspects, the template strand comprises a nucleic acid modification capable of enhancing nucleic acid cleavage. For example, the nucleic acid modification capable of enhancing nucleic acid cleavage can be modification that facilitate chemical, photo, or enzymatic cleavage. Exemplary modification that can enhance cleavage include, but are not limited to, 5’-phosphate groups, 2’-OH nucleotides (e.g., RNA nucleotides), nucleotides comprising a methylated nucleobase; abasic nucleotides, acyclic nucleotides, cleavable linker/spacers, and any combinations thereof. In some embodiments, the template strand comprises a nucleic acid modification capable of enhancing enzymatic cleavage.

[0088] In some embodiments of any of the aspects, the template strand comprises a phosphate group at the 5’-end. For example, the template strand comprises a 5'-monophosphate; 5'- diphosphate or a 5 '-triphosphate at the 5 ’-end.

[0089] In some embodiments any of the aspects, the template strand is RNA.

[0090] In some embodiments of any of the aspects, the template strand comprises a modification capable of terminating nucleic acid polymerization. For example, at least one of the first stem domain, the second stem domain and the linker domain of the template strand comprises a modification capable of terminating nucleic acid polymerization.

[0091] Exemplary modifications capable of terminating nucleic acid polymerization include, but are not limited to, a spacer (e.g., a C3-spacer, a triethylene glycol spacer, etc...), an abasic nucleotide, an acyclic nucleotide, a modified nucleobase nucleotide, a non-natural nucleotide (e.g. , iso-dG and iso-dC), CNVK, CNVD, and any combinations thereof.

[0092] In some embodiments of any one of the aspects, the template strand comprises a modification capable of terminating nucleic acid polymerization, wherein said modification is in the first stem domain of the template strand.

[0093] In some embodiments of any one of the aspects, the template strand comprises a modification capable of terminating nucleic acid polymerization, wherein said modification is in the second stem domain of the template strand.

[0094] In some embodiments of any one of the aspects, the template strand comprises a modification capable of terminating nucleic acid polymerization, wherein said modification is in the linker domain linking the first and second stem domains of the template strand.

[0095] In some embodiments of any one of the aspects, the template strand comprises a modification capable of terminating nucleic acid polymerization, wherein said modification is present between the linker domain linking the first and second stem domains of the template strand and either first stem domain or the second stem domain.

[0096] In some embodiments of any of the aspects, template strand comprises a barcode sequence/domain or a nucleotide sequence complementary to a barcode sequence/domain. It is noted that the barcode sequence/domain can be a discrete domain or overlap, partially or fully with the hybridization domain and/or, the second domain, the first stem domain, the second domain and/or a linker domain of the template strand. [0097] The barcode sequence/domain of the template strand can be of any desired length. For example, the barcode sequence/domain of the template strand can be 1 or more nucleotides. Accordingly, in some embodiments of any one of the aspects, the barcode sequence/domain of the primer strand has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the second domain of the primer strand has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[0098] In some embodiments of any of the aspects, a domain, e.g., the hybridization domain and/or, the second domain, the first stem domain, the second domain and/or a linker domain of the template strand comprises a sequence of nucleotides that lacks one of A, T, C or G.

[0099] In some embodiments of any of the aspects, the template strand does not comprise a double-stranded region. In some other embodiments of any of the aspects, the template strand comprises a double-stranded region. For example, the template strand is in form of a hairpin. In some embodiments of any of the aspects, the template strand is in form of a self-folding hairpin. [00100] In some embodiments of any of the aspects, the template strand is not in form of a hairpin.

[00101] A template strand provided herein can be any length that permit hybridization to a primer strand and/or an extension strand provided herein. Generally, the template strand is 25-300 nucleotides in length. In some embodiments of any of the aspects, the template strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50-250, 50-200, 50-150 or 50-100 nucleotides. In some embodiments of any of the aspects, the template strand has a length of 25- 100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100-150 or 100-200 nucleotides. For example, the template strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49. 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,

65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides.

Extension strand

[00102] The methods, compositions, and kits provided herein include an extension nucleic acid strand, also referred to as an extension strand herein.

[00103] In some embodiments of any one of the aspects, the extension strand comprises a hybridization domain. The hybridization domain can be present in anywhere in the extension strand. For example, the hybridization domain can be at the 5 ’-end, the 3 ’-end or at an internal position of the extension strand. In some embodiments of any one of the aspects, the hybridization domain is at the 5 ’-end of the extension strand. Generally, the hybridization domain of the extension strand is substantially complementary to a hybridization domain, e.g., the second hybridization domain of a template nucleic acid strand provided herein.

[00104] Without limitations, the hybridization domain of the extension strand can be of any desired length. For example, the hybridization domain of the extension strand can be of length sufficient to allow hybridization with a template strand. Thus, in some embodiments of any one of the aspects, the hybridization domain of the extension strand has a length of 2-35, 2-30, 2-25, 2- 20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15- 40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the hybridization domain of the extension strand has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides. [00105] In some embodiments of any one of the aspects, the extension strand comprises a second domain. Optionally, a linker can be present between the hybridization domain and the second domain. The second domain can be positioned 5’ or 3’ of the hybridization domain of the extension strand. Preferably, the second domain is 3’ of the hybridization domain.

[00106] Without limitations, the second domain of the extension strand can be of any desired length. For example, the second domain of the extension strand can be 1 or more nucleotides. Accordingly, in some embodiments of any one of the aspects, the second domain of the extension strand has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the second domain of the primer strand has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00107] In some embodiments of any of the aspects, extension strand comprises a barcode sequence/domain. It is noted that the barcode sequence/domain can be a discrete domain or overlap, partially or fully with the hybridization domain and/or, the second domain of the extension strand.

[00108] The barcode sequence/domain of the extension strand can be of any desired length. For example, the barcode sequence/domain of the extension strand can be 1 or more nucleotides. Accordingly, in some embodiments of any one of the aspects, the barcode sequence/domain of the extension strandhas a length of2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5- 10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20- 30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. For example, the barcode sequence/domain of the extension strand has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides. [00109] In some embodiments of any one of the aspects, the extension strand can comprise a nucleic acid modification, e.g., a nucleic acid modification selected from the group consisting of modified intemucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof. Exemplary nucleic acid modifications described herein. The nucleic acid modification can be located anywhere in the extension strand. For example, the nucleic acid modification can be at the 5 ’ -end, the 3 ’ -end or at an internal position of the extension strand. Generally, the primer strand comprises a 5 ’-OH group at the 5 ’-end.

[00110] In some embodiments of any one of the aspects, the extension strand comprises a nucleic acid modification, wherein said modification is in the hybridization domain.

[00111] In some embodiments of any one of the aspects, the extension strand comprises a nucleic acid modification, wherein said modification is in the second domain.

[00112] In some embodiments of any one of the aspects, the extension strand comprises a nucleic acid modification, wherein said modification is present at position between the hybridization domain and the second domain.

[00113] In some embodiments of any one of the aspects, the extension strand comprises a cross- linking moiety. When present, the cross-linking moiety can be present at any position of the extension strand. For example, the cross-linking moiety can be or at a terminus of the primer nucleic acid strand. In other words, the cross-linking moiety can be at the 5’-end, 3 ’-end or at an internal position of the extension strand.

[00114] In some embodiments of any of the aspects, the cross-linking moiety can be at the 5’- end of the primer strand.

[00115] In some embodiments of any of the aspects, the cross-linking moiety is present at an internal position of the extension strand. For example, the cross-linking moiety is present in the hybridization domain of the extension strand.

[00116] In some embodiments of any one of the aspects, the extension strand comprises a photo cross linking moiety. For example, a photo-cross linking moiety selected from the group consisting of 3-Cyanovinylcarbazole (CNVK) nucleotide; 5-bromo deoxycytosine; 5-iodo deoxycytosine; 5- bromo deoxyurdine (Bromo dU); 5-iodo deoxyuridine; and nucleotides comprising an aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluorinated aryl azide (FAB-dUMP) or diazirine (DB- dUMP), psoralen, 4-thio-dT (S4dT), and the like.

[00117] In some embodiments of any one of the aspects, the extension strand comprises a chemical-cross linking moiety comprising a chemical functional group for linking. Exemplary chemical functional group for cross-linking include, but are not limited to, azide, alkyne, tetrazine, DBCO, thiol, amine, carbonyl, carboxyl group, maleimide, next generation maleimide, carboiimide, NHS-ester, sulfo-NHS, and the like. [00118] In some embodiments of any one of the aspects, the extension strand comprises a one member of a binding pair. The other member of the binding pair can be present in a primer strand described herein. For example, one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[00119] In some embodiments of any of the aspects, a domain, e.g., the hybridization domain, the second domain, and/or the barcode sequence/domain of the extension strand comprises a sequence of nucleotides that lacks one of A, T, C or G.

[00120] In some embodiments, provided herein is a use of an extension strand for creating combinatorial barcodes to assign unique identities to a target binding molecule provided herein. [00121] The primer strand provided herein can be single or double stranded. In embodiments of any of the aspects, the primer strand is single-stranded.

[00122] The extension strand can be any length that permits, if needed, hybridization with a template stand. For example, the extension strand is generally 25-300 nucleotides in length. In some embodiments of any of the aspects, the extension strand has a length of 25-300, 25-250, 25- 200, 25-150, 25-100, 25-50, 50-300, 50-250, 50-200, 50-150 or 50-100 nucleotides. In some embodiments of any of the aspects, the extension strand has a length of 25-100, 30-50, 40-60, SO O, 60-80, 70-90, 80-100, 100-125, 100-150 or 100-200 nucleotides. For example, the extension strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,

45, 46, 47, 48, 49. 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,

71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,

97, 98, 99 or 100 nucleotides.

[00123] The extension strand provided herein can be single or double stranded. In some embodiments of any of the aspects, the extension stand is single stranded.

[00124] In some embodiments of any of the aspects, the extension strand comprises a double- stranded region. For example, the extension strand is in form of a hairpin. In some embodiments of any of the aspects, the extension strand is in form of a self-folding hairpin.

[00125] In some embodiments of any of the aspects, the extension strand does not comprise a double-stranded region.

[00126] In some embodiments of any of the aspects, the extension strand comprises a hybridization domain and a second domain, optionally linked by a linker domain. The hybridization domain is substantially complementary to a second hybridization domain of a template strand, where the first hybridization domain of the template strand is substantially complementary to a hybridization domain of a different extension strand. [00127] In some embodiments of any of the aspects, the extension strand comprises a hybridization domain and a second domain, optionally linked by a linker domain. The hybridization domain is substantially complementary to a second hybridization domain of a template strand, where the first hybridization domain of the template strand comprises a nucleotide sequence substantially identical to a nucleotide sequence of the second domain.

Nucleic acid modifications

[00128] In some embodiments of any of the aspects, the nucleic acids, e.g, the primer strand, the template strand and/or the extension strand provided herein comprises a nucleic acid modification. Exemplary nucleic acid modifications include, but are not limited to, nucleobase modifications, sugar modifications, inter-sugar linkage modifications, conjugates (e.g., ligands), and any combinations thereof.

[00129] Exemplary modified nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine and uracil, such as 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6- azapyrimi dines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5- alkyluracil, 7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6, N6-dimethyladenine, 2,6- diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5- nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5- methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5- methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5- methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2- methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I, ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613.

[00130] In some embodiments, modified nucleobase can be selected from the group consisting of inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2- (alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine,

2-(aminopropyl)adenine, 2-(methylthio)-N^<5-(isopentenyl)adenine, 6-(alkyl)adenine,

6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine,

8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N⁶-(isopentyl)adenine, N⁶-(methyl)adenine, N⁶, N⁶-(dimethyl)adenine, 2-

(alkyl)guanine,2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine,

7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine,

8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine,

3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine,

5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N⁴-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 5-ethynyl-2'-deoxyuridine, 2- (thio)uracil,5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5- (alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(l,3-diazole-l-alkyl)uracil, 5-(cyanoalkyl)uracil, 5- (dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil- 5 -oxy acetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N³-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil,4-(thio)pseudouracil,2,4- (dithio)psuedouracil,5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-

(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-

4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil,

1 -substituted pseudouracil, 1 -substituted 2(thio)-pseudouracil, 1 -substituted 4-(thio)pseudouracil, 1 -substituted 2,4-(dithio)pseudouracil, 1 -(aminocarbonylethylenyl)-pseudouracil,

1 -(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1 -(aminocarbonylethylenyl)-

4-(thio)pseudouracil, l-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,

1 -(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1 -(aminoalkylamino-carbonylethylenyl)- 2(thio)-pseudouracil, l-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil,

1 -(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1 ,3-(diaza)-2-(oxo)-phenoxazin- 1-yl, l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, l-(aza)-2- (thio)-3-(aza)-phenthiazin-l-yl, 7-substituted l,3-(diaza)-2-(oxo)-phenoxazin-l-yl, 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, 7-substituted l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, 7- substituted 1 -(aza)-2-(thio)-3 -(aza)-phenthiazin- 1 -yl, 7-(aminoalkylhy droxy)- 1 ,3 -(diaza)-2-(oxo)- phenoxazin- 1 -yl, 7-(aminoalkylhy droxy )- 1 -(aza)-2-(thio)-3 -(aza)-phenoxazin- 1 -yl, 7-

(aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, 7-(aminoalkylhy droxy)- l-(aza)-2- (thio)-3 -(aza)-phenthiazin- 1 -y 1, 7-(guanidiniumalkylhy droxy)- 1 ,3 -(diaza)-2-(oxo)-phenoxazin- 1 - yl, 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, 7-(guanidiniumalkyl- hy droxy)- 1 ,3 -(diaza)-2-(oxo)-phenthiazin- 1 -y 1, 7-(guanidiniumalkylhy droxy)- 1 -(aza)-2-(thio)-3 - (aza)-phenthiazin-l-yl, l,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3- (methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7- (aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5- (trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6- (methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N²-substituted purines, N⁶-substituted purines, 0⁶-substituted purines, substituted 1,2,4-triazoles, and any O-alkylated or N-alkylated derivatives thereof.

[00131] Exemplary sugar modifications include, but are not limited to, 2’-Fluoro, 3 ’-Fluoro, 2’- OMe, 3’-OMe, 2’-deoxy modifications, and acyclic nucleotides, e.g., peptide nucleic acids (PNA), unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).

[00132] In some embodiments, a nucleic acid modification can include replacement or modification of an inter-sugar linkage. Exemplary inter-sugar linkage modifications include, but are not limited to, phosphotriesters, methylphosphonates, phosphoramidite, phosphorothioates, methylenemethylimino, thiodiester, thionocarbamate, siloxane, N,N'-dimethylhydrazine ( — CH2- N(CH3)-N(CH3)-), amide-3 (3'-CH₂-C(=0)-N(H)-5') and amide-4 (3'-CH₂-N(H)-C(=0)-5'), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxy methyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH₂-0-5'), formacetal (3 '-O-CEE-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenemethylimino (MMf 3'-CH₂-N(CH3)-0-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethybmino, ethers (C3’-0-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=0)-CH₂-S-C5’, C3’-0-P(0)-0-SS-C5’, C3’- CH₂-NH-NH-C5’, 3'-NHP(0)(0CH₃)-0-5' and 3'-NHP(0)(0CH₃)-0-5’

[00133] In some embodiments, nucleic acid modifications can include peptide nucleic acids (PNA), bridged nucleic acids (BNA), morpholinos, locked nucleic acids (LNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), or any other xeno nucleic acids (XNA) described in the art. Target binding molecule

[00134] In some embodiments of any of the aspects, the primer strand is attached to a target binding molecule.

[00135] As used herein, a “target binding molecule” is a molecule or moiety that binds, e.g., specifically to a target molecule. The target binding molecule can be a synthetic or natural molecule. A target binding molecule can be a biomolecule, such as a polypeptide or a polynucleotide. In some embodiments, a target-binding molecule is a polypeptide. In some embodiments, a target binding molecule is a protein (e.g. , full-length protein or peptide). Non-limiting examples of target binding molecules include peptides, polypeptides, antibodies, nucleosides, nucleotides, oligonucleotides, molecular probes, receptors, ligands, hormones, vitamins, nanobodies, affibodies, cells, lipids, sugars, microorganisms, microorganism peptides and fragments thereof, epitopes, biomarkers, aptamers, nucleic acids, biomolecular complexes, enzymes, second messenger signaling molecules, nanoparticles, microparticles, extracellular matrix proteins, synthetic molecule, synthetic nanoparticles, synthetic beads or labels, etc.

[00136] In some embodiments of any of the aspects, the target binding molecule is selected from the group consisting of: an antibody, nanobody, an affibody, a receptor, a receptor ligand, an aptamer, a modified aptamer, an ankyrin-repeat protein, a peptide binder, a small molecule, and any combination thereof and any combination thereof.

[00137] In some embodiments of any of the aspects, the target binding molecule is an antibody.

[00138] Like the target binding molecule, the target molecule it binds to can be a synthetic or a biological material. Accordingly, in some embodiments of any of the aspects, the target molecule is a biological material. For example, the target molecule can be a cell.

[00139] In some embodiments of any of the aspects, the target molecule is a synthetic material.

[00140] In some embodiments of any of the aspects, the target binding molecule binds to i.e., the target molecule is a molecule selected from the group consisting of lipids, sugars, oligo- or poly saccharides, amino acids, peptides or polypeptides, nucleosides, nucleotides, oligo- or poly nucleotides, hormones, vitamins, small molecules, miRNAs, H2O2, free-radicals, metabolites, and any combinations thereof.

[00141] In some embodiments of any of the aspects, the target binding molecule binds to two or more molecules selected from the group consisting of: a lipid, a sugar, an oligo- or poly- saccharide, an amino acid, a peptide or a polypeptide, a nucleoside, a nucleotide, an oligo- or poly- nucleotide, a hormone, a vitamin, a small molecule, amiRNA, H2O2, a free-radical, metabolites, a nucleic acid, and any combinations thereof. [00142] In some embodiments of any of the aspects, the target binding molecule binds a molecule that is DNA or RNA barcoded.

Linker domain

[00143] In some embodiments of any of the aspects, a nucleic acid strand provided herein, e.g. , a primer strand, a template strand and/or an extension strand comprises one or more linker domains. Without limitations, the linker domain can be of any desired length and/or nucleotide sequence. For example, the linker domain can simply be a nucleic acid backbone linkage e.g. , phosphodiester linkage. In addition, the linkers can all be the same, all different, or some are the same and some are different.

[00144] In some embodiments of any of the aspects, the linker domain can comprise a cleavable group or moiety. For example, a photocleavable group or moiety, hydrolyzable group or moiety, redox cleavable group or moiety, phosphate-based cleavable group or moiety, acid cleavable group or moiety, ester-based cleavable group or moiety, peptide-based cleavable group or moiety, and any combinations thereof. In some embodiments, the cleavable group or moiety can comprise a disulfide bond, a tetrazine-trans-cyclooctene group, a sulfhydryl group, a nitrobenzyl group, a nitoindoline group, a bromo hydroxycoumarin group, a bromo hydroxyquinoline group, a hydroxyphenacyl group, a dimethozybenzoin group, or any combinations thereof.

[00145] Any art-recognized photocleavable group or moiety can be used. In some embodiments, the cleavable linker can comprise a photocleavable linker. Generally, a photocleavable group or moiety contains a photolabile functional group that is cleavable upon exposure to a light source (e.g., UV light) or specific wavelength. Non-limiting examples of photocleavable group or moiety can be found, for example, in US Patent Nos. 6,589,736 Bl; 7,622,279 B2; 9,371,348 B2; 7,547,530 B2; and 7,057,031 B2; and PCT Publication No. WO20 14200767, contents of all of which are incorporated herein by reference in their entirety. [00146] Without limitations, each linker domain can be independently of any desired length. For example, each linker domain can be independently of a length of one or more nucleotides. In some embodiments of any one of the aspects, each linker domain can independently have a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10- 25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-

30, 30-40, 30-35 or 35-40 nucleotides. For example, each linker domain can independently have a length of 1, 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

Annealing/hybridizing [00147] Generally, the method described herein comprises a step of annealing or hybridizing together a primer strand and a template strand, or a primer strand, a template strand and an extension strand. As used herein, the term “hybridizing”, “hybridize”, “hybridization”, “annealing”, or “anneal” are used interchangeably in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. In other words, the term “hybridization” refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double- stranded polynucleotide. The term “hybridization” may also refer to triple-stranded hybridization. The resulting (usually) double-stranded polynucleotide is a “hybrid” or “duplex.”

[00148] “Hybridization conditions” will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and even more usually less than about 200 mM. Hybridization temperatures can be as low as 5° C., but are typically greater than 22° C., more typically greater than about 30° C., and often in excess of about 37° C. Hybridizations are usually performed under stringent conditions, i.e., conditions under which a strand will hybridize to its intended target strands. Stringent conditions are sequence-dependent and are different in different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. Generally, stringent conditions are selected to be about 5° C lower than the Tm for the specific sequence at a defined ionic strength and pH. Exemplary stringent conditions include salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25° C. For example, conditions of 5*SSPE (750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C are suitable for allele-specific probe hybridizations. For stringent conditions, see for example, Sambrook, Fritsche and Maniatis, Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989) and Anderson Nucleic Acid Hybridization, 1st Ed., BIOS Scientific Publishers Fimited (1999). “Hybridizing specifically to” or “specifically hybridizing to” or like expressions refer to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture.

[00149] In some embodiments of any of the aspects, nucleic acid strands described herein, e.g., the primer strand, the template strand and/or the extension strand can be annealed together (e.g. by cooling from 80°C to 20°C over a period of time, e.g., 1 hour), or they can be combined together isothermally (e.g. at room temperature, 37°C, 46°C, etc.). [00150] In some embodiments of any one of the aspects, the method can comprise a step of changing a hybridization buffer with a buffer for nucleic acid polymerization and/or nucleic acid ligation.

Extension/polymerization

[00151] In some embodiments of the various aspects described herein, the method comprises a step of extending the template and/or a synthesized strand with a polymerase. Method of synthesizing nucleic acid strand using polymerases are well known in the art and available to one of ordinary skill in the art.

[00152] The concentration of particular strands and dNTPs in a polymerase reaction composition, method or kit can be varied depending, for example, on the particular application and kinetics required for that particular application.

[00153] The concentration of strand in a composition, method or kit described herein can be, for example, 5 nM to 1000 nM. In some embodiments of any of the aspects, the strand concentration is 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 5-125, 5-150, 5-200, 10-50, 10- 75, 10-100, 10-150, 10-200, 25-75, 25-100, 25-125 or 25-200 nM. In some embodiments of any of the aspects, the strand concentration is 10-200, 10-300, 10-400, 10-500, 10-600, 10-70, 10-800, 10- 900 or 10-100 nM. In some embodiments of any of the aspects, the strand concentration is 10, 15,

20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,

140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 nM. In some embodiments of any of the aspects, the strand concentration is 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nM. In some embodiments of any of the aspects, the strand concentration can be less than 5 nM or greater than 1000 nM. For example, the concentration of strand in a composition, method or kit described herein can be less than 10 nM or greater than 1000 nM.

[00154] The ratio of any two strands (e.g., primer strand and the template strand) can be approximately 1:1. It is noted that the ratio of any two strands can be varied. For example, the ratio of any two strands can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or more.

[00155] It is noted that kinetics of polymerization can be controlled by varying temperature, time, buffer/salt conditions, and deoxyribonucleotide triphosphate (dNTP) concentrations, for example. Polymerases, like most enzymes, are sensitive to many buffer conditions, including ionic strength, pH and types of metal ions present (e.g., sodium ions vs. magnesium ions). Thus, the temperature at which polymerization step is performed can vary from, for example, 4°C to 65°C (e.g., 4°C, 25°C, 37°C, 42°C or 65°C). In some embodiments of the various aspects, the temperature at which the polymerization step is performed is 4-25°C, 4-30°C, 4-35°C, 4-40°C, 4-

45°C, 4-50°C, 4-55°C, 4-60°C, 10-25C, 10-30°C, 10-35°C, 10-40°C, 10-45°C, 10-50°C, 10-55°C, 10-60°C, 25-30°C, 25-35°C, 25-40°C, 25-45°C, 25-50°C, 25-55°C, 25-60°C, 25-65°C, 35-40°C, 35-45°C, 35-50°C, 35-55°C, 35-60°C, or 35-65°C. In some embodiments of the various aspects, the polymerization step is performed at room temperature. In some other embodiments, the polymerization step is performed at 37 °C.

[00156] A polymerization step can be performed (incubated) for about 30 minutes to about 24 hours. In some embodiments of any of the aspects, the polymerization step can be carried out for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours or about 24 hours.

[00157] In some embodiments of the various aspects described herein, concentration of dNTPs can be, for example, 2-1000 mM. For example, concentration of the dNTPs can be 2-10 pM, 2-15 pM, 2-20 pM, 2-25 pM, 2-30 pM, 2-35 pM, 2-40 pM, 2-45 pM, 2- 50 pM, 2-55 pM, 2-60 pM, 2- 65 pM, 2-70 pM, 2-75 pM, 2-80 pM, 2-85 pM, 2-90 pM, 2-95 pM, 2-100 pM, 2-110 pM, 2-120 pM, 2-130 pM, 2-140 pM, 2-150 pM, 2-160 pM, 2-170 pM, 2-180 pM, 2-190 pM, 2-200 pM, 2- 250 pM, 2-300 pM, 2-350 pM, 2-400 pM, 2-450 pM, 2-500 pM, 2-600 pM, 2-700 pM, 2-800 pM, 2-900 pM or 2-1000 pM. For example, concentration of the dNTPs can be 2 pM, 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM, 150 pM, 155 pM, 160 pM, 165 pM, 170 pM, 175 pM, 180 pM, 185 pM, 190 pM, 195 pM or 200 pM. In some embodiments of any of the aspects, concentration of the dNTPs can be 10-20 pM, 10-30 pM, 10-40 pM, 10-50 pM, 10-60 pM, 10-70 pM, 10-80 pM, 10-90 pM or 10-100 pM. It is noted that dNTP variants can also be used.

[00158] In some embodiments of the various aspects described herein, polymerization step comprises incubating the reaction under conditions that result in nucleic acid polymerization, strand displacement and annealing, for a time sufficient to produce a synthesized strand.

[00159] In some embodiments of any of the aspects, the polymerase is a DNA polymerase (DNAP), such as a DNA polymerase having DNA strand displacement activity (a strand displacing polymerase). “Strand displacement” describes the ability to displace downstream DNA encountered during synthesis. Examples of polymerases having DNA strand displacement activity that can be used as provided herein include, without limitation, phi29 DNA polymerase (e.g., NEB #M0269), Bst DNA polymerase, large fragment (e.g., NEB #M0275), or Bsu DNA polymerase, large fragment ( e.g . , NEB #M0330). Other polymerases having strand displacement activity can be used.

[00160] In some embodiments of any of the aspects, the polymerase is a RNA polymerase.

[00161] In some embodiments of the various aspects described herein, the polymerase is phi29

DNA polymerase. In such embodiments, the reaction conditions can be as follows: reaction buffer {e.g., 50 mM Tris-HCl, 10 mM MgCh, 10 mM (NH4)2S04, 4 mM DTT) supplement with purified bovine serum albumin (BSA), pH 7.5, incubated at 30°C.

[00162] In some embodiments of any of the aspects, the polymerase is Bst DNA polymerase, large fragment. In such embodiments, the reaction conditions can be as follows: IX reaction buffer (e.g, 20 mM Tris-HCl, 10 mM (NH₄)₂S04, 10 mM KC1, 2 mM MgS0₄, 0.1% TRITON® X-100), pH 8.8, incubated at 65°C.

[00163] In some embodiments of any of the aspects, the polymerase is Bsu DNA polymerase. In such embodiments, the reaction conditions can be as follows: reaction buffer (e.g. , 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCh, 1 mM DTT), pH 7.9, incubated at 37°C.

[00164] In some embodiments, the polymerase is Bst DNA polymerase Bsu, Bst 2.0, Bst 3.0, Bsu, phi29, SD polymerase, any combination or fragment thereof.

[00165] Exemplary reaction conditions that can be used in the methods provided herein include but are not limited to: Thermopol® buffer; NEB® buffers 1,2, 3, 4; CutSmart® buffer; Isothermal Amplification Buffer; and the like. Custom buffers can be made with 0.5 to 2X PBS; 5 to 200 mM Tris-HCl; 5-200 mM Potassium Acetate; 5-200 mM Magnesium Acetate; 5-200 mM Tris-Acetate; or 5-200 mM Bis-Tris-Propane-HCl can be used with the addition one or all of these additives to modulate the enzyme activity (e.g., 1-50 mM KC1, 1-20 mM MgS04, 1-20 mM MgC12, 1-5 mM DTT, 0-500 ug/ml BSA, 1-500 NaCl, 0.01% to 0.5% Triton X-100 at pH values of 6-9.5). The buffer can also include dNTPs (e.g, dATP, dCTP, dGTP and dTTP). When only 2-3 types of dNTPs are used, the omitted nucleotides serve as stoppers for the polymerase action, optionally with functional modifications.

Linking with extension strand

[00166] In some embodiments of the various aspects described herein, the method comprises a step of linking one nucleic acid strand with a second nucleic acid strand. Generally, said linking comprises ligation, cross-linking, or binding interaction. Further, said linking can comprise enzymatic ligation, chemical ligation, photo-crosslinking, or interaction binding. It is noted that method and reagents for linking nucleic acid strands are well known in the art and available to one of ordinary skill in the art. [00167] In some embodiments of any of the aspects, said linking comprises linking 3 ’-end of the primer nucleic acid strand with 5 ’-end of the extension nucleic acid strand.

[00168] In some embodiments of any of the aspects, said linking comprises linking 5 ’-end of the primer nucleic acid strand with 3 ’-end of the extension nucleic acid strand.

[00169] Linking can be achieved by ligases. Non-limiting examples of ligases that can be used include SplintR Ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, E. coli DNA Ligase, Taq DNA ligase, HiFi Taq DNA ligase, and 9°N DNA Ligase. Complementary end modifications that result in affinity binding ( e.g ., biotin-streptavidin) or chemical ligation can be used to link the extension strand provided herein. For example, click chemistry linkage utilizing internal or end modifications such as azide, alkyne, tetrazine, DBCO, or crosslinking of end modified strands via maleimide, next generation maleimide, carbomide, NHS-ester, sulfo-NHS, thiol, amine, carbonyl, carboxyl, functional groups with or without linkers (e.g., spacers or PEG linkers) or additional crosslinkers can also be used.

Removing the template and/or excess extension strands

[00170] In some embodiments of the various aspects described herein, the method comprises a step of removing the template and/or excess extension strands. Methods of removing nucleic acid strands are well known in the art and available to one of ordinary skill in the art.

[00171] In some embodiments of any of the aspects, said the template and/or excess extension strands comprises dehybridizing the template strand from the primer and/or synthesized strand. This can be done by heating the reaction mixture to a high temperature. For example, the reaction mixture could be heated to a temperature higher than the melting temperature of the template strand when bound to a fully complementary second strand. Generally, dehybridizing comprises heating to a temperature of about 55°C or higher, about 60°C or higher, about 65°C or higher, about 70°C or higher, about 75°C or higher, about 80°C or higher, about 85°C or higher, about 90°C or higher, about 95°C or higher.

[00172] In some embodiments of the various aspects, dehybridizing comprises heating to a temperature of about 65°C to about 95°C. For example, heating to a temperature of about 70°C to about 95°C, about 75°C to about 95°C, about 80°C to about 95°C, about 85°C to about 95°C. [00173] In some embodiments of the various aspects, some embodiments of the various aspects, dehybridizing comprises heating to a temperature of about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, about 80°C, about 81°C, about 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, or about 95°C. [00174] In some embodiments of any of the aspects, said dehybridizing comprises contacting a complex comprising the template and the synthesized strand with an agent that reduces and/or inhibits hydrogen bonding between two nucleic acid strands. Exemplary such agents include, but are not limited to, formamide, DMSO, or divalent ion chelators. The reaction can be heated to further facilitate dehybridization. For example, the agent can be added to the reaction mixture and the reaction heated to a temperature of about 40°C or more, about 45°C or more, about 50°C or more, about 55°C or more, about 60°C or more, about 65°C or more, about 70°C or more, about 75°C or more, about 76°C or more, about 77°C or more, about 78°C or more, about 79°C or more, about 80°C or more, about 81°C or more, about 82°C or more, about 83 °C or more, about 84°C or more, about 85°C or more, about 86°C or more, about 87°C or more, about 88°C or more, about 89°C or more, about 90°C or more, about 91°C or more, about 92°C or more, about 93 °C or more, about 94°C or more, or about 95°C or more.

[00175] In some embodiments of any of the aspects, said removing the template strand comprises hybridizing or annealing the template strand with a competing nucleic acid strand having a sequence complementary to the template strand. For example, the template strand can comprise on its 3’- terminus a toehold domain for the competing strand, and optionally the toehold domain comprises a nucleotide sequence that is not complementary to the primer strand and/or the synthesized stand. [00176] Alternatively, or in addition to dehybridization, the template and/or the excess extension strands can be cleaved. Methods of removing one nucleic acid strands, e.g., one strand of a double- stranded nucleic acid well known in the art and available to one of ordinary skill in the art. Exemplary such methods include enzymatic cleavage, chemical cleavage and/or photo-cleavage. Thus, in some embodiments of the various aspects, the step of removing the template and/or excess extension strand comprises cleaving the template strand and/or excess extension strands by enzymatic cleavage. The enzymatic cleavage can be by a nuclease.

[00177] In some embodiments of any of the aspects, the step of removing the template and/or excess extension strand comprises cleaving the template strand and/or excess extension strands with an exonuclease. In some embodiments of any one of the aspects, exonuclease is a 5 ’->3’ exonuclease. Exemplary exonucleases include, but are not limited to, T7 exonuclease, lambda exonuclease, Exonuclease VIII, T5 exonuclease, RecJf, and the like.

[00178] In some embodiments of any of the aspects, the step of removing the template and/or excess extension strand comprises cleaving the template strand and/or excess extension strands with an endonuclease. Exemplary endonucleases include, but are not limited to, RNase HI, RNAse HII, endonuclease PI, endonuclease IV, uracil DNA glycosylase, and endonuclease VIII, and the like. [00179] In some embodiments of any of the aspects, the step of removing the template and/or excess extension strand comprises cleaving the template strand and/or excess extension strands with an RNase. Exemplary RNases include, but are not limited to, XRN-1, RNase If, Monarch RNase A, RNase H, RNase HI, RNase HII, and the like.

[00180] It is noted that kinetics of enzymatice cleavage can be controlled by varying temperature, time, and buffer/salt conditions, for example. Nuclease, like most enzymes, are sensitive to many buffer conditions, including ionic strength, pH and types of metal ions present ( e.g ., sodium ions vs. magnesium ions). Thus, the temperature at which enzymatic cleavage is performed can vary from, for example, 4°C to 65°C (e.g., 4°C, 25°C, 37°C, 42°C or 65°C). In some embodiments of the various aspects, the temperature at which the enzymatic cleavage step is performed is 4-25°C, 4-30°C, 4-35°C, 4-40°C, 4-45°C, 4-50°C, 4-55°C, 4-60°C, 10-25C, 10-30°C, 10-35°C, 10-40°C, 10-45°C, 10-50°C, 10-55°C, 10-60°C, 25-30°C, 25-35°C, 25-40°C, 25-45°C, 25-50°C, 25-55°C, 25-60°C, 25-65°C, 35-40°C, 35-45°C, 35-50°C, 35-55°C, 35-60°C, or 35-65°C. In some embodiments of the various aspects, the enzymatic cleavage step is performed at room temperature. In some other embodiments, the enzymatic cleavage step is performed at 37°C. [00181] The enzymatic cleavage step can be performed for about 30 minutes to about 24 hours. In some embodiments of any of the aspects, the enzymatic cleavage step can be carried out for about 10 min, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours or about 24 hours.

[00182] The cleavage efficiency can be improved with some exonucleases (e.g, lamda exonuclease) and the template strand can be phosphorylated on the 5’ end. Exemplary buffer formulations for enzymatic cleavage include but are not limited to: Thermopol, NEB buffers 1,2, 3, 4, CutSmart, or customs buffers made with 0.5 to 2X PBS or 5 to 200 mM Tris-HCl or 5-200 mM Potassium Acetate or 5-200 mM Magnesium Acetate, 5-200 mM Tris-Acetate or 5-200mM bis-tris-propance HCL. The buffers can be used with one or more of the following additives to modulate enzyme activity: 1-50 mM KC1, 1-20 mM MgS04, 1-20 mM MgC12, 1-5 mM DTT, 0- 500 pg/ml BSA, 1-500 NaCl, 0.01% to 0.5% Triton X-100 at pH values of 6-9.5.

Isolation/purification

[00183] It is noted that the polymerization and/or the linking step and the removing the template and/or the excess extension strands can be carried out in the same vessel or in separate vessel. For example, the target binding molecule bound with template strand can be isolated or purified from the reaction mixture prior to removing the template strand. This isolating/purifying step can comprise affinity purification, e.g., using a ligand or tag linked to one of the strands. [00184] In some embodiments of any of the aspects, the template strand comprises a ligand, e.g., a ligand for isolation/purification. The ligand can be a ligand amenable for affinity purification. Exemplary ligands for affinity purification include, but are not limited to, biotin, antigens, receptor ligands, strep-tag, strep-tactin and derivatives, and other peptide, hapten, dye- based tags-anti-Tag combinations such as SpyCatcher-SpyTag, His-Tag, Fc Tag, Digitonin, GFP, FAM, haptens, SNAP-TAG. HRP, FFAG, HA, myc, glutathione S-transferase (GST), maltose binding protein (MBP), small molecules, and the like.

[00185] It is noted that the ligand, e.g., the ligand for isolation/purification can be attached to the template strand at any position of the template strand. For example, the ligand, e.g., the ligand for isolation/purification can be attached to the template strand at 5 ’-end, 3 ’end or and internal position. In some embodiments of any one of the aspects, the ligand, e.g., the ligand for isolation/purification is linked to the 5 ’-end of the template strand.

[00186] The ligand, e.g., the ligand for isolation/purification can be attached to the template strand directly, e.g., by a bond or a linker.

Detectable labels

[00187] The nucleic acid strands provided herein can be modified with a detectable label. For example, the synthesized strand can comprise a detectable label. Without wishing to be bound by a theory, such a detectable label can facilitate detection. As used herein, the term “detectable label” refers to a composition capable of producing a detectable signal indicative of the presence of a target. Detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. [00188] A wide variety of fluorescent reporter dyes are known in the art. Typically, the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine or other like compound. [00189] Exemplary fluorophores 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); 5-TAMRA (5-Carboxytetramethylrhodamine); 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 SITSA; Aequorin (Photoprotein); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 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 (Y66H); 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; Chromomycin A; CMFDA; Coelenterazine ; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidm; CPM Methylcoumarm; 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 (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-l 6-ASP); DIDS; Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosm; Erythrosm ITC; Ethidium homodimer- 1 (EthD-1); Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4; Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl 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); bitrawhite 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; MagdalaRed (Phloxin B); Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; 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 Iavin E8G; Oregon Green™; Oregon Green 488-X; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosanibne (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; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 ; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primubne; Procion Yellow; Propidium Iodid (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; Tetracycline; Tetramethylrhodamine ; Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine RedR; 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; TnColor (PE-Cy5); TRITC (TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamme; 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.

[00190] Other exemplary detectable labels include 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. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference.

[00191] Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photo-detector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with an enzyme substrate and detecting the reaction product produced by the action of the enzyme on the enzyme substrate, and calorimetric labels can be detected by visualizing the colored label.

[00192] In some embodiments, the detectable label is a fluorophore or a quantum dot. Without wishing to be bound by a theory, using a fluorescent reagent can reduce signal-to-noise in the imaging/readout, thus maintaining sensitivity.

[00193] In some embodiments, a label can be configured to include a “smart label”, which is undetectable when conjugated with the nucleic acid template, the guide strand, and/or the synthesized nucleic acid sequence, but produces a color change when released.

[00194] Acrydite modifications can also be made to the nucleic acid template, a guide strand, and/or the synthesized nucleic acid sequence. Acrydite modifications can permit the oligonucleotides to be used in reactions with nucleophiles such as thiols (e.g, microarrays) or incorporated into gels (e.g, polyacrylamide). Accordingly, in some embodiments, the nucleic acid template, a guide strand, and/or the synthesized nucleic acid sequence comprises one or more acrydite nucleosides. The acrydite nucleoside can be at the 3’- end, 5-end, and/or at an internal position of the nucleic acid template, the guide strand, and/or the synthesized nucleic acid sequence. [00195] Any modifications to the nucleic acid template strand, primer strand, extension strand, and/or the synthesized nucleic acid strand provided herein that permit purification, extraction, quantification of expression, binding, electrophoresis, and the like, can be made.

Compositions

[00196] In another aspect, provided herein is a composition comprising one or more components, e.g., strands and reagents described herein. Exemplary compositions provided herein are generally depicted in FIGs. 3A-3H. Generally, the composition comprises a primer nucleic acid strand, a template nucleic acid strand, and optionally an extension nucleic acid strand. In certain embodiments, such compositions are intended for therapeutic and/or diagnostic applications. In certain embodiments, such compositions are intended for research use. The compositions can be useful in the methods described herein.

[00197] In some embodiments of any of the aspects, the composition comprises: a primer nucleic acid strand attached to a target binding molecule and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain is at the 3 ’-end of the primer strand. The template strand comprises a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain. The hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand. Optionally, neither the primer strand nor the template strand comprises a hairpin structure.

[00198] In some embodiments of any one of the aspects, the composition comprises a primer nucleic acid stand attached to a target binding molecule, an extension nucleic acid strand, and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain is at a terminus of the primer strand. The extension strand comprises a hybridization domain (b), a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c). The template strand comprises a first hybridization domain (a*); and a second hybridization domain (b*). The hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand. The hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the second hybridization domain (b*) of the template nucleic acid strand.

[00199] In some embodiments of any one of the aspects, the composition comprises: a primer nucleic acid stand is attached to a target binding molecule and an extension nucleic acid strand comprising a barcode sequence/domain and, optionally, a hybridization domain. At least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety, and/or one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[00200] In some embodiments of any one of the aspects, the composition comprises: a primer nucleic acid strand attached to a target binding molecule, and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain (a) is at 3 ’-terminus of the primer strand. The template strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker domain between the first stem domain and the second stem domain, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure. At least one of the first stem domain (b*), the second stem domain (b) and the linker domain of the template strand comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure. The hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand. Optionally, the template strand is in form of a hairpin.

[00201] A composition described herein can further comprise one or more reagents/components for nucleic acid polymerization and/or linking. Accordingly, in some embodiments of the various aspects described herein, the composition further comprises a polymerase. For example, a DNA polymerase and/or an RNA polymerase. In some embodiments of any of the aspects, the polymerase is a polymerase having strand displacing activity. In some embodiments of any of the aspects, the polymerase is a reverse transcriptase. In some embodiments of any one of the aspects, the composition comprises a reverse transcriptase and a DNA polymerase.

[00202] In some embodiments of any of the aspects, the composition further comprises a nuclease. For example, the composition further comprises an exonuclease, e.g., a 5’->3’ exonuclease, an endonuclease and/or an RNase.

[00203] In some embodiments of any of the aspects, the composition further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and/or buffers.

Kits

[00204] In yet another aspect, provided herein is a kit comprising one or more components, e.g. , strands and reagents described herein. Exemplary components are depicted in FIGs. 3A-3H. Generally, the kit comprises a primer nucleic acid strand, a template nucleic acid strand, and optionally an extension nucleic acid strand. In certain embodiments, such kits are intended for therapeutic and/or diagnostic applications. In certain embodiments, such kits are intended for research use. The kit can be useful in the methods described herein.

[00205] In some embodiments of any of the aspects, the kit comprises: a primer nucleic acid strand attached to a target binding molecule and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain is at the 3 ’-end of the primer strand. The template strand comprises a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain. The hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand. Optionally, neither the primer strand nor the template strand comprises a hairpin structure. [00206] In some embodiments of any one of the aspects, the kit comprises a primer nucleic acid stand attached to a target binding molecule, an extension nucleic acid strand, and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain is at a terminus of the primer strand. The extension strand comprises a hybridization domain (b), a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c). The template strand comprises a first hybridization domain (a*); and a second hybridization domain (b*). The hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand. The hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the second hybridization domain (b*) of the template nucleic acid strand. [00207] In some embodiments of any one of the aspects, the kit comprises: a primer nucleic acid stand is attached to a target binding molecule and an extension nucleic acid strand comprising a barcode sequence/domain and, optionally, a hybridization domain. At least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety, and/or one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[00208] In some embodiments of any one of the aspects, the kit comprises: a primer nucleic acid strand attached to a target binding molecule, and a template nucleic acid strand. The primer strand comprises a hybridization domain (a), optionally the hybridization domain (a) is at 3’- terminus of the primer strand. The template strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker domain between the first stem domain and the second stem domain, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure. At least one of the first stem domain (b*), the second stem domain (b) and the linker domain of the template strand comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure. The hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand. Optionally, the template strand is in form of a hairpin.

[00209] A kit described herein can further comprise one or more reagents/components for nucleic acid polymerization, nucleic acid linking, and/or nucleic acid strand cleavage/removal. Accordingly, in some embodiments of the various aspects described herein, the kit further comprises a polymerase. For example, a DNA polymerase and/or an RNA polymerase. In some embodiments of any of the aspects, the polymerase is a polymerase having strand displacing activity. In some embodiments of any of the aspects, the kit comprises a reverse transcriptase. In some embodiments of any one of the aspects, the kit comprises a reverse transcriptase and a DNA polymerase.

[00210] In some embodiments of any of the aspects, the kit further comprises a nuclease. For example, the composition kit comprises an exonuclease, e.g., a 5 ’->3’ exonuclease, an endonuclease and/or an RNase.

[00211] In some embodiments of any of the aspects, the kit further comprises one or more reagents for nucleic acid polymerization by a polymerase, such as deoxyribonucleotide triphosphates (dNTPs), salt and/or buffers.

[00212] In some embodiments of any of the aspects, the kit further comprises a light source, e.g. , for photo cross-linking.

[00213] In some embodiments of any of the aspects, the kit further comprises instructions for use.

[00214] Aspects of the disclosure can be described with the following numbered Embodiments: [00215] Embodiment 1: A method comprising: (a) annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, wherein the primer nucleic acid stand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises a hybridization domain (a), wherein the template nucleic acid strand comprises a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain, wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand; (b) extending the primer nucleic acid strand from its 3 ’-end by a polymerase to synthesize a nucleic acid strand; and (c) removing the template nucleic acid strand to produce a synthesized single-strand nucleic acid, and wherein the synthesized strand is attached to the target binding molecule.

[00216] Embodiment 2: The method of Embodiment 1, further comprising prior to step (c): (d) annealing or hybridizing the synthesized strand with a new template nucleic acid strand, wherein the new template strand comprises a hybridization domain (al*), a second domain (bl*), and optionally a linker domain between the hybridization domain and the second domain, wherein the hybridization domain (al*), of the new template strand comprises a nucleotide sequence having substantial identity to the second domain (b*) of the previous template strand; (e) extending the synthesized nucleic acid strand from its 3 ’-end by a polymerase to synthesize a new nucleic acid strand; and (f) optionally, repeating steps (d) and (e).

[00217] Embodiment 3: The method of Embodiment 1 or 2, further comprising after step (c): (d) annealing or hybridizing the synthesized strand with a new template nucleic acid strand, wherein the new template strand comprises a hybridization domain (al*), a second domain (bl*), and optionally a linker domain between the hybridization domain and the second domain, wherein the hybridization domain (al*), of the new template strand comprises a nucleotide sequence having substantial identity to the second domain (b*) of the previous template strand, extending the synthesized nucleic acid strand from its 3 ’-end by a polymerase to synthesize a new nucleic acid strand; (e) removing the new template nucleic acid strand to produce a synthesized new single strand nucleic acid; and (f) optionally, repeating steps (d)-(f).

[00218] Embodiment 4: A method comprising: (a) annealing or hybridizing together a primer nucleic acid strand, an extension nucleic acid strand and a template nucleic acid strand, wherein the primer nucleic acid stand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises at one end a hybridization domain (a), wherein the extension nucleic acid strand comprises a hybridization domain (b), wherein the template nucleic acid strand comprises a first hybridization domain (a*); and a second hybridization domain (b*), wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand, and wherein the hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand; (b) ligating an end of the primer nucleic acid strand with an end of the extension nucleic acid strand; and (c) removing the template nucleic acid strand to produce a synthesized single-strand nucleic acid, and wherein the synthesized strand is attached to the target binding molecule.

[00219] Embodiment 5: The method of Embodiment 4, further comprising prior to step (c): (d) annealing or hybridizing together the synthesized strand, a new extension nucleic acid strand and a new template nucleic acid strand, wherein the new extension nucleic acid strand comprises a hybridization domain (bl), a second domain (cl), and optionally a linker domain linking the hybridization domain (bl) and the second domain (cl), wherein the new template nucleic acid strand comprises a first hybridization domain (al*); and a second hybridization domain (bl*), wherein the first hybridization domain (al *) of the template new nucleic acid strand is substantially complementary to the second domain (c) of the previous extension nucleic acid strand, and wherein the hybridization domain (bl) of the new extension nucleic acid strand is substantially complementary to the second hybridization domain (bl*) of the new template nucleic acid strand; (e) ligating an end of the primer nucleic acid strand with an end of the extension nucleic acid strand; and (f) optionally, repeating steps (d) and (e).

[00220] Embodiment 6: The method of Embodiment 5, further comprising after step (c): (d) annealing or hybridizing together the synthesized strand, a new extension nucleic acid strand and a new template nucleic acid strand, wherein the new extension nucleic acid strand comprises a hybridization domain (bl), a second domain (cl), and optionally a linker domain linking the hybridization domain (bl) and the second domain (cl), wherein the new template nucleic acid strand comprises a first hybridization domain (al*); and a second hybridization domain (bl*), wherein the first hybridization domain (al *) of the template new nucleic acid strand is substantially complementary to the second domain (c) of the previous extension nucleic acid strand, and wherein the hybridization domain (bl) of the new extension nucleic acid strand is substantially complementary to the second hybridization domain (bl*) of the new template nucleic acid strand; (e) ligating an end of the primer nucleic acid strand with an end of the extension nucleic acid strand; and (f) removing the template nucleic acid strand to produce a synthesized new single-strand nucleic acid, and (g) optionally, repeating steps (d)-(f).

[00221] Embodiment 7: A method comprising: (a) linking a primer nucleic acid strand with an extension nucleic acid strand to produce a synthesized strand, wherein the primer stand is attached to a target binding molecule, wherein the extension nucleic acid strand comprises a barcode sequence/domain and, optionally, a hybridization domain, and wherein: (i) at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety; and/or (ii) one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair; and (b) optionally, removing any excess extension strands, and (c) wherein the synthesized strand is attached to the target binding molecule. [00222] Embodiment 8: The method of Embodiment 7, further comprising prior to step (b): (c)linking the synthesized strand with a new extension nucleic acid strand to produce a new synthesized strand, wherein the new extension nucleic acid strand comprises a barcode sequence/domain and, optionally, a hybridization domain; and (d) optionally, repeating step (c). [00223] Embodiment 9: The method of Embodiment 7 or 8, further comprising after step (b): (c) linking the synthesized strand with a new extension nucleic acid strand to produce a new synthesized strand, wherein the new extension nucleic acid strand comprises a barcode sequence/domain and, optionally, a hybridization domain; (d) optionally, repeating step (c); and (e) optionally, removing any excess extension strands.

[00224] Embodiment 10: A method comprising: (a) annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, wherein the primer strand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises a hybridization domain (a), wherein the template nucleic acid strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker domain between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker domain comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, wherein the hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand; (b) extending the target nucleic acid strand from its 3 ’-end by a polymerase having strand displacing activity to produce a synthesized strand; and (c) optionally, removing any excess template nucleic acid strand, and wherein the synthesized strand is attached to the target binding molecule.

[00225] Embodiment 11: The method of Embodiment 10, further comprising prior to step (c): (d) annealing or hybridizing synthesized strand with a new template nucleic acid strands, wherein the new template nucleic acid strand comprises in 3’ to 5’ direction: a hybridization domain (al*), a first stem domain (bl*), a second stem domain (bl), and a linker domain between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker domain comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, wherein the hybridization domain (al*) of the new template nucleic acid strand comprises a sequence having substantial identity to first stem domain of the previous template strand; (e) extending the target nucleic acid strand from its 3 ’-end by a polymerase having strand displacing activity to produce a synthesized strand; and (f) optionally, repeating steps (d) and (e).

[00226] Embodiment 12: The method of Embodiment 10 or 11, further comprising after step (c): (d) annealing or hybridizing synthesized strand with a new template nucleic acid strands, wherein the new template nucleic acid strand comprises in 3’ to 5’ direction: a hybridization domain (al*), a first stem domain (bl*), a second stem domain (bl), and a linker domain between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker domain comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, wherein the hybridization domain (al*) of the new template nucleic acid strand comprises a sequence having substantial identity to first stem domain of the previous template strand; (e) extending the target nucleic acid strand from its 3 ’-end by a polymerase having strand displacing activity to produce a synthesized strand; and (f) optionally, removing any excess template strands; and (g) optionally, repeating steps (d) - (f).

[00227] Embodiment 13 : The method of any of the preceding Embodiments, wherein the primer strand comprises a barcode sequence/domain.

[00228] Embodiment 14: The method of any one of the preceding Embodiments, wherein the primer strand is attached to the target binding molecule via its 5 ’-end. [00229] Embodiment 15 : The method of any one of the preceding Embodiments, wherein the primer strand is attached to the target binding molecule via its 3 ’-end.

[00230] Embodiment 16: The method of any one of the preceding Embodiments, wherein the primer strand is single-stranded.

[00231] Embodiment 17: The method of any of the preceding Embodiments, wherein the primer strand comprises a second domain.

[00232] Embodiment 18 : The method of any of the preceding Embodiments, wherein the second domain of the primer strand is 5’ of the hybridization domain of the primer strand.

[00233] Embodiment 19: The method of any of the preceding Embodiments, wherein the second domain of the primer strand is 3’ of the hybridization domain of the primer strand.

[00234] Embodiment 20: The method of any one of the preceding Embodiments, wherein the primer strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50-250, 50- 200, 50-150 or 50-100 nucleotides.

[00235] Embodiment 21: The method of any one of the preceding Embodiments, wherein the primer strand has a length of 25-100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100-150 or 100-200 nucleotides.

[00236] Embodiment 22: The method of any one of the preceding Embodiments, wherein the primer strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, 99 or 100 nucleotides.

[00237] Embodiment 23 : The method of any one of the preceding Embodiments, wherein the template strand comprises a barcode sequence/domain.

[00238] Embodiment 24: The method of any one of the preceding Embodiments, wherein the template strand is in form of a hairpin.

[00239] Embodiment 25: The method of any one of the preceding Embodiments, wherein the template strand is not in form of a hairpin.

[00240] Embodiment 26: The method of any one of the preceding Embodiments, wherein the template strand does not comprise a double-stranded region.

[00241] Embodiment 27: The method of any one of the preceding Embodiments, wherein the hybridization domain of the template nucleic acid strand is 3’ to the second domain of the template nucleic acid strand.

[00242] Embodiment 28: The method of any one of the preceding Embodiments, wherein the hybridization domain of the template nucleic acid strand is 5’ to the second domain of the template nucleic acid strand. [00243] Embodiment 29: The method of any one of the preceding Embodiments, wherein the first hybridization domain of the template nucleic acid strand is 3’ to the second hybridization domain of the template nucleic acid strand.

[00244] Embodiment 30: The method of any one of the preceding Embodiments, wherein the first hybridization domain of the template nucleic acid strand is 5’ to the second hybridization domain of the template nucleic acid strand.

[00245] Embodiment 31 : The method of any one of the preceding Embodiments, wherein the template nucleic acid comprises a barcode or a nucleotide sequence complementary to a barcode sequence/domain.

[00246] Embodiment 32: The method of any one of the preceding Embodiments, wherein the primer strand comprises a nucleic acid modification selected from the group consisting of modified intemucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof. [00247] Embodiment 33: The method of any one of the preceding Embodiments, wherein the template strand comprises a nucleic acid modification selected from the group consisting of modified intemucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof.

[00248] Embodiment 34: The method of any one of the preceding Embodiments, wherein the template strand comprises a nucleic acid modification capable of enhancing nucleic acid cleavage. [00249] Embodiment 35 : The method of any one of the preceding Embodiments, wherein the template strand comprises a nucleic acid modification selected from the group consisting of: a 5’- phosphate group; (b) a 2’-OH nucleotide (RNA nucleotide); (c) a nucleotide comprising a methylated nucleobase; (d) an abasic nucleotide; (e) an acyclic nucleotide; (f) a cleavable linker/spacer; and (g) any combinations of (a)-(f).

[00250] Embodiment 36: The method of any one of the preceding Embodiments, wherein the template strand comprises a phosphate group at the 5 ’-end.

[00251] Embodiment 37: The method of any one of the preceding Embodiments, wherein the template strand comprises a 5'-monophosphate; 5 '-diphosphate or a 5'-triphosphate at the 5’-end. [00252] Embodiment 38: The method of any one of the preceding Embodiments, wherein the template strand is RNA.

[00253] Embodiment 39: The method of any one any one of the preceding Embodiments, wherein the template strand comprises a modification capable of terminating nucleic acid polymerization.

[00254] Embodiment 40: The method of any one of the preceding Embodiments, wherein said modification capable of terminating nucleic acid polymerization is selected from the group consisting of: a spacer, an abasic nucleotide, an acyclic nucleotide, a modified nucleobase nucleotide, a non-natural nucleotide, CNVK, CNVD, an inverted dT modification, an inverted nucleobase, and any combinations thereof.

[00255] Embodiment 41: The method of any one of the preceding Embodiments, wherein the spacer is a C3 -spacer or a tri ethylene glycol spacer.

[00256] Embodiment 42: The method of any one of the preceding Embodiments, wherein the non-natural nucleotide is an iso-dG or an iso-dC.

[00257] Embodiment 43: The method of any one of the preceding Embodiments, wherein one or more domains utilize a 3 -letter code.

[00258] Embodiment 44: The method of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is in the first stem domain. [00259] Embodiment 45: The method of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is in the second stem domain. [00260] Embodiment 46: The method of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is in the linker.

[00261] Embodiment 47: The method of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is between the linker and one of the first or second stem domain.

[00262] Embodiment 48: The method of any one of the preceding Embodiments, wherein the template strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50-250, 50-200, 50-150 or 50-100 nucleotides.

[00263] Embodiment 49: The method of any one of the preceding Embodiments, wherein the template strand has a length of 25-100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100- 150 or 100-200 nucleotides.

[00264] Embodiment 50: The method of any one of the preceding Embodiments, wherein the template strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, 99 or 100 nucleotides.

[00265] Embodiment 51: The method of any of the preceding Embodiments, wherein the extension strand comprises a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c).

[00266] Embodiment 52: The method of any one of the preceding Embodiments, wherein the hybridization domain of the extension strand is 5’ of the second domain of the extension strand. [00267] Embodiment 53: The method of any one of the preceding Embodiments, wherein the hybridization domain of the extension strand is 3 ’ of the second domain of the extension strand. [00268] Embodiment 54: The method of any one of the preceding Embodiments, wherein the extension strand comprises a barcode sequence/domain.

[00269] Embodiment 55: The method of any one of the preceding Embodiments, wherein at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross- linking moiety.

[00270] Embodiment 56: The method of any one of the preceding Embodiments, wherein said cross-linking moiety is near or at a terminus of the primer nucleic acid strand or the extension nucleic acid strand.

[00271] Embodiment 57: The method of any one the preceding Embodiments, wherein said cross-linking moiety is a photo-cross linking moiety or chemical cross-linking moiety.

[00272] Embodiment 58: The method of any one of the preceding Embodiments, wherein the photo-cross linking moiety is selected from the group consisting of: psoralen, bromo- dU, 4-thio- dT (S4dT), 3-Cyanovinylcarbazole nucleoside (CNVK), 3-Cyanovinylcarbazole with D-threoninol (CNVD).

[00273] Embodiment 59: The method of any one of the preceding Embodiments, wherein said chemical cross-linking moiety comprises a chemical functional group selected from the group consisting of: maleimide, next generation maleimide, carbomide, NHS-ester, sulfo-NHS, azide, alkyne, tetrazine, DBCO, thiol, amine, carbonyl, carboxyl group, and any combinations thereof. [00274] Embodiment 60: The method of any one of the preceding Embodiments, wherein one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[00275] Embodiment 61: The method of any one of the preceding Embodiments, wherein the members of the binding pair are near or at a terminus of the template nucleic acid strand and the extension nucleic acid strand.

[00276] Embodiment 62: The method of any one of the preceding Embodiments or Embodiment 48, wherein the binding pair comprises biotin and avidin, biotin and streptavidin, a ligand receptor pair, neutravidin, strep-tag, strep-tactin, hapten, SpyTag, His-Tag, Fc Tag, digitonin, GFP, FAM, SNAP-TAG, horseradish peroxidase (HRP) tag, FLAG tag, HA tag, myc, glutathione S-transferase (GST), maltose binding protein (MBP), small molecule tags, or any combinations thereof.

[00277] Embodiment 63: The method of any one of the preceding Embodiments, wherein at least one of the hybridization domain of the primer strand and the hybridization domain of the extension nucleic acid strand comprises the cross-linking moiety.

[00278] Embodiment 64: The method of any y one of the preceding Embodiments, wherein one of the hybridization domain of the primer nucleic acid strand and the hybridization domain of the extension nucleic acid strand comprises one member of the binding pair and the other of the hybridization domain of the primer nucleic acid strand and the hybridization domain of the extension nucleic acid strand comprises second member of the binding pair.

[00279] Embodiment 65: The method of any one of the preceding Embodiments, wherein the extension strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50-250, 50-200, 50-150 or 50-100 nucleotides.

[00280] Embodiment 66: The method of any one of the preceding Embodiments, wherein the extension strand has a length of 25-100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100- 150 or 100-200 nucleotides.

[00281] Embodiment 67: The method of any one of the preceding Embodiments, wherein extension strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides.

[00282] Embodiment 68: The method of any one of the preceding Embodiments, wherein the extension stand is single stranded.

[00283] Embodiment 69: The method of any one of the preceding Embodiments, wherein the extension strand comprises a double-stranded region.

[00284] Embodiment 70: The method of any one of the preceding Embodiments, wherein the extension strand is in form of a hairpin.

[00285] Embodiment 71: The method of any one of the preceding Embodiments, wherein the target binding molecule is a synthetic or natural molecule.

[00286] Embodiment 72: The method of any one of the preceding Embodiments, wherein the target binding molecule is selected from the group consisting of: an antibody, a nanobody, an affibody, a receptor, a receptor ligand, an aptamer, a modified aptamer, an ankyrin-repeat protein, a peptide binder, a small molecule, and any combination thereof.

[00287] Embodiment 73 : The method of any one of the preceding Embodiments, wherein the target binding molecule is an antibody.

[00288] Embodiment 74: The method of any one of the preceding Embodiments, wherein the target binding molecule binds to a synthetic or a biological material.

[00289] Embodiment 75: The method of any one of the preceding Embodiments, wherein the target binding molecule binds to a cell.

[00290] Embodiment 76: The method of any one of the preceding Embodiments, wherein the target binding molecule binds to one or more molecules selected from the group consisting of: a lipid, a sugar, an oligo- or poly- saccharide, an amino acid, a peptide or a polypeptide, a nucleoside, a nucleotide, an oligo- or poly- nucleotide, a hormone, a vitamin, a small molecule, a miRNA, H2O2, a free-radical, metabolites, a nucleic acid, and any combinations thereof.

[00291] Embodiment 77: The method of any one of the preceding Embodiments, wherein the target binding molecule binds to two or more molecules selected from the group consisting of: a lipid, a sugar, an oligo- or poly- saccharide, an amino acid, a peptide or a polypeptide, a nucleoside, a nucleotide, an oligo- or poly- nucleotide, a hormone, a vitamin, a small molecule, a miRNA, H2O2, a free-radical, metabolites, a nucleic acid, and any combinations thereof.

[00292] Embodiment 78: The method of any one of the preceding Embodiments, wherein the target binding molecule binds a molecule that is DNA or RNA barcoded.

[00293] Embodiment 79: The method of any one of the preceding Embodiments, wherein each hybridization domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15- 20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00294] Embodiment 80: The method of any one of the preceding Embodiments, wherein each hybridization domain independently has alength of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00295] Embodiment 81 : The method of any one of the preceding Embodiments, wherein each second domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20- 40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00296] Embodiment 82: The method of any one of the preceding Embodiments, wherein each second domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00297] Embodiment 83: The method of any one of the preceding Embodiments, wherein each linker domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20- 40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00298] Embodiment 84: The method of any one of the preceding Embodiments, wherein each linker domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00299] Embodiment 85: The method of any one of the preceding Embodiments, wherein the toehold domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20- 40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. [00300] Embodiment 86: The method of any one of the preceding Embodiments, wherein the toehold domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00301] Embodiment 87: The method of any one of the preceding Embodiments, wherein the first stem domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5- 25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00302] Embodiment 88: The method of any one of the preceding Embodiments, wherein the first stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00303] Embodiment 89: The method of any one of the preceding Embodiments, wherein the second stem domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15- 20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00304] Embodiment 90: The method of any one of the preceding Embodiments, wherein the second stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00305] Embodiment 91: The method of any one of the preceding Embodiments, wherein the stem domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5- 20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20- 40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00306] Embodiment 92: The method of any one of the preceding Embodiments, wherein the stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00307] Embodiment 93 : The method of any one of the preceding Embodiments, wherein each barcode sequence/domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15- 25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. [00308] Embodiment 94: The method of any one of the preceding Embodiments, wherein each barcode sequence/domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00309] Embodiment 95: The method of any one of the preceding Embodiments, wherein said linking comprises ligation, cross-linking, or binding interaction.

[00310] Embodiment 96: The method of any one of the preceding Embodiments, wherein said linking comprises enzymatic ligation, chemical ligation, photo-crosslinking, or interaction binding. [00311] Embodiment 97: The method of any one of the preceding Embodiments, wherein said linking comprises linking 3 ’-end of the primer nucleic acid strand with 5 ’-end of the extension nucleic acid strand.

[00312] Embodiment 98: The method of any one of the preceding Embodiments, wherein said linking comprises linking 5 ’-end of the primer nucleic acid strand with 3 ’-end of the extension nucleic acid strand.

[00313] Embodiment 99: The method of any one of the preceding Embodiments, wherein said removing the template strand comprises dehybridizing the template strand from the primer and/or synthesized strand.

[00314] Embodiment 100: The method of any one of the preceding Embodiments, wherein said dehybridizing comprises contacting a complex comprising the template and the synthesized strand with formamide, DMSO, or a divalent ion chelator.

[00315] Embodiment 101 : The method of any one of the preceding Embodiments, wherein said dehybridizing comprises a temperature change.

[00316] Embodiment 102: The method of any one of the preceding Embodiments, wherein said dehybridizing comprises heating a complex comprising the template strand and the primer and/or synthesized strand.

[00317] Embodiment 103 : The method of any one of the preceding Embodiments, wherein said removing the template strand comprises hybridizing or annealing the template strand with a competing nucleic acid strand having a sequence complementary to the template strand.

[00318] Embodiment 104: The method of any one of the preceding Embodiments, wherein the template strand comprises on its 3 ’-terminus a toehold domain for the competing strand, and optionally the toehold domain comprises a sequence that is not complementary to the primer strand. [00319] Embodiment 105: The method of any one of the preceding Embodiments, wherein said removing the template strand comprises cleaving the template strand.

[00320] Embodiment 106: The method of any one of the preceding Embodiments, wherein said removing the template strand comprises enzymatic cleavage, photo-cleavage or chemical cleavage of the template strand.

[00321] Embodiment 107: The method of any one of the preceding Embodiments, wherein said removing the template strand comprises cleaving the template nucleic acid strand with a nuclease. [00322] Embodiment 108: The method of any one of the preceding Embodiments, wherein the nuclease is an exonuclease.

[00323] Embodiment 109: The method of any one of the preceding Embodiments, wherein the exonuclease is selected from the groups consisting of T7 exonuclease, lambda exonuclease, Exonuclease VIII, T5 exonuclease, RecJf, and any combinations thereof. [00324] Embodiment 110: The method of any one of the preceding Embodiments, wherein the nuclease is an endonuclease.

[00325] Embodiment 111: The method of any one of the preceding Embodiments, wherein the endonuclease is RNase HI, RNAse HP, endonuclease PI, endonuclease IV, uracil DNA glycosylase, and endonuclease VIII, and any combinations thereof.

[00326] Embodiment 112: The method of any one of the preceding Embodiments, wherein the nuclease is an RNase.

[00327] Embodiment 113: The method of any one of the preceding Embodiments, wherein the RNase is selected from the group consisting of XRN-1, RNase If, Monarch RNase A, RNase H, RNase HI, RNase HP, and any combinations thereof.

[00328] Embodiment 114: The method of any one of the preceding Embodiments, wherein the synthesized strand comprises a detectable label.

[00329] Embodiment 115: The method of any one of the preceding Embodiments, wherein the method comprises a step of isolating/purifying the target binding molecule bound to the template strand prior to removing the template strand.

[00330] Embodiment 116: The method of any one of the preceding Embodiments, wherein the method comprises a step of isolating/purifying the target binding molecule bound to the template strand by affinity purification prior to removing the template strand.

[00331] Embodiment 117: The method of any one of the preceding Embodiments, wherein the template strand comprises a ligand.

[00332] Embodiment 118: A composition comprising: (a) a primer nucleic acid strand attached to a target binding molecule and comprising at its 3 ’-end a hybridization domain (a); and (b) a template nucleic acid strand comprising a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain, and wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand, and, optionally, wherein neither the primer strand nor the template strand comprises a hairpin structure.

[00333] Embodiment 119: A composition comprising: (a) a primer nucleic acid stand is attached to a target binding molecule and comprising at one terminus a hybridization domain (a) and optionally a second domain; (b) an extension nucleic acid strand comprising a hybridization domain (b), a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c); and (c) a template nucleic acid strand comprises a first hybridization domain (a*); and a second hybridization domain (b*), and wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand, and wherein the hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand.

[00334] Embodiment 120: A composition comprising: (a) a primer nucleic acid stand is attached to a target binding molecule; and (b) an extension nucleic acid strand comprising a barcode sequence/domain and, optionally, a hybridization domain, and wherein at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety, and/or wherein one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[00335] Embodiment 121 : A composition comprising: (a) a primer nucleic acid strand, wherein the primer strand is attached to a target binding molecule and comprises at its 3 ’-end a hybridization domain (a); and (b) a template nucleic acid strand, wherein the template strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, and wherein the hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand.

[00336] Embodiment 122: The composition of any of the preceding Embodiments, wherein the primer strand comprises a barcode sequence/domain.

[00337] Embodiment 123 : The composition of any one of the preceding Embodiments, wherein the primer strand is attached to the target binding molecule via its 5 ’-end.

[00338] Embodiment 124: The composition of any one of the preceding Embodiments, wherein the primer strand is attached to the target binding molecule via its 3 ’-end.

[00339] Embodiment 125: The composition of any one of the preceding Embodiments, wherein the primer strand is single-stranded.

[00340] Embodiment 126: The composition of any of the preceding Embodiments, wherein the primer strand comprises a second domain.

[00341] Embodiment 127: The composition of any of the preceding Embodiments, wherein the second domain of the primer strand is 5’ of the hybridization domain of the primer strand.

[00342] Embodiment 128: The composition of any of the preceding Embodiments, wherein the second domain of the primer strand is 3’ of the hybridization domain of the primer strand. [00343] Embodiment 129: The composition of any one of the preceding Embodiments, wherein the primer strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50-250, 50-200, 50-150 or 50-100 nucleotides.

[00344] Embodiment 130: The composition of any one of the preceding Embodiments, wherein the primer strand has alength of 25-100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100- 150 or 100-200 nucleotides.

[00345] Embodiment 131: The composition of any one of the preceding Embodiments, wherein the primer strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

94, 95, 96, 97, 98, 99 or 100 nucleotides.

[00346] Embodiment 132: The composition of any one of the preceding Embodiments, wherein the template strand comprises a barcode sequence/domain.

[00347] Embodiment 133: The composition of any one of the preceding Embodiments, wherein the template strand is in form of a hairpin.

[00348] Embodiment 134: The composition of any one of the preceding Embodiments, wherein the template strand is not in form of a hairpin.

[00349] Embodiment 135: The composition of any one of the preceding Embodiments, wherein the template strand does not comprise a double-stranded region.

[00350] Embodiment 136: The composition of any one of the preceding Embodiments, wherein the hybridization domain of the template nucleic acid strand is 3’ to the second domain of the template nucleic acid strand.

[00351] Embodiment 137: The composition of any one of the preceding Embodiments, wherein the hybridization domain of the template nucleic acid strand is 5’ to the second domain of the template nucleic acid strand.

[00352] Embodiment 138: The composition of any one of the preceding Embodiments, wherein the first hybridization domain of the template nucleic acid strand is 3’ to the second hybridization domain of the template nucleic acid strand.

[00353] Embodiment 139: The composition of any one of the preceding Embodiments, wherein the first hybridization domain of the template nucleic acid strand is 5’ to the second hybridization domain of the template nucleic acid strand.

[00354] Embodiment 140: The composition of any one of the preceding Embodiments, wherein the template nucleic acid comprises a barcode or a nucleotide sequence complementary to a barcode sequence/domain. [00355] Embodiment 141 : The composition of any one of the preceding Embodiments, wherein the primer strand comprises a nucleic acid modification selected from the group consisting of: modified intemucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof.

[00356] Embodiment 142: The composition of any one of the preceding Embodiments, wherein the template strand comprises a nucleic acid modification selected from the group consisting of modified intemucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof.

[00357] Embodiment 143 : The composition of any one of the preceding Embodiments, wherein the template strand comprises a nucleic acid modification capable of enhancing nucleic acid cleavage.

[00358] Embodiment : 144 The composition of any one of the preceding Embodiments, wherein the template strand comprises a nucleic acid modification selected from the group consisting of: (a) a 5’-phosphate group; (b) a 2’-OH nucleotide (RNA nucleotide); (c) a nucleotide comprising a methylated nucleobase; (d) an abasic nucleotide; (e) an acyclic nucleotide; (f) a cleavable linker/spacer; and (g) any combinations of (a)-(f).

[00359] Embodiment 145: The composition of any one of the preceding Embodiments, wherein the template strand comprises a phosphate group at the 5 ’-end.

[00360] Embodiment 146: The composition of any one of the preceding Embodiments, wherein the template strand comprises a 5'-monophosphate; 5 '-diphosphate or a 5'-triphosphate at the 5’- end.

[00361] Embodiment : 147 The composition of any one of the preceding Embodiments, wherein the template strand is RNA.

[00362] Embodiment 148: The composition of any one any one of the preceding Embodiments, wherein the template strand comprises a modification capable of terminating nucleic acid polymerization.

[00363] Embodiment 149: The composition of any one of the preceding Embodiments wherein said modification capable of terminating nucleic acid polymerization is selected from the group consisting of: a spacer, an abasic nucleotide, an acyclic nucleotide, psoralen, bromo- dU, 4-thio-dT (S4dT), 3-Cyanovinylcarbazole nucleoside (CNVK), 3-Cyanovinylcarbazole with D-threoninol (CNVD), a modified nucleobase nucleotide, a non-natural nucleotide, an inverted dT modification, an inverted nucleobase, and any combinations thereof.

[00364] Embodiment 150: The composition of any one of the preceding Embodiments, wherein the spacer is a C3 -spacer or a tri ethylene glycol spacer. [00365] Embodiment 151: The composition of any one of the preceding Embodiments, wherein the non-natural nucleotide is an iso-dG or an iso-dC.

[00366] Embodiment 152: The composition of any one of the preceding Embodiments, wherein one or more domains utilize a 3 -letter code.

[00367] Embodiment 153: The composition of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is in the first stem domain. [00368] Embodiment 154: The composition of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is in the second stem domain. [00369] Embodiment 155: The composition of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is in the linker.

[00370] Embodiment 156: The composition of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is between the linker and one of the first or second stem domain.

[00371] Embodiment 157: The composition of any one of the preceding Embodiments, wherein the template strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50- 250, 50-200, 50-150 or 50-100 nucleotides.

[00372] Embodiment 158: The composition of any one of the preceding Embodiments, wherein the template strand has a length of 25-100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100-150 or 100-200 nucleotides.

[00373] Embodiment 159: The composition of any one of the preceding Embodiments, wherein the template strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

94, 95, 96, 97, 98, 99 or 100 nucleotides.

[00374] Embodiment 160: The composition of any of the preceding Embodiments, wherein the extension strand comprises a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c).

[00375] Embodiment 161 : The composition of any one of the preceding Embodiments, wherein the hybridization domain of the extension strand is 5’ of the second domain of the extension strand. [00376] Embodiment 162: The composition of any one of the preceding Embodiments, wherein the hybridization domain of the extension strand is 3 ’ of the second domain of the extension strand. [00377] Embodiment 163 : The composition of any one of the preceding Embodiments, wherein the extension strand comprises a barcode sequence/domain. [00378] Embodiment 164: The composition of any one of the preceding Embodiments, wherein at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety.

[00379] Embodiment 165: The composition of any one of the preceding Embodiments, wherein said cross-linking moiety is near or at a terminus of the primer nucleic acid strand or the extension nucleic acid strand.

[00380] Embodiment 166: The composition of any one the preceding Embodiments, wherein said cross-linking moiety is a photo-cross linking moiety or chemical cross-linking moiety. [00381] Embodiment 167: The composition of any one of the preceding Embodiments, wherein the photo-cross linking moiety is selected from the group consisting of: psoralen, bromo- dU, 4- thio-dT (S4dT), 3-Cyanovinylcarbazole nucleoside (CNVK), 3-Cyanovinylcarbazole with D- threoninol (CNVD).

[00382] Embodiment 168: The composition of any one of the preceding Embodiments, wherein said chemical cross-linking moiety comprises a chemical functional group selected from the group consisting of: maleimide, next generation maleimide, carbomide, NHS-ester, sulfo-NHS, azide, alkyne, tetrazine, DBCO, thiol, amine, carbonyl, carboxyl group, and any combinations thereof. [00383] Embodiment 169: The composition of any one of the preceding Embodiments, wherein one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[00384] Embodiment 170: The composition of any one of the preceding Embodiments, wherein the members of the binding pair are near or at a terminus of the template nucleic acid strand and the extension nucleic acid strand.

[00385] Embodiment 171: The composition of any one of the preceding Embodiments or 48, wherein the binding pair comprises biotin and avidin, biotin and streptavidin, a ligand receptor pair, neutravidin, strep-tag, strep-tactin, hapten, SpyTag, His-Tag, Fc Tag, digitonin, GFP, FAM, SNAP- TAG, horseradish peroxidase (HRP) tag, FLAG tag, HA tag, myc, glutathione S-transferase (GST), maltose binding protein (MBP), small molecule tags, or any combinations thereof.

[00386] Embodiment 172: The composition of any one of the preceding Embodiments, wherein at least one of the hybridization domain of the primer strand and the hybridization domain of the extension nucleic acid strand comprises the cross-linking moiety.

[00387] Embodiment 173 : The composition of any one of the preceding Embodiments, wherein one of the hybridization domain of the primer nucleic acid strand and the hybridization domain of the extension nucleic acid strand comprises one member of the binding pair and the other of the hybridization domain of the primer nucleic acid strand and the hybridization domain of the extension nucleic acid strand comprises second member of the binding pair.

[00388] Embodiment 174: The composition of any one of the preceding Embodiments, wherein the extension strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50- 250, 50-200, 50-150 or 50-100 nucleotides.

[00389] Embodiment 175: The composition of any one of the preceding Embodiments, wherein the extension strand has a length of 25-100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100-150 or 100-200 nucleotides.

[00390] Embodiment 176: The composition of any one of the preceding Embodiments, wherein extension strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, 99 or 100 nucleotides.

[00391] Embodiment 177: The composition of any one of the preceding Embodiments, wherein the extension stand is single stranded.

[00392] Embodiment 178: The composition of any one of the preceding Embodiments, wherein the extension strand comprises a double-stranded region.

[00393] Embodiment 179: The composition of any one of the preceding Embodiments, wherein the extension strand is in form of a hairpin.

[00394] Embodiment 180: The composition of any one of the preceding Embodiments, wherein the target binding molecule is a synthetic or natural molecule.

[00395] Embodiment 181: The composition of any one of the preceding Embodiments, wherein the target binding molecule is selected from the group consisting of: an antibody, a nanobody, an affibody, a receptor, a receptor ligand, an aptamer, a modified aptamer, an ankyrin-repeat protein, a peptide binder, a small molecule, and any combination thereof.

[00396] Embodiment 182: The composition of any one of the preceding Embodiments, wherein the target binding molecule is an antibody.

[00397] Embodiment 183 : The composition of any one of the preceding Embodiments, wherein the target binding molecule binds to a synthetic or a biological material.

[00398] Embodiment 184: The composition of any one of the preceding Embodiments, wherein the target binding molecule binds to a cell.

[00399] Embodiment 185: The composition of any one of the preceding Embodiments, wherein the target binding molecule binds to a molecule selected from the group consisting of: a lipid, a sugar, an oligo- or poly- saccharide, an amino acid, a peptide or a polypeptide, a nucleoside, a nucleotide, an oligo- or poly- nucleotide, a hormone, a vitamin, a small molecule, a miRNA, H2O2, a free-radical, metabolites, a nucleic acid, and any combinations thereof.

[00400] Embodiment 186: The composition of any one of the preceding Embodiments, wherein the target binding molecule binds to two or more molecules selected from the group consisting of: a lipid, a sugar, an oligo- or poly- saccharide, an amino acid, a peptide or a polypeptide, a nucleoside, a nucleotide, an oligo- or poly- nucleotide, a hormone, a vitamin, a small molecule, a miRNA, H2O2, a free-radical, metabolites, a nucleic acid, and any combinations thereof.

[00401] Embodiment 187: The composition of any one of the preceding Embodiments, wherein the target binding molecule binds a molecule that is DNA or RNA barcoded.

[00402] Embodiment 188: The composition of any one of the preceding Embodiments, wherein each hybridization domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15- 25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. [00403] Embodiment 189: The composition of any one of the preceding Embodiments, wherein each hybridization domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00404] Embodiment 190: The composition of any one of the preceding Embodiments, wherein each second domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15- 20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00405] Embodiment 191 : The composition of any one of the preceding Embodiments, wherein each second domain independently has a length of2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00406] Embodiment 192: The composition of any one of the preceding Embodiments, wherein each linker domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5- 25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00407] Embodiment 193 : The composition of any one of the preceding Embodiments, wherein each linker domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00408] Embodiment 194: The composition of any one of the preceding Embodiments, wherein the toehold domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5- 25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. [00409] Embodiment 195: The composition of any one of the preceding Embodiments, wherein the toehold domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00410] Embodiment 196: The composition of any one of the preceding Embodiments, wherein the first stem domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15- 20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00411] Embodiment 197: The composition of any one of the preceding Embodiments, wherein the first stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00412] Embodiment 198: The composition of any one of the preceding Embodiments, wherein the second stem domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5- 30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. [00413] Embodiment 199: The composition of any one of the preceding Embodiments, wherein the second stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00414] Embodiment 200: The composition of any one of the preceding Embodiments, wherein the stem domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20- 40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00415] Embodiment 201 : The composition of any one of the preceding Embodiments, wherein the stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00416] Embodiment 202: The composition of any one of the preceding Embodiments, wherein each barcode sequence/domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5- 35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. [00417] Embodiment 203 : The composition of any one of the preceding Embodiments, wherein each barcode sequence/domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00418] Embodiment 204: The composition of any one of the preceding Embodiments, wherein the template strand comprises a ligand.

[00419] Embodiment 205: The composition of any one of the preceding Embodiments, wherein the composition further comprises a polymerase. [00420] Embodiment 206: The composition of any one of the preceding Embodiments, wherein the polymerase is a DNA polymerase.

[00421] Embodiment 207: The composition of any one of the preceding Embodiments, wherein the polymerase is a polymerase having strand displacing activity.

[00422] Embodiment 208: The composition of any one of the preceding Embodiments, wherein the polymerase is an RNA polymerase.

[00423] Embodiment 209: The composition of any one of the preceding Embodiments, wherein the polymerase is a reverse transcriptase.

[00424] Embodiment 210: The composition of any one of the preceding Embodiments, wherein the composition further comprises a nuclease.

[00425] Embodiment 211: The composition of any one of the preceding Embodiments, wherein the nuclease is an exonuclease, e.g., a 5’->3’ exonuclease.

[00426] Embodiment 212: The composition of any one of the preceding Embodiments, wherein the nuclease is an endonuclease.

[00427] Embodiment 213: The composition of any one of the preceding Embodiments, wherein the nuclease is an RNase.

[00428] Embodiment 214: The composition of any one of the preceding Embodiments, wherein the composition further comprises one or more reagents for nucleic acid polymerization by a polymerase.

[00429] Embodiment 215: The composition of any one of the preceding Embodiments, wherein the composition further comprises deoxyribonucleotide triphosphates (dNTPs).

[00430] Embodiment 216: The composition of any one of the preceding Embodiments, wherein the composition further comprises a salt.

[00431] Embodiment 217: The composition of any one of the preceding Embodiments, wherein the composition further comprises a buffer.

[00432] Embodiment 218: A kit comprising: (a) a primer nucleic acid strand attached to a target binding molecule and comprising at its 3 ’-end a hybridization domain (a); and (b) a template nucleic acid strand comprising a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain, and wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand, and, optionally, wherein neither the primer strand nor the template strand comprises a hairpin structure.

[00433] Embodiment 219: A kit comprising: (a) a primer nucleic acid stand is attached to a target binding molecule and comprising at one terminus a hybridization domain (a) and optionally a second domain; (b) an extension nucleic acid strand comprising a hybridization domain (b), a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c); and (c) a template nucleic acid strand comprises a first hybridization domain (a*); and a second hybridization domain (b*), and wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand, and wherein the hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand.

[00434] Embodiment 220: A kit comprising: (a) a primer nucleic acid stand is attached to a target binding molecule; and (b) an extension nucleic acid strand comprising a barcode sequence/domain and, optionally, a hybridization domain, and wherein at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety, and/or wherein one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[00435] Embodiment 221 : A kit comprising: (a) a primer nucleic acid strand, wherein the primer strand is attached to a target binding molecule and comprises at its 3 ’-end a hybridization domain (a); and (b) a template nucleic acid strand, wherein the template strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, and wherein the hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand.

[00436] Embodiment 222: The kit of any of the preceding Embodiments, wherein the primer strand comprises a barcode sequence/domain.

[00437] Embodiment 223: The kit of any one of the preceding Embodiments, wherein the primer strand is attached to the target binding molecule via its 5 ’-end.

[00438] Embodiment 224: The kit of any one of the preceding Embodiments, wherein the primer strand is attached to the target binding molecule via its 3 ’-end.

[00439] Embodiment 225: The kit of any one of the preceding Embodiments, wherein the primer strand is single-stranded.

[00440] Embodiment 226: The kit of any of the preceding Embodiments, wherein the primer strand comprises a second domain. [00441] Embodiment 227: The kit of any of the preceding Embodiments, wherein the second domain of the primer strand is 5’ of the hybridization domain of the primer strand.

[00442] Embodiment 228: The kit of any of the preceding Embodiments, wherein the second domain of the primer strand is 3’ of the hybridization domain of the primer strand.

[00443] Embodiment 229: The kit of any one of the preceding Embodiments, wherein the primer strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50-250, 50- 200, 50-150 or 50-100 nucleotides.

[00444] Embodiment 230: The kit of any one of the preceding Embodiments, wherein the primer strand has a length of 25-100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100-150 or 100-200 nucleotides.

[00445] Embodiment 231: The kit of any one of the preceding Embodiments, wherein the primer strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

95, 96, 97, 98, 99 or 100 nucleotides.

[00446] Embodiment 232: The kit of any one of the preceding Embodiments, wherein the template strand comprises a barcode sequence/domain.

[00447] Embodiment 233: The kit of any one of the preceding Embodiments, wherein the template strand is in form of a hairpin.

[00448] Embodiment 234: The kit of any one of the preceding Embodiments, wherein the template strand is not in form of a hairpin.

[00449] Embodiment 235: The kit of any one of the preceding Embodiments, wherein the template strand does not comprise a double-stranded region.

[00450] Embodiment 236: The kit of any one of the preceding Embodiments, wherein the hybridization domain of the template nucleic acid strand is 3’ to the second domain of the template nucleic acid strand.

[00451] Embodiment 237: The kit of any one of the preceding Embodiments, wherein the hybridization domain of the template nucleic acid strand is 5’ to the second domain of the template nucleic acid strand.

[00452] Embodiment 238: The kit of any one of the preceding Embodiments, wherein the first hybridization domain of the template nucleic acid strand is 3’ to the second hybridization domain of the template nucleic acid strand.

[00453] Embodiment 239: The kit of any one of the preceding Embodiments, wherein the first hybridization domain of the template nucleic acid strand is 5’ to the second hybridization domain of the template nucleic acid strand. [00454] Embodiment 240: The kit of any one of the preceding Embodiments, wherein the template nucleic acid comprises a barcode or a nucleotide sequence complementary to a barcode sequence/domain.

[00455] Embodiment 241: The kit of any one of the preceding Embodiments, wherein the primer strand comprises a nucleic acid modification selected from the group consisting of: modified intemucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof. [00456] Embodiment 242: The kit of any one of the preceding Embodiments, wherein the template strand comprises a nucleic acid modification selected from the group consisting of modified intemucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof.

[00457] Embodiment 243: The kit of any one of the preceding Embodiments, wherein the template strand comprises a nucleic acid modification capable of enhancing nucleic acid cleavage. [00458] Embodiment 244: The kit of any one of the preceding Embodiments, wherein the template strand comprises a nucleic acid modification selected from the group consisting of: (a) a 5 ’-phosphate group; (b) a 2’ -OH nucleotide (RNA nucleotide); (c) a nucleotide comprising a methylated nucleobase; (d) an abasic nucleotide; (e)an acyclic nucleotide; (f) a cleavable linker/spacer; and (g) any combinations of (a)-(f).

[00459] Embodiment 245: The kit of any one of the preceding Embodiments, wherein the template strand comprises a phosphate group at the 5 ’-end.

[00460] Embodiment 246: The kit of any one of the preceding Embodiments, wherein the template strand comprises a 5'-monophosphate; 5 '-diphosphate or a 5 '-triphosphate at the 5’-end. [00461] Embodiment 247: The kit of any one of the preceding Embodiments, wherein the template strand is RNA.

[00462] Embodiment 248: The kit of any one any one of the preceding Embodiments, wherein the template strand comprises a modification capable of terminating nucleic acid polymerization. [00463] Embodiment 249: The kit of any one of the preceding Embodiments wherein said modification capable of terminating nucleic acid polymerization is selected from the group consisting of: a spacer, an abasic nucleotide, an acyclic nucleotide, a modified nucleobase nucleotide, a non-natural nucleotide, CNVK, CNVD, an inverted dT modification, an inverted nucleobase, and any combinations thereof.

[00464] Embodiment 250: The kit of any one of the preceding Embodiments, wherein the spacer is a C3 -spacer or a tri ethylene glycol spacer.

[00465] Embodiment 251 : The kit of any one of the preceding Embodiments, wherein the non natural nucleotide is an iso-dG or an iso-dC. [00466] Embodiment 252: The kit of any one of the preceding Embodiments, wherein one or more domains utilize a 3 -letter code.

[00467] Embodiment 253: The kit of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is in the first stem domain. [00468] Embodiment 254: The kit of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is in the second stem domain. [00469] Embodiment 255: The kit of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is in the linker.

[00470] Embodiment 256: The kit of any one of the preceding Embodiments, wherein the modification capable of terminating nucleic acid polymerization is between the linker and one of the first or second stem domain.

[00471] Embodiment 257: The kit of any one of the preceding Embodiments, wherein the template strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50-250, 50-200, 50-150 or 50-100 nucleotides.

[00472] Embodiment 258: The kit of any one of the preceding Embodiments, wherein the template strand has a length of 25-100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100- 150 or 100-200 nucleotides.

[00473] Embodiment 259: The kit of any one of the preceding Embodiments, wherein the template strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

[00474] Embodiment 260: The kit of any of the preceding Embodiments, wherein the extension strand comprises a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c).

[00475] Embodiment 261: The kit of any one of the preceding Embodiments, wherein the hybridization domain of the extension strand is 5’ of the second domain of the extension strand. [00476] Embodiment 262: The kit of any one of the preceding Embodiments, wherein the hybridization domain of the extension strand is 3’ of the second domain of the extension strand. [00477] Embodiment 263: The kit of any one of the preceding Embodiments, wherein the extension strand comprises a barcode sequence/domain.

[00478] Embodiment 264: The kit of any one of the preceding Embodiments, wherein at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety. [00479] Embodiment 265: The kit of any one of the preceding Embodiments, wherein said cross-linking moiety is near or at a terminus of the primer nucleic acid strand or the extension nucleic acid strand.

[00480] Embodiment 266: The kit of any one the preceding Embodiments, wherein said cross- linking moiety is a photo-cross linking moiety or chemical cross-linking moiety.

[00481] Embodiment 267: The kit of any one of the preceding Embodiments, wherein the photo-cross linking moiety is selected from the group consisting of: psoralen, bromo- dU, 4-thio- dT (S4dT), 3-Cyanovinylcarbazole nucleoside (CNVK), 3-Cyanovinylcarbazole with D-threoninol (CNVD).

[00482] Embodiment 268: The kit of any one of the preceding Embodiments, wherein said chemical cross-linking moiety comprises a chemical functional group selected from the group consisting of: maleimide, next generation maleimide, carbomide, NHS-ester, sulfo-NHS, azide, alkyne, tetrazine, DBCO, thiol, amine, carbonyl, carboxyl group, and any combinations thereof. [00483] Embodiment 269: The kit of any one of the preceding Embodiments, wherein one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

[00484] Embodiment 270: The kit of any one of the preceding Embodiments, wherein the members of the binding pair are near or at a terminus of the template nucleic acid strand and the extension nucleic acid strand.

[00485] Embodiment 271: The kit of any one of the preceding Embodiments, wherein the binding pair comprises biotin and avidin, biotin and streptavidin, a ligand receptor pair, neutravidin, strep-tag, strep-tactin, hapten, SpyTag, His-Tag, Fc Tag, digitonin, GFP, FAM, SNAP-TAG, horseradish peroxidase (HRP) tag, FLAG tag, HA tag, myc, glutathione S-transferase (GST), maltose binding protein (MBP), small molecule tags, or any combinations thereof.

[00486] Embodiment 272: The kit of any one of the preceding Embodiments, wherein at least one of the hybridization domain of the primer strand and the hybridization domain of the extension nucleic acid strand comprises the cross-linking moiety.

[00487] Embodiment 273 : The kit of any one of the preceding Embodiments, wherein one of the hybridization domain of the primer nucleic acid strand and the hybridization domain of the extension nucleic acid strand comprises one member of the binding pair and the other of the hybridization domain of the primer nucleic acid strand and the hybridization domain of the extension nucleic acid strand comprises second member of the binding pair. [00488] Embodiment 274: The kit of any one of the preceding Embodiments, wherein the extension strand has a length of 25-300, 25-250, 25-200, 25-150, 25-100, 25-50, 50-300, 50-250, 50-200, 50-150 or 50-100 nucleotides.

[00489] Embodiment 275: The kit of any one of the preceding Embodiments, wherein the extension strand has a length of 25-100, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, 100-125, 100- 150 or 100-200 nucleotides.

[00490] Embodiment 276: The kit of any one of the preceding Embodiments, wherein extension strand has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,

45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,

97, 98, 99 or 100 nucleotides.

[00491] Embodiment 277: The kit of any one of the preceding Embodiments, wherein the extension stand is single stranded.

[00492] Embodiment 278: The kit of any one of the preceding Embodiments, wherein the extension strand comprises a double-stranded region.

[00493] Embodiment 279: The kit of any one of the preceding Embodiments, wherein the extension strand is in form of a hairpin.

[00494] Embodiment 280: The kit of any one of the preceding Embodiments, wherein the target binding molecule is a synthetic or natural molecule.

[00495] Embodiment 281: The kit of any one of the preceding Embodiments, wherein the target binding molecule is selected from the group consisting of: an antibody, a nanobody, an affibody, a receptor, a receptor ligand, an aptamer, a modified aptamer, an ankyrin-repeat protein, a peptide binder, a small molecule, and any combination thereof.

[00496] Embodiment 282: The kit of any one of the preceding Embodiments, wherein the target binding molecule is an antibody.

[00497] Embodiment 283 : The kit of any one of the preceding Embodiments, wherein the target binding molecule binds to a synthetic or a biological material.

[00498] Embodiment 284: The kit of any one of the preceding Embodiments, wherein the target binding molecule binds to a cell.

[00499] Embodiment 285 : The kit of any one of the preceding Embodiments, wherein the target binding molecule binds to a molecule selected from the group consisting of: a lipid, a sugar, an oligo- or poly- saccharide, an amino acid, a peptide or a polypeptide, a nucleoside, a nucleotide, an oligo- or poly- nucleotide, a hormone, a vitamin, a small molecule, a miRNA, H2O2, a free-radical, metabolites, a nucleic acid, and any combinations thereof. [00500] Embodiment 286: The composition of any one of the preceding Embodiments, wherein the target binding molecule binds a molecule that is DNA or RNA barcoded.

[00501] Embodiment 287: The kit of any one of the preceding Embodiments, wherein each hybridization domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15- 20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00502] Embodiment 288: The kit of any one of the preceding Embodiments, wherein each hybridization domain independently has alength of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00503] Embodiment 289: The kit of any one of the preceding Embodiments, wherein each second domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20- 40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00504] Embodiment 290: The kit of any one of the preceding Embodiments, wherein each second domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00505] Embodiment 291: The kit of any one of the preceding Embodiments, wherein each linker domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20- 40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00506] Embodiment 292: The kit of any one of the preceding Embodiments, wherein each linker domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00507] Embodiment 293: The kit of any one of the preceding Embodiments, wherein the toehold domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20- 40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00508] Embodiment 294: The kit of any one of the preceding Embodiments, wherein the toehold domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00509] Embodiment 295: The kit of any one of the preceding Embodiments, wherein the first stem domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5- 20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20- 40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. [00510] Embodiment 296: The kit of any one of the preceding Embodiments, wherein the first stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00511] Embodiment 297: The kit of any one of the preceding Embodiments, wherein the second stem domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15- 20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00512] Embodiment 298: The kit of any one of the preceding Embodiments, wherein the second stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00513] Embodiment 299: The kit of any one of the preceding Embodiments, wherein the stem domain independently has alength of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5- 15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20- 35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides.

[00514] Embodiment 300: The kit of any one of the preceding Embodiments, wherein the stem domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, 25, 30, 35 or 40 nucleotides.

[00515] Embodiment 301: The kit of any one of the preceding Embodiments, wherein each barcode sequence/domain independently has a length of 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15- 25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35 or 35-40 nucleotides. [00516] Embodiment 302: The kit of any one of the preceding Embodiments, wherein each barcode sequence/domain independently has a length of 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35 or 40 nucleotides.

[00517] Embodiment 303: The kit of any one of the preceding Embodiments, wherein the template strand comprises a ligand.

[00518] Embodiment 304: The kit of any one of the preceding Embodiments, wherein the kit further comprises a polymerase.

[00519] Embodiment 305: The kit of any one of the preceding Embodiments, wherein the polymerase is a DNA polymerase.

[00520] Embodiment 306: The kit of any one of the preceding Embodiments, wherein the polymerase is a polymerase having strand displacing activity.

[00521] Embodiment 307: The kit of any one of the preceding Embodiments, wherein the polymerase is an RNA polymerase. [00522] Embodiment 308: The kit of any one of the preceding Embodiments, wherein the polymerase is a reverse transcriptase.

[00523] Embodiment 309: The kit of any one of the preceding Embodiments, wherein the kit further comprises a nuclease.

[00524] Embodiment 310: The kit of any one of the preceding Embodiments, wherein the kit further comprises a polymerase with internal nuclease activity.

[00525] Embodiment 311: The kit of any one of the preceding Embodiments, wherein the nuclease is an exonuclease.

[00526] Embodiment 312: The kit of any one of the preceding Embodiments, wherein the exonuclease is a 5 ’->3’ exonuclease.

[00527] Embodiment 313: The kit of any one of the preceding Embodiments, wherein the nuclease is an endonuclease.

[00528] Embodiment 314: The kit of any one of the preceding Embodiments, wherein the endonuclease is selected from the group consisting of: RNase HI, RNAse HP, endonuclease III, endonuclease IV, uracil DNA glycosylase, and endonuclease VIII, and any combinations thereof. [00529] Embodiment 315: The kit of any one of the preceding Embodiments, wherein the nuclease is an RNase.

[00530] Embodiment 316: The kit of any one of the preceding Embodiments, further comprising a polymerase with internal RNase activity.

[00531] Embodiment 317: The kit of any one of the preceding Embodiments, wherein the kit further comprises one or more reagents for nucleic acid polymerization by a polymerase.

[00532] Embodiment 318: The kit of any one of the preceding Embodiments, wherein the kit further comprises deoxyribonucleotide triphosphates (dNTPs).

[00533] Embodiment 319: The kit of any one of the preceding Embodiments, wherein the kit further comprises a salt.

[00534] Embodiment 320: The kit of any one of the preceding Embodiments, wherein the kit further comprises a buffer.

[00535] Embodiment 321: The kit of any one of the preceding Embodiments, wherein the kit further comprises a light source for photo cross-linking.

[00536] Embodiment 322: The kit of any one of the preceding Embodiments, wherein the kit further comprises instructions for use.

Some selected definitions

[0002] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments of the aspects provided herein, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0003] Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0004] As used herein, “nucleic acid” means DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof.

[00537] As used herein, the term “oligonucleotide” is intended to include, but is not limited to, a single-stranded DNA or RNA molecule, typically prepared by synthetic means. Nucleotides of the present invention will typically be the naturally-occurring nucleotides such as nucleotides derived from adenosine, guanosine, uridine, cytidine and thymidine. When oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exists in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term “double-stranded” as used herein is also meant to include those form which include such structural features as bulges and loops (see Stryer, Biochemistry, Third Ed. (1988), incorporated herein by reference in its entirety for all purposes).

[0005] The term “substantially identical” means two or more nucleotide sequences have at least 65%, 70%, 80%, 85%, 90%, 95%, or 97% identical nucleotides. In some embodiments, “substantially identical” means two or more nucleotide sequences have the same identical nucleotides.

[0006] As used herein the term “complementary” generally refers to the potential for a hybridized pairing or binding interaction between two sets of nucleic acids. Complementary nucleic acids are capable of binding to one another through hydrogen bond pairing according to canonical Watson-Crick base pairing and non-Watson-Crick base pairing ( e.g ., Wobble base pairing and Hoogsteen base pairing). In some embodiments, two sets of nucleic acids may be 100% complementary to one another. In other embodiments, two sets of nucleic acids may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides that are not complementary. In other embodiments, two sets of nucleic acids may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% complementary. In some embodiments, two sets of nucleic acids are complementary so long as they are capable of forming a stable or transient complex. “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogsteen base pairing.

[00538] As used herein, the term “hybridization domain(s)” generally refers to either a portion of a strand having complementary for hybridizing with a second strand.

[00539] A “polymerase” refers to an enzyme that performs template-directed synthesis of polynucleotides, e.g., DNA and/or RNA. The term encompasses both the full length polypeptide and a domain that has polymerase activity. DNA polymerases are well-known to those skilled in the art, including but not limited to DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. Additional examples of commercially available 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.), and phi29 DNA polymerase (New England Biolabs® Inc.). Polymerases include both DNA-dependent polymerases and RNA-dependent polymerases such as reverse transcriptase. At least five families of DNA-dependent DNA polymerases are known, although most fall into families A, B and C. There is little or no sequence similarity among the various families. Most family A polymerases are single chain proteins that can contain multiple enzymatic functions including polymerase, 3' to 5' exonuclease activity and 5' to 3' exonuclease activity. Family B polymerases typically have a single catalytic domain with polymerase and 3' to 5' exonuclease activity, as well as accessory factors. Family C polymerases are typically multi-subunit proteins with polymerizing and 3' to 5' exonuclease activity. In A. coli, three types of DNA polymerases have been found, DNA polymerases I (family A), II (family B), and PI (family C). In eukaryotic cells, three different family B polymerases, DNA polymerases a, d, and e, are implicated in nuclear replication, and a family A polymerase, polymerase g, is used for mitochondrial DNA replication. Other types of DNA polymerases include phage polymerases. Similarly, RNA polymerases typically include eukaryotic RNA polymerases I, II, and III, and bacterial RNA polymerases as well as phage and viral polymerases. RNA polymerases can be DNA-dependent and RNA- dependent.

[00540] It is noted that reagents, such as strand displacing DNA or RNA polymerases, and methods for synthesizing nucleic acid sequences from nucleic acid templates are well known in the art and are amenable to the invention. See, for example, US20050277146A1, US20100035303A1, and W02006030455A1, contents of all of which are incorporated herein by reference in their entirety.

[00541] As used herein, the terms “protein” and “polypeptide” are used interchangeably to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids ( e.g ., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[00542] The term “antibody” broadly refers to any immunoglobulin (Ig) molecule and immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen binding site that immunospecifically bind an antigen) comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Non-limiting embodiments of which are discussed below, and include but are not limited to a variety of forms, including full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a human antibody, a humanized antibody, a single chain antibody, a Fab, a F(ab’), a F(ab’)2, a Fv antibody, fragments produced by a Fab expression library, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, abispecific antibody, a functionally active epitope-binding fragment thereof, bifunctional hybrid antibodies ( e.g ., Lanzavecchia et al, Eur. J. Immunol. 17, 105 (1987)) and single chains ( e.g ., Huston et al, Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al, Science 242, 423-426 (1988), which are incorporated herein by reference) and/or antigen-binding fragments of any of the above (See, generally, Hood et al, Immunology, Benjamin, N.Y., 2ND ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference). It is to be understood that the antibody can be a polyclonal antibody or monoclonal antibody. In addition, the antibody can be a human antibody and/or a humanized antibody.

[00543] As used herein, the term “agent” refers to any substance, chemical constituent, chemical molecule of synthetic or biological origin.

[00544] As used herein, “contacting” refers to any suitable means for delivering, or exposing, an agent, or nucleic acid provided herein to at least one component as provided herein (e.g. , sample, a target binding molecule, etc.). In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.

[00545] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein,

“reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder. [00546] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[00547] The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[00548] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[00549] As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. [00550] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those provided herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[00551] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00552] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. [00553] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[00554] It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., provided herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. The invention is further illustrated by the following example, which should not be construed as further limiting.

EXAMPLES

EXAMPLE 1: METHODS AND COMPOSITIONS FOR NEW NUCLEIC ACID BARCODE GENERATION OR BARCODE SWAPPING ON BIOMOLECULES [00555] DNA barcoding of molecular probes such as antibodies enable simultaneous multiplexed detection of biomolecules. Several recent methods utilize DNA-barcoded antibodies for multiplexed imaging or profiling of cells (Exchange-PAINT¹, Immuno-SABER², CODEX³, cell hashing and CITE-Seq⁴). However, in the multiplexed workflows, generation of the biomolecules or probes tagged with different sequences to create barcoded libraries constitutes the bottleneck step. To overcome this challenge, we herein disclose methods and compositions to generate a new barcode from an existing nucleic acid moiety on a biomolecule. The main approach relies on enzymatic synthesis of nucleic acid barcodes directly on biomolecules by a polymerase using RNA or DNA strands as template by simultaneous and subsequent removal of the template strand. We also describe many alternative approaches.

[00556] The methods provided herein can be used to synthesize various single-stranded or double-stranded barcodes from a universal primer sequence or a pre-existing nucleotide sequence that can be as short as a single nucleotide, or swap one barcode with another barcode (i.e. remap). In some embodiments, they can also be used to make a copy of an RNA or DNA sequence on the biomolecule.

[00557] The barcode swapping capability makes high throughput production of barcoded biomolecule libraries easy and accessible. As DNA barcoding becomes a go-to strategy for multiplexed detection, the remapping approach provides a means to utilize universally barcoded reagents and remap them with custom sequences in smaller batches, which can be adapted and used for different assays and applications including multiplexed imaging, diagnostics, cell hashing, single-cell sequencing, immunoassays. This capability streamlines the process of commercializing such reagents as opposed to manufacturing each reagent with custom barcodes for each different application and panel. The newly barcoded biomolecule can be optionally purified or cleaned up via chromatography, column-based methods including concentration with membrane of different size cut-offs, dialysis, heat inactivation, pull down, buffer exchange, nuclease treatment, protease treatment, detergent treatment to remove or inactivate the molecules in the reaction mixture.

Exemplary methods

[00558] Provided herein are multiple approaches that can be utilized to append a single-stranded nucleic acid sequence by enzymatic synthesis using a template nucleic acid. The template does not become a part of the barcoded biomolecular complex as it either dissociates from the newly synthesized barcode via branch migration and toehold mediated displacement ⁵ or is cleaved via the action of nuclease that can be added after the synthesis or could be included in the synthesis reaction. For alternative uses the template strand can be left as part of the complex or can be partially cleaved/digested to create a double-stranded or partially double stranded barcode. FIGS. 2B-2E depicts the end products of the barcode remapping of the barcode show in FIG. 2A. [00559] FIGS. 3A-3H depict several exemplary alternative methods that can be used for barcode remapping.

Extension of the DNA barcode via Primer Exchange Reactions

[00560] The Primer Exchange Reaction (PER) method uses a catalytic template structure ( e.g . a self-folding hairpin) to append a new sequence domain onto a single-stranded primer sequence (FIG. 2A)⁶. Briefly, a primer can bind to a short single-stranded overhang on the template structure (e.g. a* in FIG. 3A), and then a strand displacing polymerase copies a new sequence domain (e.g. b in FIG. 3A) onto the 3 ’ end of the primer, until it reaches a stopper (indicated by black dots in FIG. 3A). The template structure then competes with the newly synthesized domain through competitive branch migration process ⁷, and the primer can ultimately become fully dissociated from the template. The primer can then optionally undergo further extension steps if another catalytic hairpin with a 3’ short single-stranded overhang that has a full or partial complementarity to the 3’ end of the primer sequence. The 5’ domain of the hairpin molecule encodes the sequence that will be appended onto the primer. By using multiple hairpins in a cascade, longer and complex sequences can be appended onto the primer via sequential rounds of PER.

[00561] In this embodiment, the primer domain is directly or indirectly tethered to the biomolecule (such as protein, lipid, RNA, DNA, sugar moiety) or probe (such as primary antibodies, secondary antibodies, nanobodies, affibodies, antibody fragments, Fab, F(ab’)2, Fc domains, ligands, Protein A/G, receptors or molecular complexes). Minimally, catalytic hairpin(s), strand displacing polymerase (such as Bst DNA polymerase - full length or large fragment, Bst 2.0, Bst 3.0, Bsu, phi29, SD polymerase), dNTPs are co-incubated with the biomolecule-primer in an aqueous reaction buffer that optionally includes Mg ions. In some embodiments, a 3 -letter code can be utilized where one of the 4 nucleotides is omitted from the reaction mixture (for example, dGTP can be omitted from the dNTP mix), where the missing nucleotide may be used as a stopper ⁶. The reaction mixture can optionally include sponge strands that will capture the trace amounts of the omitted nucleotide that might be found as a contamination in the reaction mixtures. In some embodiments, a pre incubation can be performed to clean up the reaction mixture before the addition of the biomolecule.

[00562] In some embodiments, a 5’ to 3’ directed exonuclease (such as T7 exonuclease, Exonuclease VIII, lambda exonuclease, T5 exonuclease, RecJf) can be used to remove the excess hairpin sequence. The exonuclease can be added during the reaction for simultaneous extension and cleavage. Alternatively, it can be added to the reaction mixture as a second step. In some embodiments, the exonuclease can be included in the reaction mixture in the inactive form and can be activated upon temperature change or addition of catalysts or co-factors such as Mg ions.

Extension of the DNA barcode via polymerase and exonuclease activity.

[00563] As an alternative approach, a single-stranded DNA molecule with a full or partial complementarity to the 3 ’ primer on the biomolecule is used for extension of the primer by a DNA polymerase (such as Taq, OneTaq, Q5U, Phusion, Q5 high-fidelity, DNA Polymerase I, Klenow, T4, Therminator, Tth DNA Polymerase and any other alternative DNA polymerase). This is achieved by incubating the barcoded biomolecule minimally with a DNA polymerase, dNTPs, and a template strand in an aqueous buffer (such as phosphate buffered saline or alternative) (FIG. 3B). [00564] This way, a new double-stranded barcode sequence can be created on the biomolecule. Optionally, after extension of the primer with a new sequence domain, the template strand can be removed by exonuclease-based cleavage with directional nucleases, such as T7 exonuclease, Exonuclease VIII, lambda exonuclease, T5 exonuclease, RecJf, which has 5’ to 3’ exonuclease activity. The extended DNA barcode is protected from the nuclease action via the tether in its 5’ (i.e. the end modification or conjugation site to the biomolecule), while the template strand is cleaved away. This enables remapping the primer to a new single-stranded DNA barcode. The cleavage can be induced by addition of co-factors (like Mg, Mn ions) or change of temperature. Template strand may optionally carry a 5’ Phosphate group to aid in exonuclease based cleavage.

Extension of the DNA barcode via polymerase using a DNA template with modified bases (abasic sites - endonuclease IV, RNA bases- USER)

[00565] The template DNA can also be rendered to susceptible to enzymatic cleavage by endonucleases via incorporation of one or multiple unconventional, unnatural or modified nucleotides, such as abasic sites, nicks or intercalated RNA bases, such as a uracil base, or methylated RNA (FIG. 3C). These designs allow the template DNA to be cleaved at these sites by the action of endonucleases such as RNase HI, RNase HP, endonuclease III, endonuclease IV, Uracil DNA glycosylase and Endonuclease VIII (or the USER combination). The cleavage can optionally be induced by addition of co-factors or change of temperature.

Extension of the DNA barcode using an RNA template via polymerase and RNAse H [00566] Instead of a DNA template, an RNA strand can be used as a template for the extension as above. In this case a DNA polymerase that can process an RNA template ( i.e . reverse transcriptases) (such as ProtoScript II Reverse Transcriptase, Tth polymerase, HIV-1 Reverse transcriptase). In some embodiments, an RNA-specific nuclease, such as XRN-1, RNase If Monarch RNase A, RNase H, HI, HII and others, can be used to specifically cleave the RNA template, to leave behind the biomolecule with the extended or re-mapped single-stranded DNA barcode after or during DNA synthesis (FIG. 3D). RNase cleave can also be done simultaneously, especially when RNase H is used which specifically cleave the RNA template that is hybridized to the DNA. It can also be used in combination with other RNAses that can be added simultaneously or after the DNA synthesis. The cleavage can be induced by addition of co-factors or change of temperature.

[00567] Light-based template removal after synthesis: The template strands can also be cleaved at alternative moieties such as a photocleavable spacers which can be excised upon illumination. [00568] Other strategies for template strand removal: To create a new single-stranded barcode, the template strand can also be removed off the newly synthesized strand via dehybridizing additives such as formamide or DMSO, divalent ion chelators such as EDTA, EGTA or by temperature variations. Alternatively, or additionally, it could be displaced by a competing strand that mediates displacement of the template off the extended primer via branch migration and toehold mediated displacement ⁵. In some embodiments these competing/sponge strands can be added to the reaction mix at high concentrations to attain efficient displacement after the synthesis.

Barcode swap by crosslinking

[00569] As an alternative to appending a new sequence onto the primer site on the biomolecule by direct synthesis, crosslinking methods could be utilized to covalently anchor a new barcode sequence as shown in FIGS. 3E-3F.

[00570] This could be achieved by incorporating a photocrosslinking moiety (such as CNVK, CNVD, psoralen) in the original barcode/primer on the biomolecule. In this case sequence swapping can be achieved by sequence-specific hybridization of the new nucleic acid sequence (previously referred as template) onto the original one. Alternatively, the modified base can be incorporated in the template strand (FIG. 3E).

[00571] A modified nucleic acid on the primer strand or template strand or both can be reacted to create a partially double stranded structure or in both the primer and template strand (FIG. 3F). Example functional groups that can be carried on the strands could be any of the common reactive chemistries such as azide, alkyne, tetrazine, DBCO, thiol, amine, carbonyl, carboxyl, maleimide, next generation maleimide, carbomide, NHS-ester, sulfo-NHS, functional groups with or without linkers (such as spacers or PEG linkers) or additional crosslinkers. The modifications can alternatively be placed at the end of one or both of the strands.

Barcode swap by binding

[00572] The remapping can be achieved by a binding interaction.

[00573] Hybridization between the complementary domains of the primer and template. [00574] Binding of two high affinity moieties on the primer and template strands (such as biotin/avidin-streptavidin, peptide/nanobody/affibody, aptamer), where one part of the pair is on the primer strand and the other one is on the template strand (FIG. 3G)

Direct ligation of a new barcode onto the existing sequence on the biomolecule [00575] A DNA ligase-mediated reaction could be utilized to ligate the free end of the original barcode to the new barcode strand (FIG. 3H). The reaction can be run in a non-templated manner by incubating the biomolecule with the original barcode with DNA ligase and the template strand in the reaction buffer. Alternatively, the reaction can be performed in a templated manner where a template strand is used to bridge the end of the old and new strands to create a temporary double stranded structure. DNA ligase can then ligate the two strands together. The template strand used as a bridge can then be removed via the action of nucleases as described above.

[00576] References

1. Jungmann, R. el al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313-318 (2014).

2. Saka, S. K. et al. Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues. Nat. Biotechnol. 37, 1080-1090 (2019).

3. Goltsev, Y. et al. Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging. Cell 174, 968-981. el5 (2018).

4. Stoeckius, M. etal. Simultaneous epitope and transcriptome measurement in single cells. Nat. Methods 14, 865-868 (2017).

5. Zhang, D. Y. & Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131, 17303-17314 (2009).

6. Kishi, J. Y., Schaus, T. E., Gopalkrishnan, N., Xuan, F. & Yin, P. Programmable autonomous synthesis of single-stranded DNA. Nat. Chem. 10, 155-164 (2018). 7. Lee, C. S., Davis, R. W. & Davidson, N. A physical study by electron microscopy of the terminally repetitious, circularly permuted DNA from the coliphage particles of Escherichia coh 15. J Mol. Biol. 48, 1-22 (1970).

EXAMPLE 2: ANTIBODY BARCODE SWAPPING

[00577] Methods for single hairpin (FIG. 4A) or multiple hairpin/ repeated extension (FIG.4B) of the barcodes for barcode swapping on antibodies were performed. An antibody (IgG) band shift on a polyacrylamide gel (PAGE) by electrophoresis was observed using the method outlined in FIGS. 4A and 4B (FIG. 4C). The 9-nt original barcode on the antibody is swapped either to a 19 nucleotide barcode by hairpin-templated extension via Primer Exchange reaction, as shown in panel FIG. 4A, or is extended to a long concatemer of several b units by use of a 2 hairpin system as shown in FIG. 4B.

[00578] Methods of DNA-templated (FIG. 5A) or RNA-templated (FIG. 5B) barcode swap were performed. An antibody (IgG) band shift on a polyacrylamide gel (PAGE) by electrophoresis was observed using the method outlined in FIG. 5A-5B (FIG. 5C). The 9-nt original barcode on the antibody is swapped either to a new 30 nucleotide barcode by DNA-templated or RNA templated extension.

[00579] Different reaction conditions (buffer type, polymerase concentration, incubation time, sequential vs. Simultaneous clean-up) were also tested for DNA or RNA templated barcode swapping and nuclease based clean-up for single-stranded barcode generation (FIG. 6).

[00580] Integrated barcode swap and purification was also performed with one step purification of ssDNA barcode swapped antibodies (FIGS. 7A and 7B). An antibody (IgG) band shift on a polyacrylamide gel (PAGE) by electrophoresis was observed using the method outlined in FIGS. 7 A and 7B (FIG. 7C).

[00581] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of providing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

CLAIMS What is claimed is:

1. A method comprising:

(a) annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, wherein the primer nucleic acid stand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises a hybridization domain (a), wherein the template nucleic acid strand comprises a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain, wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand;

(b) extending the primer nucleic acid strand from its 3 ’-end by a polymerase to synthesize a nucleic acid strand; and

(c) removing the template nucleic acid strand to produce a synthesized single-strand nucleic acid, and wherein the synthesized strand is attached to the target binding molecule.

2. A method comprising:

(a) annealing or hybridizing a primer nucleic acid strand with a template nucleic acid strand, wherein the primer strand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises a hybridization domain (a), wherein the template nucleic acid strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker domain between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker domain comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, wherein the hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand;

(b) extending the target nucleic acid strand from its 3 ’-end by a polymerase having strand displacing activity to produce a synthesized strand; and

(c) optionally, removing any excess template nucleic acid strand, and wherein the synthesized strand is attached to the target binding molecule.

3. A method comprising:

(a) annealing or hybridizing together a primer nucleic acid strand, an extension nucleic acid strand and a template nucleic acid strand, wherein the primer nucleic acid stand is attached to a target binding molecule, wherein the primer nucleic acid strand comprises at one end a hybridization domain

(a), wherein the extension nucleic acid strand comprises a hybridization domain (b), wherein the template nucleic acid strand comprises a first hybridization domain (a*); and a second hybridization domain (b*), wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand, and wherein the hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand;

(b) ligating an end of the primer nucleic acid strand with an end of the extension nucleic acid strand; and

4. A method comprising:

(a) linking a primer nucleic acid strand with an extension nucleic acid strand to produce a synthesized strand, wherein the primer stand is attached to a target binding molecule, wherein the extension nucleic acid strand comprises a barcode sequence/domain and, optionally, a hybridization domain, and wherein: (i) at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety; and/or (ii) one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair; and

(b) optionally, removing any excess extension strands, and wherein the synthesized strand is attached to the target binding molecule.

5. The method of claim 4, wherein said linking comprises ligation, cross-linking, or binding interaction.

6. The method of claim 3 or 4, wherein the extension strand comprises a barcode sequence/domain.

7. The method of claim 3 or 4, wherein at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety.

8. The method of claim 3 or 4, wherein the extension strand is in form of a hairpin.

9. The method of any one of claims 1-4, wherein the primer strand and/or the template strand comprises a barcode sequence/domain.

10. The method of any one of claims 1-4, wherein the template strand comprises a nucleic acid modification capable of enhancing nucleic acid cleavage, or the template strand comprise a modification capable of terminating nucleic acid polymerization, or the template strand is RNA, or the template strand is in form of a hairpin.

11. The method of any one of claims 1-4, wherein one or more domains utilize a 3 -letter code.

12. The method of any one of claim 1-4, wherein said removing the template strand comprises: (i) dehybridizing the template strand from the primer and/or synthesized strand; (ii) hybridizing or annealing the template strand with a competing nucleic acid strand having a sequence complementary to the template strand; or (iii) cleaving the template strand.

13. The method of claim 12, wherein the template strand comprises a toehold domain for the competing strand, and optionally the toehold domain comprises a sequence that is not complementary to the primer strand.

14. The method of any one of claim 1-14, wherein the synthesized strand comprises a ligand and/or a detectable label.

15. A composition comprising: a. a primer nucleic acid strand attached to a target binding molecule and comprising at its 3 ’-end a hybridization domain (a); and b. a template nucleic acid strand comprising a hybridization domain (a*), a second domain (b*), and optionally a linker domain between the hybridization domain and the second domain, and wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand, and, optionally, wherein neither the primer strand nor the template strand comprises a hairpin structure.

16. A composition comprising: (a) a primer nucleic acid strand, wherein the primer strand is attached to a target binding molecule and comprises at its 3 ’-end a hybridization domain (a); and

(b) a template nucleic acid strand, wherein the template strand comprises in 3’ to 5’ direction: a hybridization domain (a*), a first stem domain (b*), a second stem domain (b), and a linker between the first stem domain and the second stem domain, wherein at least one of the first stem domain, the second stem domain and the linker comprises a modification capable of terminating nucleic acid polymerization, wherein the first and second stem domains are substantially complementary to each other and capable of forming a double-stranded structure, and wherein the hybridization domain (a) of the target nucleic acid strand is substantially complementary to the hybridization domain (a*) of the template nucleic acid strand.

17. A composition comprising:

(a) a primer nucleic acid stand is attached to a target binding molecule and comprising at one terminus a hybridization domain (a) and optionally a second domain;

(b) an extension nucleic acid strand comprising a hybridization domain (b), a second domain (c), and optionally a linker domain linking the hybridization domain (b) and the second domain (c); and

(c) a template nucleic acid strand comprises a first hybridization domain (a*); and a second hybridization domain (b*), and wherein the hybridization domain (a) of the primer nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand, and wherein the hybridization domain (b) of the extension nucleic acid strand is substantially complementary to the first hybridization domain (a*) of the template nucleic acid strand.

18. A composition comprising: a. a primer nucleic acid stand is attached to a target binding molecule; and b. an extension nucleic acid strand comprising a barcode sequence/domain and, optionally, a hybridization domain, and wherein at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety, and/or wherein one of the primer nucleic acid strand and the extension nucleic acid strand comprises one member of a binding pair and the other of the primer nucleic acid strand and the extension nucleic acid strand comprises second member of the binding pair.

19. The composition of any one of claims 15-18, wherein the extension strand comprises a barcode sequence/domain.

20. The composition of any one of claims 15-18, wherein at least one of the primer nucleic acid strand and the extension nucleic acid strand comprises a cross-linking moiety.

21. The composition of any one of claims 15-18, wherein the extension strand is in form of a hairpin.

22. The composition of any one of claims 15-18, wherein the primer strand and/or the template strand comprises a barcode sequence/domain.

23. The composition of any one of claims 15-18, wherein the template strand comprises a nucleic acid modification capable of enhancing nucleic acid cleavage, or the template strand comprise a modification capable of terminating nucleic acid polymerization, or the template strand is RNA, or the template strand is in form of a hairpin.

24. The composition of any one of claims 15-18, wherein one or more domains utilize a 3-letter code.

25. The composition of any one of claims 15-18, wherein the template strand comprises a ligand or a detectable label.

26. The composition of any one of claims 15-18, wherein the composition further comprises one or more reagents for nucleic acid polymerization by a polymerase.

27. The composition of any one of claims 15-18, wherein the composition further comprises a nuclease.

28. The composition of any one of claims 15-18, wherein the composition further comprises a buffer.

29. A kit comprising a composition of any one of claims 15-28.

30. The kit of claim 28, wherein the kit further comprises a light source for photo cross-linking.