Compositions and Methods for Synthesizing Nucleic Acids

Abstract

Provided herein are compositions, kits, and methods for synthesizing nucleic acids. Further provided herein are compositions and methods for synthesizing strands of nucleic acid across different nucleic acid backbones hybridized together using strand-displacing polymerases. TIFF2022518489000003.tif156147

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62 / 796,982, filed on Jan. 25, 2019, and U.S. Provisional Application No. 62 / 845,539, filed on May 9, 2019, the entire contents of both of which are hereby incorporated by reference in their entirety.

[0002] Government Support This invention was made with government support under Grant Nos. 1317291 and 1729397 awarded by the National Science Foundation, and under Grant No. GM133052 awarded by the National Institutes of Health, and under Contracts Nos. N00014 - 16 - 1 - 2410 and N00014 - 18 - 1 - 2549 awarded by the Office of Naval Research, Department of Defense. The government has certain rights in this invention.

[0003] Technical Field The present disclosure relates to compositions and methods for the synthesis of nucleic acids using strand - displacing polymerases.

Background Art

[0004] Background The ability to synthesize any sequence of nucleic acids, particularly DNA, has revolutionized the ways in which scientists can study and manipulate biology. Currently, whole genomes can be efficiently sequenced, but the technology for synthesizing genomes lags behind. Synthetic single-stranded sequences (“oligos”) are typically chemically synthesized through cyclic coupling steps. However, oligonucleotides are typically not chemically synthesized beyond 200 bases due to the limitations of chemical coupling efficiency. Thus, synthetic methods for assembling larger fragments of single-stranded and double-stranded sequences have been developed, including enzymatic assembly that uses sequential or simultaneous combinations of multiple enzyme activities (e.g., restriction enzyme digestion and ligation, or isothermal Gibson assembly that combines 5’ exonuclease, 3’ extension activity of DNA polymerase, and DNA ligase activity), non-enzymatic twin primer assembly, and chemical assembly (e.g., click DNA assembly). These can be effective for generating longer sequences of single-stranded and double-stranded DNA, but sequences of hundreds of thousands of bases must be further assembled in vivo from shorter fragments using yeast vectors. A simple and robust new synthetic method that can generate long DNA sequences could have important and achievable applications in molecular biology, genome engineering, nanotechnology, and polymer-based data storage.

[0005] Accordingly, a need has remained in the art for compositions and methods for synthesizing nucleic acids of a predetermined sequence of any length, polymer sequences of a predetermined length, or sequences containing a predetermined number of repeats. The present disclosure addresses some of these needs. SUMMARY OF THE INVENTION

[0006] Summary Provided herein are compositions and methods for synthesizing nucleic acid strands across different nucleic acid backbones that are hybridized together using a strand-displacing polymerase. The compositions and methods described herein for synthesizing strands across nucleic acid junctions can effectively decouple the assembly step of the nucleic acid template from the synthesis step. This provides, among other things, the ability to sequentially perform robust annealing of guide strands that can hybridize in a predictable manner close to thermodynamically optimal conditions, followed by polymerization to create new transcripts. The compositions and methods described herein can enable scaling to arbitrarily long sequences. Furthermore, the entire assembly and synthesis process can occur isothermally, including at room temperature, which enables a generalized workflow for the synthesis of arbitrarily long sequences under relatively mild conditions.

[0007] The methods and compositions provided herein are based in part on the discovery that DNA polymerase displacement can be exploited to synthesize any desired DNA sequence by using a particular nucleic acid template secondary structure (FIG. 1A). Generally and without limitation, a nucleic acid template can include oligonucleotides hybridized together to form one or several junctions that prevent a DNA polymerase from straightforwardly synthesizing a nucleic acid sequence across a guide strand having complementary nucleotides, but that enable the DNA polymerase to displace and overcome the junction. Polymerization can then continue on a second guide strand and copy along the new backbone.

[0008] By hybridizing together multiple oligonucleotides, called guide strands, cross-junction synthesis can be cascaded to form longer sequences (FIG. 2). Multiple guide strands can be hybridized to constitute a nucleic acid template of any length. The nucleic acid template can be used to synthesize a nucleic acid sequence of any desired length.

[0009] Accordingly, in one aspect, a nucleic acid template is provided herein. For example, a nucleic acid template for use in the synthesis of nucleic acid sequences by the methods described herein. Generally, the nucleic acid template includes a region, such as a double-stranded region, that can block the progression of strand-displacing polymerase. The double-stranded region, along with the sequences at the 3' and 5' ends, may also be referred to herein as a synthetic domain or synthetic region.

[0010] As shown in FIG. 1B, an exemplary cross-junction (100) includes, in the 3' to 5' direction, a synthetic region (101), a double-stranded region (102), and another synthetic region (103). The 3' end (104) of the double-stranded region (102) has a sequence substantially similar to the nucleic acid sequence of the synthetic region (103) at the 5' end of the duplex. The double-stranded region (102) further includes a blocking domain (105) at the 5' end of region (104), where the blocking domain (105) can block the strand-displacing activity of the polymerase. The cross-junction can be a single continuous polynucleotide or the cross-junction can be prepared by annealing two separate guide strands (106 and 107).

[0011] The nucleic acid template can include any number of cross-junctions. For example, the nucleic acid template can include two or more, such as 3, 4, 5, 6, 7, 8, 9, 10 or more cross-junctions. When the nucleic acid template includes two or more cross-junctions, they can be linked together via a nucleic acid linker. For example, the 5' end of the first cross-junction can be linked to the 3' end of the second cross-junction. Non-limitingly, the linker can be of any desired length and / or nucleotide sequence. For example, the linker can simply be a nucleic acid backbone linkage, such as a phosphodiester linkage. Further, the nucleic acid linkers can all be the same, all be different, or some can be the same and some can be different.

[0012] Furthermore, when the nucleic acid template contains two or more cross-junctions, the two or more cross-junctions can all be the same, some can be the same, some can be different, and / or all can be different. For example, the synthetic regions (101) of two or more cross-junctions can have substantially identical nucleotide sequences; the synthetic regions (103) of two or more cross-junctions can have substantially identical nucleotide sequences; and / or the synthetic region (101) of the first cross-junction can have a nucleotide sequence substantially identical to the synthetic region (103) of the second cross-junction.

[0013] A cross-junction can be prepared from two separate nucleic acid strands, for example, a guide strand. Thus, in some embodiments, the nucleic acid template comprises, in the 3' to 5' direction, a first guide strand comprising a first synthetic region, a second synthetic region, a first junction domain, and a first blocking region; and, in the 3' to 5' direction, a second guide strand comprising a second blocking region, a second junction domain, and a third synthetic region. The first junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the third synthetic region, and the first junction domain and the second junction domain are substantially complementary to each other to form a double-stranded region. The first blocking region and the second blocking region together form a first blocking domain that blocks the strand displacement activity of the polymerase. Without limitation, the regions and domains described above can each independently comprise any nucleotide sequence, provided that the nucleotide sequence of the first junction domain of the first guide strand and the nucleotide sequence of the third synthetic region of the second guide strand are substantially identical, and the nucleotide sequences of the first junction domain of the first guide strand and the second junction domain of the second guide strand are substantially complementary to each other to form a double-stranded region. Without limitation, the nucleic acid template can comprise any number of guide strands.

[0014] In another aspect, provided herein is a method for forming a nucleic acid template. The method includes the step of annealing a first guide strand and a second guide strand. The first guide strand includes, in the 3’ to 5’ direction, a first synthetic region, a second synthetic region, a first junction domain, and a first blocking region. The second guide strand includes, in the 3’ to 5’ direction, a second blocking region, a second junction domain, and a third synthetic region, wherein the first junction domain includes a nucleotide sequence that is substantially identical to the nucleotide sequence of the third synthetic region, and the first junction domain and the second junction domain are substantially complementary to each other and form a double-stranded region. The first blocking region and the second blocking region together form a blocking domain that blocks the strand displacement activity of a polymerase.

[0015] In another aspect, provided herein is a reaction mixture comprising the nucleic acid template provided herein.

[0016] In yet another aspect, provided herein is a reaction mixture that can be utilized to generate a guide strand by enzymatically or chemically adding a junction region onto an existing nucleic acid strand. For example, a reaction mixture that can be utilized to generate a guide strand by enzymatically or chemically adding a blocking region onto an existing nucleic acid strand.

[0017] In another aspect, provided herein is a kit comprising the nucleic acid template provided herein.

[0018] In yet another aspect, provided herein is a kit comprising components or a reaction mixture for generating the nucleic acid template described herein from a nucleic acid sequence (partial guide strand) by enzymatically or chemically adding a junction region and / or a blocking region onto an existing nucleic acid strand.

[0019] The present specification further provides a method for synthesizing a nucleic acid sequence. Generally, the method includes the step of extending a nucleotide sequence from the 3′ end of a primer annealed to a nucleic acid template using a DNA polymerase having strand displacement activity. If the primer has not yet been annealed to the nucleic acid template, the method can include the step of annealing or hybridizing the primer to the nucleic acid template.

[0020] In some embodiments, the method for synthesizing a nucleic acid sequence includes the step of extending a nucleotide sequence from the 3′ end of a primer annealed to a guide strand using a DNA polymerase having strand displacement activity, and the step of continuously adding additional guide strands to the synthesis reaction, wherein the nucleotide sequence is extended prior to adding each additional guide strand.

[0021] The present specification provides the use of a nucleic acid template for generating combinatorial barcodes to assign identities specific to a target population, wherein the target population is a surface location / compartment, matrix, biomolecule, molecular library, or biological material (e.g., vesicles, cells, tissues, organoids, droplets, liposomes, small molecules, beads), and optionally, the use includes the step of partitioning the target population prior to addition of the guide strand, and the step of pooling the partitioned target populations after addition of the guide strand. BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 5-1

Figure 5-2

Figure 6-1

Figure 6-2

Figure 7-1

Figure 7-2

Figure 8-1

Figure 8-2

Figure 9-1

Figure 9-2

Figure 10

Figure 11

Figure 12

Figure 13A

Figure 13B-1

Figure 13B-2

Figure 13C-1

Figure 13C-2

Figure 14

Figure 15-1

Figure 15-2

Figure 16-1

Figure 16-2

Figure 17

Figure 18-1

Figure 18-2

Figure 18-3

Figure 19A-1

Figure 19A-2

Figure 19B

Figure 20A

Figure 20B

Figure 21A-1

Figure 21A-2

Figure 21A-3

Figure 21A-4

Figure 21A-5

Figure 21B-1

Figure 21B-2

Mode for Carrying Out the Invention

[0023] Detailed Description The basic strategy for cross-junction synthesis is depicted in FIG. 1. Shown are two guide strands (also called template strands) hybridized together to form a junction, and a primer bound in front of that junction on the first template strand (left). A strand-displacing polymerase is used to copy the x domain (also called the junction domain herein) until it reaches a stopper (shown in black and also called the blocking domain herein). Thereafter, the new and old x domains compete in a random walk branch migration process (Lee et al., 1970). Eventually, the new x domain binds to the exposed x* domain (right, also called the synthesis region herein) on the second template strand, and thus the junction can be successfully crossed. Polymerization then continues on the second template strand and can copy along the new backbone.

[0024] Accordingly, in one aspect, a nucleic acid template is provided herein. As used herein, "nucleic acid template" or "template nucleic acid" refers to a nucleic acid comprising at least two guide strands (106 and 107) that form at least one cross-junction (100). Without limitation, the nucleic acid template can include any number of cross-junctions (100), i.e., the nucleic acid template can include any number of guide strands to form the desired number of cross-junctions. Multiple guide strands can hybridize to form a complete nucleic acid template that facilitates the synthesis of the desired nucleic acid sequence. Figures 1A-2 provide guidance on the structure of an exemplary nucleic acid template based on the secondary structure and hybridization of the first and second guide strands. The nucleic acid template can be designed based on the desired nucleotide sequence to be synthesized.

[0025] As disclosed herein, the nucleic acid template includes one or more cross-junctions. Generally, each cross-junction includes a first guide strand and a second guide strand in the 3' to 5' direction. The first guide strand includes, in the 3' to 5' direction, optionally a first blocking region, optionally a first junction domain, a first synthesis region, a second synthesis region, a second junction domain, and a second blocking region. The second guide strand includes, in the 3' to 5' direction, a first blocking region, a first junction domain, a first synthesis region, a second synthesis region, optionally a second junction domain, and optionally a second blocking region. The second blocking region of the first guide strand and the first blocking region of the second guide strand together form a blocking domain that blocks the strand displacement activity of the polymerase. The second junction domain of the first guide strand includes a nucleotide sequence that is substantially complementary to the nucleotide sequence of the first junction domain of the second guide strand to form a double-stranded region. The second junction domain of the first guide strand includes a nucleotide sequence that is substantially identical to the nucleotide sequence of the first synthesis region of the second guide strand.

[0026] When the nucleic acid template contains two or more cross junctions, the first guide strand of the first cross junction can be the second guide strand of the second cross junction. Similarly, the second guide strand of the first cross junction can be the first guide strand of the second cross junction.

[0027] It is noted that the guide strands described herein may include a barcode domain. For example, the second guide strand of a junction can include a barcode domain at its 5' end. In some embodiments, one of the first or second synthetic regions of the guide strand includes a barcode domain.

[0028] As used herein, "barcode domain" refers to a portion of a strand, such as a guide strand, that includes a nucleic acid sequence that indicates information or data. Non-limiting examples include spatial information, sequences, molecular sequences, experiment / batch numbers, binary codes, random barcodes for generating unique molecular identifiers (UMIs), or any combination thereof. The barcode domain sequence can be predefined by a barcode library. The barcode domain can be any suitable sequence. Bit values can be assigned to the barcode domain. For example, bit values can be independently assigned to each barcode domain. It is noted that the bit values are not limited to 0 and 1. In some embodiments, the barcode domain sequence can be 0 nucleotides in length. For example, the absence of a barcode sequence can provide information (e.g., a 0 bit value can be assigned to the absence of a barcode, and a 1 bit value can be indicated by a barcode domain of 1 or more nucleotides in length).

[0029] In some embodiments, the barcode domain is immobilized on a substrate surface. In some embodiments, the barcode is immobilized in a predetermined pattern. In some embodiments, the barcode domain indicates spatial information.

[0030] The guide strands described herein may also include primer sequences. For example, the first guide strand of a junction may include a primer sequence at its 3' end. In another non-limiting example, the second guide strand of a junction may include a primer sequence at its 5' end.

[0031] In some embodiments, a nucleic acid template includes a first guide strand and a second guide strand. The first guide strand includes, in the 3' to 5' direction, a first synthesis region, a second synthesis region, a first junction domain, and a first blocking region. The second guide strand includes, in the 3' to 5' direction, a second blocking region, a second junction domain, and a third synthesis region. The first junction domain includes a nucleotide sequence that is substantially identical to the nucleotide sequence of the third synthesis region, and the first junction domain and the second junction domain are substantially complementary to each other and form a double-stranded region. The first blocking region and the second blocking region together form a first blocking domain that blocks the strand displacement activity of polymerase.

[0032] In some embodiments, the second guide strand further includes a fourth synthesis region at the 5' end of the third synthesis region.

[0033] As used herein, the terms "guide strand" or "template strand" are used interchangeably to refer to a portion of a nucleic acid template and serve as the basic building blocks of the nucleic acid template. Some guide strands can be combined to construct a nucleic acid template that is used to synthesize a desired output nucleic acid sequence. A guide strand can include, for example, in the 3' to 5' direction, a first synthesis region, a second synthesis region, a first junction domain, and a first blocking region. A guide strand can also include, in the 3' to 5' direction, a second blocking region, a second junction domain, and a third synthesis region. For reference, some exemplary guide strands are depicted in FIG. 2. A set of guide strands (template strands) can be used to assemble a full-length template nucleic acid for use in nucleic acid synthesis.

[0034] As used herein, "synthetic region" refers to a portion of the template strand (guide strand) that is copied into the nucleic acid sequence being synthesized. Without limitation, each synthetic region can independently comprise any desired nucleotide sequence. In other words, each synthetic region can independently be of any length. For example, each synthetic region can be from 1 nucleotide to thousands of nucleotides in length. In some embodiments, each synthetic region is 1,000 nucleotides or less, 750 nucleotides or less, 500 nucleotides or less, 400 nucleotides or less, 300 nucleotides or less, 250 nucleotides or less, 200 nucleotides or less, 150 nucleotides or less, 100 nucleotides or less in length, or 50 nucleotides or less in length. Without limitation, a synthetic region can be just 1 nucleotide.

[0035] Without limitation, the double-stranded region of the cross-junction can independently comprise any desired nucleotide sequence or number of base pairs. In other words, the double-stranded region can independently be of any length. For example, each double-stranded region can be from 1 base pair to dozens of base pairs in length. In some embodiments, the double-stranded region is independently at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 nucleotides or base pairs in length.

[0036] As used herein, "junction domain" refers to a portion of the guide strand that can hybridize with a portion of a second guide strand to form a duplex, and the nucleic acid sequence generated from the nucleic acid template comprising the guide strand comprises: (i) a sequence complementary to the nucleotide sequence of the junction domain; or (ii) a sequence complementary to the reverse complement of the nucleotide sequence of the junction domain. In other words, the nucleic acid sequence generated from the nucleic acid template comprising the guide strand comprises either a sequence synthesized from the junction domain sequence or the reverse complement of the junction domain sequence.

[0037] Optionally, each junction domain can independently contain any desired nucleotide sequence or number of nucleotides. In other words, each junction domain can independently be of any length. For example, each junction domain can be from 1 nucleotide to dozens of nucleotides in length. In some embodiments, each junction domain can independently be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.

[0038] As described above, the junction domain of the first guide strand can hybridize with the junction domain of the second domain to form a double-stranded structure. Optionally, each double-stranded region can independently contain any desired number of base pairs. In other words, each double-stranded region can independently be of any length. For example, each double-stranded region can be from 1 base pair to dozens of base pairs in length. In some embodiments, each double-stranded region can independently be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or base pairs in length.

[0039] As used herein, a "blocking domain" refers to a portion of a nucleic acid template that can prevent or stop a polymerase from moving along the backbone of the template strand and / or from continuing to copy the template strand. For example, a blocking domain can be a double-stranded region having high stability, such as a super-stable double-stranded region where the polymerase cannot separate the two strands. Methods for forming super-stable double-stranded nucleic acids are well known in the art and suitable for the present invention. For example, a blocking domain can contain one or more modified nucleotides known in the art for increasing the T m of a double-stranded nucleic acid. Exemplary such nucleotides include, but are not limited to, locked nucleic acid (LNA), 2'-O-methoxy-ethyl (2'-MOE) nucleotides, 2,6-diaminopurine, G-clamp (an analog of C having four hydrogen bonds), and guanidinium G-clamp nucleotides.

[0040] In some embodiments, the blocking domain may include a covalent cross - link between two guide strands. It is noted that the covalent cross - link may include a situation where the nucleotide at the 5' end of the first guide strand is covalently linked to the nucleotide at the 3' end of the second template strand via, for example, an oligonucleotide or a single nucleic acid backbone linkage (e.g., a phosphodiester bond). Generally, the covalent cross - link between two guide strands excludes the strands that can result in one continuous sequence from the two guide strands. Thus, in some embodiments, at least one (or both) of the nucleotides involved in the covalent cross - link is not a terminal nucleotide. In other words, at least one (or both) of the nucleotides involved in the cross - link is not the first or the last nucleotide of the template strand.

[0041] Non - limitingly, each blocking domain can independently include any desired nucleotide sequence or number of base pairs. In other words, each blocking region can independently be of any length. For example, each blocking domain can be from 1 base pair to dozens of base pairs in length. In some embodiments, each blocking domain can independently be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides or base pairs in length.

[0042] In some embodiments, at least one of the blocking domains includes a homopolymer stretch. For example, the blocking domain includes a stretch of polyA, polyT, polyC or polyG.

[0043] As used herein, "blocking region" refers to a portion of a template strand that forms a blocking domain with a second template strand. Without limitation, a blocking region can independently comprise any desired nucleotide sequence or number of nucleotides. In other words, each blocking region can independently be of any length. For example, each blocking region can be from 1 nucleotide to several tens of nucleotides in length. In some embodiments, each blocking region can independently be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. It is noted that a blocking region can be a single nucleotide.

[0044] In some embodiments, at least one blocking region within a blocking domain comprises a modified nucleotide that includes a modification for cross-linking with another blocking region in the blocking domain.

[0045] As used herein, the term "cross-linking segment" refers to a portion of a nucleic acid template that comprises a modified nucleotide that cross-links to another oligonucleotide upon exposure to a phase change stimulus (e.g., ultraviolet light, temperature change, pH change, etc.) and enables assembly of a complete nucleic acid template sequence. Figure 4A provides an example of a cross-linking segment of a nucleic acid template.

[0046] Monomers and modified nucleotides for cross-linking of oligonucleotides are well known in the art. For example, photoreactive nucleotides are well known in the art for cross-linking oligonucleotides. Thus, in some embodiments, the blocking region can include a photoreactive nucleotide, i.e., a nucleotide having a photoreactive group. Exemplary photoreactive nucleotides include 3-cyanovinylcarbazole (CNVK) nucleotides; 5-bromodeoxycytosine; 5-iododeoxycytosine; 5-bromodeoxyuridine; 5-iododeoxyuridine; and nucleotides containing aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluoroaryl azide (FAB-dUMP) or diazirine (DB-dUMP), but are not limited thereto. In some embodiments, the blocking region includes CNVK as a photoreactive nucleotide.

[0047] In some embodiments of any aspect, the cross-linking segment includes 3-cyanovinylcarbazole.

[0048] For example, the first and second blocking regions can be covalently linked to each other. Thus, in some embodiments, the first or second blocking region includes a cross-linking segment with the other blocking region to form the first blocking domain. For example, the cross-linking segment can include 3-cyanovinylcarbazole for cross-linking.

[0049] Exemplary secondary structures of the nucleic acid template and the guide strands that make up the nucleic acid template are shown in FIGS. 1A-2. The secondary structure allows DNA polymerase to displace from the first guide strand to the second guide strand, the third guide strand, the fourth guide strand, the fifth guide strand, etc. until the output nucleic acid sequence is achieved. Different structure prediction models can result in different predicted structures, and even the same model can result in different predicted structures when different baseline parameters such as temperature, ionic strength, etc. are used.

[0050] That is, when a change in one nucleotide in the base pairing structure of the guide strand in the nucleic acid template is accompanied by a compensatory change in the complementary nucleotide that maintains the base pairing ability, the structure and thus the function of the guide strand can be maintained. That is, some guide strands can tolerate a certain degree of sequence variation and still retain DNA polymerase displacement activity. Furthermore, cleavage or subsequences of the nucleic acid template or guide strand as described herein can also retain displacement activity, provided that the cleavage does not alter the intramolecular base pairing required for the secondary structure of the guide strand or nucleic acid template. The examples herein provide a working demonstration of nucleic acid templates that specifically displace DNA polymerase.

[0051] It is contemplated that the reverse, complementary, reverse-complementary or cleavage sequences of the nucleic acid templates described herein can also maintain the secondary structure of the template. Secondary structure models can be used to predict the stability of the guide strand in the nucleic acid template. Maintenance or improvement in the modified guide strand for generating nucleic acid output sequences, e.g., compensatory or non-compensatory changes made based on the predicted structure, should be experimentally tested.

[0052] In some aspects of any aspect, one or more regions of the nucleic acid template utilize a three-letter code. As used herein, "three-letter code" means that the region contains only three of the four nucleic acid bases, i.e., adenine, thymine / uracil, guanine, and cytosine, or only three of their modified forms. For example, the three-letter code can comprise or consist of nucleic acid bases selected from one of the following: (i) adenine, thymine / uracil, and guanine; (ii) adenine, thymine / uracil, and cytosine; (iii) adenine, guanine, and cytosine; or (iv) thymine / uracil, guanine, and cytosine.

[0053] In some embodiments of any aspect described herein, the nucleic acid template and / or the synthesized nucleic acid sequence comprises a nucleic acid modification. For example, at least one of the synthetic region, the junction domain, and / or the blocking domain can comprise a nucleic acid modification. Exemplary nucleic acid modifications include, but are not limited to, nucleobase modifications, sugar modifications, sugar-sugar linkage modifications, conjugates (e.g., ligands), and any combination thereof.

[0054] Exemplary modified nucleobases include inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, as well as 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-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 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-azapyrimidines as well as N-2, N-6 and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazole, 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-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N 4-Acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine, or an O-alkylated base, among others, but not limited thereto. Further purines and pyrimidines include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

[0055] In some embodiments, the modified nucleobase is inosine, xanthine, hypoxanthine, nebularine, isoguanosine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(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, N 6 -(isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6-(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, N 4 -(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-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(Methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 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-(aminocarbonylethenyl)-pseudouracil, 1-(aminocarbonylethenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethenyl)-2,4-(dithio)pseudouracil, 1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaz)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaz)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxo)-naphthalene, inosine, xanthine, hypoxanthine, nubraline, tubercidin, isoguanicin, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbolstyryl, 5-(methyl)isocarbolstyryl, 3-(methyl)-7-(propynyl)isocarbolstyryl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbolstyryl, 7-(propynyl)isocarbolstyryl, 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 pyrimidine, N, 2 -substituted purine, N 6 -substituted purine, O 6 -substituted purine, substituted 1,2,4-triazole, and any O-alkylated or N-alkylated derivatives thereof may be selected from the group consisting of.

[0056] Exemplary sugar modifications include, but are not limited to, 2'-fluoro, 3'-fluoro, 2'-OMe, 3'-OMe, 2'-deoxy modification, and acyclic nucleotides, such as peptide nucleic acid (PNA), unlocked nucleic acid (UNA), or glycol nucleic acid (GNA).

[0057] In some embodiments, the nucleic acid modification may include replacement or modification of the sugar-sugar bond. Exemplary sugar-sugar bond modifications include phosphotriester, methylphosphonate, phosphoramidate, phosphorothioate, methylene methylimino, thiodiester, thiocarbamate, siloxane, N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH 2 -C(=O)-N(H)-5') and amide-4 (3'-CH 2 -N(H)-C(=O)-5'), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonic acid ester, thioformacetal (3'-S-CH 2 -O-5'), formacetal (3'-O-CH 2 -O-5'), oxime, methyleneimino, methylenecarbonylamino, methylene methylimino (MMI, 3'-CH 2 -N(CH 3 )-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether (C3’-O-C5’), thioether (C3’-S-C5’), thioacetamide (C3’-N(H)-C(=O)-CH 2-S-C5’, C3’-O-P(O)-O-SS-C5’, C3’-CH 2 -NH-NH-C5’, 3'-NHP(O)(OCH 3 )-O-5' and 3'-NHP(O)(OCH 3 )-O-5’ are included, but not limited to these.

[0058] In some embodiments, the nucleic acid modification can include peptide nucleic acid (PNA), bridged nucleic acid (BNA), morpholino, locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), or any other xeno nucleic acid (XNA) described in the art.

[0059] In some embodiments of various aspects, the nucleic acid template, guide strand, and / or synthesized nucleic acid sequence can be modified at the 3’ and / or 5’ ends. For example, a label, fluorophore, tag, or cap can be added to the 3’ and / or 5’ ends of the nucleic acid template, guide strand, and / or synthesized nucleic acid sequence.

[0060] In some aspects of various scenarios, the nucleic acid template, guide strand, and / or synthesized nucleic acid sequence can be modified with a linker or spacer, for example, at internal positions, at the 3' and / or 5' ends. Without wishing to be bound by theory, the linker or spacer can be used to link the nucleic acid template, guide strand, and / or synthesized nucleic acid sequence to a moiety such as a solid support or a label. In some aspects, the linker or spacer can be selected from the group consisting of photocleavable linkers, hydrolyzable linkers, redox-cleavable linkers, phosphate-cleavable linkers, acid-cleavable linkers, ester-cleavable linkers, peptide-cleavable linkers, and any combination thereof. In some aspects, the cleavable linker can include a disulfide bond, a tetrazine-trans-cyclooctene group, a sulfhydryl group, a nitrobenzyl group, a nitroindoline group, a bromohydroxycoumarin group, a bromohydroxyquinoline group, a hydroxyphenacyl group, a dimethozybenzoin group, or any combination thereof.

[0061] Any photocleavable linker recognized in the art can be used. In some aspects, the cleavable linker can include a photocleavable linker. Generally, a photocleavable linker contains a photosensitive functional group that can be cleaved upon exposure to a light source (e.g., UV light) or a specific wavelength. Non-limiting examples of photocleavable spacers can be found, for example, in U.S. Patent Nos. 6,589,736 B1; 7,622,279 B2; 9,371,348 B2; 7,547,530 B2; and 7,057,031 B2; and PCT Publication No. WO2014200767, the entire contents of all of which are incorporated herein by reference in their entirety.

[0062] In some aspects of various scenarios, the nucleic acid template, guide strand, and / or synthesized nucleic acid sequence can be modified with a detectable label, for example, at internal positions, at the 3' and / or 5' ends. Without wishing to be bound by theory, such detectable labels can facilitate detection. As used herein, the term "detectable label" refers to a composition capable of generating a detectable signal indicating 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 agents, bioluminescent agents, and the like. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.

[0063] A wide variety of fluorescent reporter dyes are known in the art. Typically, fluorophores are aromatic or heteroaromatic compounds and can be pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine, or other similar compounds.

[0064] Exemplary fluorophores include 1,5 IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein (pH 10); 5-carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-carboxyfluorescein); 5-hydroxytryptamine (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; acriflavine; acriflavine foilgen SITSA; aequorin (luminescent protein); Alexa Fluor 350 (trademark); Alexa Fluor 430 (trademark); Alexa Fluor 488 (trademark); Alexa Fluor 532 (trademark); Alexa Fluor 546 (trademark); Alexa Fluor 568 (trademark); Alexa Fluor 594 (trademark); Alexa Fluor 633 (trademark); Alexa Fluor 647 (trademark); Alexa Fluor 660 (trademark); Alexa Fluor 680 (trademark); alizarin complexone; alizarin red; allophycocyanin (APC); AMC, AMCA-S; AMCA (aminomethylcoumarin); AMCA-X; aminoactinomycin D; aminocoumarin; aniline blue; Anthrocyl stearate; APC-Cy7; APTS; astrazon brilliant red 4G; astrazon orange R; astrazon red 6B; astrazon yellow 7 GLL; atabrine; ATTO-TAG (trademark) CBQCA; ATTO-TAG (trademark) FQ; auramine;Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyl Oxadiazole); BCECF (High pH); BCECF (Low pH); Berberine Sulfate; β-Lactamase; BFP Blue Shift GFP (Y66H); BG-647; Bimane; Bisbenzamide; Blankophor FFG; Blankophor 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 Fl; 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 Sulfoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green-1 Ca; 2+ Dye; Calcium Green-2 Ca 2+ ; Calcium Green-5N Ca 2+ ; Calcium Green-C18 Ca 2+; Calcium Orange; CalcoFluor White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue (trademark); Cascade Yellow; Catecholamine; CFDA; CFP - Cyan Fluorescent Protein; Chlorophyll; Chromomycin A; Chromomycin A; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazine O; Coumarin phalloidin; CPM Methylcoumarin; CTC; Cy2 (trademark); Cy3.1 8; Cy3.5 (trademark); Cy3 (trademark); Cy5.1 8; Cy5.5 (trademark); Cy5 (trademark); Cy7 (trademark); Cyan GFP; Cyclic AMP Fluorosensor (FiCRhR); d2; Dabsyl; Dansyl; Dansylamine; Dansylcadaverine; Dansyl chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein diacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); DIDS; Dihydrorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1); Oocyanin; Europium(III) chloride; Europium; EYFP; Fast Blue; FDA; Foilgen (Paraosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4; Fluorescein diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43 (trademark); FM 4-46; Fura Red (trademark) (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 shift (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (Fluorogold); Hydroxytryptamine; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intralight Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurdan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; Lucifer Yellow; Mag Green; Magdala Red (Floxine B); Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanine; Methoxycoumarin; MitoTracker Green FM; MitoTracker Orange; MitoTracker Red; Mitomycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronin Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Norepinephrine; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Iavin E8G; Oregon Green (trademark); Oregon Green 488-X; Oregon Green (trademark) 488; Oregon Green (trademark) 500; Oregon Green (trademark) 514; Pacific Blue; Pararosaniline (Fuchsin); PE-Cy5; PE-Cy7;PerCP; PerCP-Cy5.5; PE-Texas Red (Red 613); Fluorescein 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; Primuline; Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronin; Pyronin B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; 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 Phalloidin; 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 (trademark); sgBFP (trademark) (Super-Glow BFP); sgGFP (trademark); sgGFP (trademark) (Super-Glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulfonate); SPQ (6-Methoxy-N-(3-Sulfopropyl)-Quinolinium); Stilbene; Sulfhorhodamine B can C; Sulfhorhodamine G Extra; Tetracycline; Tetramethylrhodamine; Texas Red (trademark); Texas Red-X (trademark) Conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R;Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC (Tetramethylrhodamine Isothiocyanate); Toluidine Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3, but are not limited to these. Many suitable forms of these fluorescent compounds are available and can be used.;

[0065] Other exemplary detectable labels include luminescent and bioluminescent markers (e.g., biotin, luciferase (e.g., bacterial, firefly, click beetle, etc.), luciferin, and aequorin), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., galactosidase, glucuronidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase), and calorimetric labels, e.g., colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads. Patents teaching the use of such labels include U.S. Patent 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.;

[0066] Means for detecting such labels are well known to those skilled in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, and fluorescent markers can be detected using a photodetector for detecting emitted light. Enzyme labels are typically detected by providing an enzyme substrate to the enzyme and detecting the reaction product generated by the action of the enzyme on the enzyme substrate, and calorimetric labels can be detected by visualizing the colored label.

[0067] In some embodiments, the detectable label is a fluorophore or a quantum dot. Without wishing to be bound by theory, using a fluorescent reagent can reduce the signal-to-noise ratio in imaging / readout and thus maintain sensitivity.

[0068] In some embodiments, the label can be configured to include a "smart label" that is undetectable when conjugated to a nucleic acid template, guide strand, and / or synthesized nucleic acid sequence, but causes a color change when released.

[0069] Acrydite modification can also be performed on nucleic acid templates, guide strands, and / or synthesized nucleic acid sequences. Acrydite modification can enable the oligonucleotide to be used in reactions with nucleophiles such as thiols (e.g., microarrays) or incorporated into gels (e.g., polyacrylamide). Thus, in some embodiments, the nucleic acid template, guide strand, and / or synthesized nucleic acid sequence includes one or more acrydite nucleosides. The acrydite nucleoside can be at the 3'-end, 5'-end, and / or internal position of the nucleic acid template, guide strand, and / or synthesized nucleic acid sequence.

[0070] Any modification to the nucleic acid templates, guide strands, and / or synthesized nucleic acid sequences provided herein that enables purification, extraction, quantification of expression, binding, electrophoresis, etc. can also be performed.

[0071] As disclosed herein, a nucleic acid template can include any desired number of cross-junctions, i.e., a nucleic acid template can include more than two guide strands. For example, a nucleic acid template can include two cross-junctions, e.g., a nucleic acid template can include three guide strands. Thus, in some embodiments, the second guide strand further includes, at its 5' end and in the 3' to 5' direction, a third junction domain and a third blocking region, and the nucleic acid template further includes a third guide strand. The third guide strand includes, in the 3' to 5' direction, a fourth blocking region, a fourth junction domain, and a fifth synthesis region. The third junction domain includes a nucleotide sequence that is substantially identical to the nucleotide sequence of the fifth synthesis region, where the third junction domain and the fourth junction domain are substantially complementary to each other to form a double-stranded region, and where the third blocking region and the fourth blocking region together form a second blocking domain that blocks the strand displacement activity of the polymerase.

[0072] In some embodiments of any aspect, the third guide strand further includes a sixth synthesis region at the 5' end of the fifth synthesis region.

[0073] As disclosed herein, the third or fourth blocking regions can be covalently linked to each other. Thus, one of the third or fourth blocking regions can include a cross-linking segment with the other blocking region to form the second blocking domain. In some embodiments, the cross-linking segment includes 3-cyanovinylcarbazole for cross-linking.

[0074] In some embodiments of any aspect, the nucleic acid template includes three cross-junctions. For example, the nucleic acid template includes four guide strands. Thus, in some embodiments, the third guide strand further includes, at its 5' end, in the 3' to 5' direction, a fifth junction domain and a fifth blocking region, and the nucleic acid template further includes a fourth guide strand. The fourth strand includes, in the 3' to 5' direction, a sixth blocking region, a sixth junction domain, and a seventh synthesis region. The fifth junction domain includes a nucleotide sequence substantially identical to the nucleotide sequence of the seventh synthesis region. The fourth junction domain and the fifth junction domain are substantially complementary to each other to form a double-stranded region, and here, the fifth blocking region and the sixth blocking region together form a third blocking domain that blocks the strand displacement activity of the polymerase.

[0075] In some embodiments, the fourth guide strand further includes an eighth synthesis region at the 5' end of the sixth synthesis region.

[0076] As described herein, the fifth and sixth blocking regions can be covalently linked to each other. Thus, one of the fifth or sixth blocking regions can include a cross-linking segment with the other blocking region to form the third blocking domain. In some embodiments, the cross-linking segment includes 3-cyanovinylcarbazole for cross-linking.

[0077] In some aspects of any scenario, the nucleic acid template includes four cross-junctions. For example, the nucleic acid template includes five guide strands. Thus, in some aspects, the fourth guide strand further includes, at its 5' end and in the 3' to 5' direction, a seventh junction domain and a seventh blocking region, and the nucleic acid template further includes a fifth guide strand. The fifth guide strand includes, in the 3' to 5' direction, an eighth blocking region, an eighth junction domain, and a ninth synthesis region. The seventh junction domain includes a nucleotide sequence that is substantially identical to the nucleotide sequence of the ninth synthesis region, and the seventh junction domain and the eighth junction domain are substantially complementary to each other to form a double-stranded region, and here, the seventh blocking region and the eighth blocking region together form a fourth blocking domain that blocks the strand displacement activity of the polymerase.

[0078] As described herein, the seventh and eighth blocking regions can be covalently linked to each other. Thus, one of the seventh or eighth blocking regions can include a cross-linking segment with the other blocking region to form the fourth blocking domain. In some aspects, the cross-linking segment includes 3-cyanovinylcarbazole.

[0079] In some aspects, all of the guide strands in the nucleic acid template can include substantially the same nucleotide sequence in the synthesis region on the 5' side of the guide strand. In some aspects, all of the guide strands in the nucleic acid template can include substantially the same nucleotide sequence in the synthesis region on the 3' side of the guide strand.

[0080] In some aspects of any scenario, the nucleic acid template, guide strand, or a portion thereof is immobilized, conjugated, or linked to a solid support or substrate. In some aspects, the nucleic acid template, guide strand, or a portion thereof is immobilized on the surface of the substrate. In some aspects, the nucleic acid template, guide strand, or a portion thereof indicates information, date, or spatial information. It is noted that the nucleic acid template, guide strand, or a portion thereof can be immobilized, conjugated, or linked to the solid support or substrate either covalently or non-covalently.

[0081] In some aspects of any scenario, the nucleic acid template, guide strand, or a portion thereof is immobilized in a predetermined pattern. In some aspects of any scenario, the predetermined pattern is a geometric shape, square, circle, or triangle. In some aspects of any scenario, the predetermined pattern includes repeating elements. In some aspects of any scenario, the predetermined pattern is asymmetric or symmetric. In some aspects of any scenario, the predetermined pattern includes spatial information and / or special information.

[0082] Non-limitingly, the solid support or substrate can exist in the form of a platform, column, filter or sheet, dish, microfluidic capture device, capillary, electrochemical reaction platform, scaffold, cartridge, resin, matrix, bead, or another solid support known in the art. The substrate can also include a biological material. Biological materials are known in the art. Non-limiting examples of biological materials include tissue, tissue section, artificial tissue, cells, patient-derived cells, primary cells, organoids, extracellular matrix, 3D biological organs, dissociated cells, live cells, fixed cells, vesicles, droplets, liposomes, and the like.

[0083] In some embodiments, the solid support or substrate comprises, but is not limited to, materials including polymers, metals, ceramics, gels, paper, or glass. The material of the solid support can further include, as non-limiting examples, polystyrene, agarose, gelatin, alginate, iron oxide, stainless steel, gold nanobeads or particles, copper, silver chloride, polycarbonate, polydimethylsiloxane, polyethylene, acrylonitrile butadiene styrene, cycloolefin polymer or cycloolefin copolymer, or Sepharose™ resin.

[0084] In some embodiments, the substrate is a hydrogel. In some embodiments, the hydrogel is a compressed hydrogel. The hydrogel may be naturally occurring, derived from a natural source, or derived from a synthetic source. The hydrogel can be any water-swellable and cross-linked polymeric material produced by the reaction of one or more monomers. The hydrogel can be a polymeric material that can swell to hold a significant proportion of water within its structure without dissolving in an aqueous solution. The hydrogel can also be any shrinkable material, such as a heat-shrinkable plastic, a viscoelastic foam, a memory foam.

[0085] In some embodiments, the solid support or substrate can further include magnetic-responsive elements such as magnetic-responsive beads. In some embodiments, the magnetic-responsive elements or beads are in the form of spheres, cubes, rectangular prisms, cylinders, cones, or any other shape described in the art.

[0086] In some embodiments, the magnetic-responsive element includes magnetite, iron(III) oxide, samarium cobalt, terfenol D, or any other magnetic element described in the art.

[0087] Synthesis of Nucleic Acid Templates In another aspect, provided herein is a method for forming a nucleic acid template described herein. Generally, the method includes annealing or hybridizing a first guide strand and a second guide strand. The first guide strand includes, in the 3' to 5' direction, a first synthetic region, a second synthetic region, a first junction domain, and a first blocking region. The second guide strand includes, in the 3' to 5' direction, a second blocking region, a second junction domain, and a third synthetic region. The first junction domain includes a nucleotide sequence that is substantially identical to the nucleotide sequence of the third synthetic region, and the first junction domain and the second junction domain are substantially complementary to each other to form a double-stranded region, and here, the first blocking region and the second blocking region together form a blocking domain that blocks the strand displacement activity of the polymerase.

[0088] In some embodiments, the first or second blocking region includes a cross-linking segment with the other region to form a blocking domain, and the method further includes the step of forming the cross-linking.

[0089] The compositions and methods described herein can be used for the synthesis of a predetermined sequence of any length. Exemplary embodiments are shown in FIG. 2. As shown in FIG. 2, by hybridizing multiple guide strands together, a cross-junction synthesis reaction can be cascaded to form a longer sequence. Each junction between the guide strands exhibits the same domain motif, such that the strand domains (b, c, d, e) copied onto the 3' end of the growing strand before reaching the stopper can reach and bind to the exposed complementary sequences (b*, c*, d*, e*) on the next guide strand across the junction. In order to enable the assembly of a longer sequence, any sequence can be added into the template region between the motif domains (shown in gray).

[0090] The compositions and methods described herein can be used for the synthesis of polymer sequences of a predetermined length. For example, the compositions and methods described herein can be used to synthesize a specific number of repeat sequences by using programmable unique hybridization domains that fix the length of the template concatemer, which has been a long-standing challenge. An exemplary embodiment is shown in FIG. 3. As shown in FIG. 3, by utilizing the specificity of DNA self-assembly, unique binding sequences promote the assembly of a predetermined length, even when the domains being synthesized are identical.

[0091] In some embodiments, cross-junctions can be assembled by using photo-reactive interstrand cross-linking bases, which can serve the dual purpose of linking two cross-junction strands together and acting as a stopper for polymerase synthesis (FIG. 4). Cross-junctions can be repeatedly assembled through cycles of hybridization, cross-linking, and washing. Cross-junctions can also be assembled and spatially addressed on a surface, and subsequent synthesis steps can still be performed on the surface.

[0092] Assembling a library of orthogonal cross-junction arrays on a surface can enable the multiplexed synthesis of orthogonal and arbitrarily long DNA of a given sequence.

[0093] In one exemplary embodiment, 3-cyanovinylcarbazole (CNVK) (Vieregg et al., 2013) base modification and a UV light source are used to cross-link DNA junctions together. Strand design can utilize a set of two or more hybridization domains (C1 and 1*, 2 and 2* in this implementation), internal barcode sequences (b1, b2, etc.) that serve as growing sequence strands, and CNVK base cross-linkers (FIG. 4A, cyan circles).

[0094] Nucleic Acid Synthesis In yet another aspect, the present disclosure provides a method for synthesizing nucleic acids using the nucleic acid templates described herein. Generally, the method involves using a DNA polymerase having strand displacement activity to extend a nucleotide sequence from the 3' end of a primer annealed or hybridized to a nucleic acid template.

[0095] In some embodiments, the method involves annealing or hybridizing a primer to a nucleic acid template. For example, the primer is annealed or otherwise hybridized to a guide strand in the nucleic acid template. In some embodiments, the primer can be annealed or hybridized to the guide strand of the nucleic acid template before assembling the full-length nucleic acid.

[0096] The nucleic acid templates for use in the methods of synthesizing nucleic acids described herein can be fully assembled prior to synthesis. For example, as shown in FIGS. 5-12 and 14-19A, all of the desired guide strands can be annealed or hybridized together to form a full-length template prior to extension from the primer. Thus, in some embodiments, the method for synthesizing nucleic acids involves annealing or hybridizing guide strands to form a full-length nucleic acid template prior to extending the nucleotide sequence from the 3' end of the primer. For example, all of the guide strands can be added to the initial reaction mixture and annealed or hybridized prior to the extension step.

[0097] Alternatively, the synthesis can be continuous. For example, the guide strand can be continuously added to the synthesis reaction over a period of time under conditions that allow for the synthesis of the desired nucleic acid sequence across the junction. Generally, synthesis begins by extending the nucleotide sequence from the 3’ end of a primer annealed or hybridized to the guide strand. The reaction substantially does not contain other guide strands of the full-length template. After a period of time, e.g., after 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes or more, the next guide strand is added. This can continue until the desired full-length template is assembled.

[0098] As shown in the exemplary embodiment in FIG. 21A, synthesis begins by annealing or hybridizing the first guide strand to the primer. The first guide strand includes, in the 3' to 5' direction, a synthesis region, a junction domain, and a blocking region. After annealing or hybridizing to the first guide strand, the primer is extended from its 3’ end. After a period of time, e.g., after 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes or more, additional new guide strands can be continuously added to the synthesis reaction.

[0099] It is noted that after adding the new guide strand to the synthesis reaction, the new guide strand is annealed or otherwise hybridized to the guide strands already in the synthesis reaction. Optionally, the complementary blocking regions are covalently cross-linked, e.g., by photocrosslinking. The synthesis is continued for a period of time, e.g., for 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes or more, and then the next guide strand is added to the reaction.

[0100] Generally, each additional guide strand, in the 3' to 5' direction, includes a first blocking region, a first junction domain, a first synthesis region, a second synthesis region, a second junction domain, and a second blocking region. The first new guide strand added to the synthesis reaction has the following characteristics: the first blocking region of the new guide strand and the blocking region of the first guide strand together form a blocking domain that blocks the strand displacement activity of the polymerase; the first junction domain of the new guide strand includes a nucleotide sequence that is substantially complementary to the nucleotide sequence of the junction domain of the first guide strand to form a double-stranded region; and the first synthesis region of the new guide strand includes a nucleotide sequence that is substantially identical to the nucleotide sequence of the junction domain of the first guide strand.

[0101] Each additional new guide strand added after the first new guide strand has the following characteristics: the first blocking region of the new guide strand and the second blocking region of the last guide strand added to the synthesis reaction together form a blocking domain that blocks the strand displacement activity of the polymerase; the first junction domain of the new guide strand includes a nucleotide sequence that is substantially complementary to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction to form a double-stranded region; and the first synthesis region of the new guide strand includes a nucleotide sequence that is substantially identical to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction.

[0102] The number of additional guide strands that can be added is not limited. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more additional guide strands can be added. In some embodiments, 75, 100, 150, 200, 250 or more additional guide strands can be added.

[0103] In some embodiments, the method further comprises adding a terminal guide strand to the reaction synthesis. The terminal guide strand comprises, in the 3' to 5' direction, a blocking region, a junction domain, a first synthesis region, optionally a second synthesis region, and optionally a primer sequence.

[0104] When one or more additional guide strands are added to the synthesis reaction, the terminal guide has the following characteristics: the blocking region of the terminal guide strand and the second blocking region of the last guide strand added to the synthesis reaction together form a blocking domain that blocks the strand displacement activity of the polymerase; the junction domain of the terminal guide strand comprises a nucleotide sequence that is substantially complementary to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction to form a double-stranded region; and the first synthesis region of the terminal guide strand comprises a nucleotide sequence that is substantially identical to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction.

[0105] When no additional guide strand is added to the synthesis, the terminal guide has the following characteristics: the blocking region of the terminal guide strand and the blocking region of the first guide strand together form a blocking domain that blocks the strand displacement activity of the polymerase; the junction domain of the terminal guide strand comprises a nucleotide sequence that is substantially complementary to the nucleic acid sequence of the junction domain of the first guide strand to form a double-stranded region; and the first synthesis region of the terminal guide strand comprises a nucleotide sequence that is substantially identical to the nucleic acid sequence of the junction domain of the first guide strand added to the synthesis reaction.

[0106] In some embodiments, the terminal guide strand comprises a primer sequence at its 5' end.

[0107] As used herein, the term "primer" is used to describe a DNA (or RNA) sequence that pairs with one strand of DNA and provides a free 3'-OH at which DNA polymerase can initiate synthesis of a deoxyribonucleotide chain. Preferably, the primer is composed of oligonucleotides. The exact length of the primer depends on many factors, including temperature and the source of the primer. For example, depending on the complexity of the target sequence, oligonucleotide primers typically contain 15 to 25 or more nucleotides, but it may contain fewer nucleotides. Short primer molecules generally require a lower temperature to form a sufficiently stable hybrid complex with the template.

[0108] "Polymerase" refers to an enzyme that performs template-directed synthesis of polynucleotides, such as DNA and / or RNA. This term encompasses both full-length polypeptides and domains having polymerase activity. DNA polymerases are well known to those skilled in the art and include, but are not limited to, DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified forms thereof. Additional examples of commercially available polymerase enzymes include, but are not limited to, the 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 transcriptases. At least five families of DNA-dependent DNA polymerases are known, most of which are classified into families A, B, and C. There is little or no sequence similarity between the various families. Most family A polymerases are single-chain proteins that can contain multiple enzyme functions including polymerase activity, 3'→5' exonuclease activity, and 5'→3' exonuclease activity. Family B polymerases typically have a single catalytic domain with polymerase activity and 3'→5' exonuclease activity, as well as accessory factors. Family C polymerases are typically multi-subunit proteins having polymerization activity and 3'→5' exonuclease activity.In Escherichia coli (E. coli), three types of DNA polymerases, DNA polymerase I (family A), II (family B), and III (family C) have been discovered. In eukaryotic cells, three different family B polymerases, DNA polymerase α, δ, and ε, are involved in nuclear replication, and polymerase γ, a family A polymerase, 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, as well as bacterial RNA polymerases, and further phage and viral polymerases. RNA polymerases can be either DNA-dependent or RNA-dependent.

[0109] 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 noted to be suitable for the present invention. See, for example, US20050277146A1, US20100035303A1, and WO2006030455A1, the entire contents of all of which are hereby incorporated by reference in their entirety.

[0110] In some embodiments of any aspect, the method further comprises the step of amplifying a nucleic acid sequence. As used herein, the term "amplifying" refers to the step of subjecting a nucleic acid sequence to conditions sufficient to effect amplification of a polynucleotide when all of the components of the reaction are intact. The components of the amplification reaction include, for example, primers, polynucleotide template, polymerase, nucleotides, and the like. The term "amplifying" typically refers to an "exponential" increase in the target nucleic acid. However, "amplifying" can also refer to a linear increase in the number of selected target sequences of a nucleic acid, as obtained by the cycle sequencing method. Methods for amplifying and synthesizing nucleic acid sequences are known in the art. See, for example, U.S. Patent Nos. 7,906,282, 8,367,328, 5,518,900, 7,378,262, 5,476,774, and 6,638,722, the entire contents of all of which are incorporated herein by reference.

[0111] In some embodiments, the step of amplifying a nucleic acid sequence comprises polymerase chain reaction (PCR). PCR is well known to those of skill in the art; see, for example, U.S. Patent Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990, the entire contents of all of which are incorporated herein by reference. Exemplary PCR reaction conditions typically include a two- or three-step cycle. A two-step cycle has a denaturation step followed by a hybridization / extension step. A three-step cycle includes a denaturation step followed by a hybridization step followed by an independent extension step.

[0112] In some embodiments, the amplification step includes an additional nucleotide sequence or template having a hairpin that is orthogonal in the amplification step. Without wishing to be bound by theory, such additional DNA hairpins can reduce or correct off-target reactions. For example, if a three-letter code is used, sequences or templates containing these additional hairpins can serve to incorporate trace amounts of unwanted nucleotides that may be present in some samples.

[0113] In some embodiments of any aspect described herein, a guide strand is added to the first reaction mixture. In some embodiments of any aspect described herein, the synthesis is continuous. In continuous synthesis, the guide strands can be added sequentially, under conditions that allow for the synthesis of the desired nucleic acid across the junction. For example, reference is made to FIG. 21A in the Examples, which provides a schematic of one embodiment of the method described herein.

[0114] To complete a series of continuous nucleic acid syntheses as described herein, a terminal barcode strand can be used. As used herein, a "terminal barcode strand" includes (i) one or more binding domains substantially identical to one template strand; and optionally (ii) a different barcode; and optionally (iii) a PCR primer sequence on the 5' end to allow for exponential amplification of the complete synthesis product. Specifically, the optional barcode of the terminal barcode strand is not complementary to the template strand. Non-limitingly, the terminal barcode strand can be added to the 3' or 5' end of the template nucleic acid. The terminal barcode strand can further include a label or fluorophore.

[0115] In another aspect, a reaction mixture containing the described nucleic acid template is provided herein.

[0116] In some embodiments of any aspect, the nucleic acid template further comprises a primer annealed or otherwise hybridized to one of the guide strands. In another embodiment, the nucleic acid template further comprises a primer annealed or hybridized to a first guide strand, a second guide strand, a third guide strand, a fourth guide strand, a fifth guide strand, a sixth guide strand, a seventh guide strand, and the like.

[0117] In some embodiments of any aspect, the reaction mixture further comprises nucleotide triphosphates or deoxynucleotide triphosphates. In some embodiments, the reaction mixture does not contain one of adenosine, thymidine / uridine, cytosine, or guanosine triphosphate or deoxynucleotide triphosphate.

[0118] In some embodiments, the reaction mixture further comprises a DNA or RNA polymerase.

[0119] In some embodiments, the reaction mixture further comprises a buffer or salts for nucleic acid synthesis. The buffer used in the reaction mixture is contemplated to be selected to provide stability to the nucleic acid template and the desired nucleic acid sequence. Methods for selecting such buffers are known to those of skill in the art and can be selected based on their properties in various conditions including the pH or temperature of the reaction being performed.

[0120] In some embodiments, the reaction mixture comprises components that can be utilized to generate guide strands by enzymatically or chemically adding junction regions onto existing nucleic acid strands. For example, a reaction mixture that can be utilized to generate guide strands by enzymatically or chemically adding blocking regions onto existing nucleic acid strands.

[0121] In one aspect, provided herein is a kit comprising a nucleic acid template as provided herein. In certain embodiments, such kits are intended for therapeutic applications. In certain embodiments, such kits are intended for research use.

[0122] In one aspect of any scenario, the kit further comprises nucleotide triphosphates or deoxynucleotide triphosphates.

[0123] In another aspect of any scenario, the kit further comprises a DNA or RNA polymerase.

[0124] In one aspect of any scenario, the kit further comprises a buffer or salts for nucleic acid synthesis.

[0125] In one aspect of any scenario, the kit further comprises a solid support as provided herein for isolating specific sequences.

[0126] In yet another scenario, provided herein is a kit comprising components or reaction mixtures for generating a nucleic acid template as described herein from a nucleic acid sequence (partial guide strand) by enzymatically or chemically adding a junction region and / or a blocking region onto an existing nucleic acid strand.

[0127] In some aspects of the various scenarios described herein, the kit further comprises instructions for use.

[0128] Methods for nucleic acid purification and analysis are known to those of skill in the art. Non-limiting examples of methods for characterizing nucleic acid synthesis include liquid chromatography, mass spectrometry, next-generation sequencing, polymerase chain reaction (PCR), gel electrophoresis, or any other method for identifying nucleoside sequences, secondary structures, chemical compositions, expression, thermodynamics, binding, or function.

[0129] Exemplary Applications The nucleic acid templates and methods provided herein enable several different applications. Non-limiting examples include, but are not limited to, those described below.

[0130] Proximity and molecular distance measurement. Since the hybridization kinetics can be adjusted so that the synthesis step occurs isothermally and even under mild (non-denaturing) conditions, the proximity of biomolecules can be determined, which can create a record indicating the distance information between labeled biomolecules. This has applications in the measurement of the distance / proximity of DNA / RNA / protein and other biomolecules of interest, and in the determination of the structure of DNA, RNA, protein, and RNA-protein complexes. This can be further used as a method for the fingerprinting of proteins and other biomolecules when the synthesis reaction is programmed to generate a different recording pattern relative to other targets.

[0131] Long-chain synthesis and DNA assembly. Since the assembly step and the synthesis step can be separated, longer sequences can be assembled than is possible with other methods.

[0132] Combinatorial synthesis for library preparation. Template concatenation can be designed to generate a programmable combinatorial set of sequences (Figure 18). If multiple options for intercalating domains are desired in the sequence library, this can be achieved simply by including the relevant guide strands, each including the option at the desired relative frequency. For example, 10 options for each of 5 different domains would require 10 * 5 = 50 guide strands instead of the usual 5 for a fixed domain, but can generate 10^5 = 100,000 possible sequences. In general, for positions p1, p2, ..., pn having numbers of possibilities x1, x3, ..., xn respectively, the total number of strands required for those positions is x1 + x2 + ... + xn, and they can generate a library of size x1 * x3 * ... * xn.

[0133] Parallel synthesis of multiple orthogonal sequences. Multiple orthogonal cross-junction synthesis reactions can be carried out together in the same reaction solution. These may or may not use the same primers or intercalating sequences.

[0134] In situ synthesis. The synthesis step is carried out in an aqueous isothermal environment, which enables in situ synthesis of any programmable strand within a biological sample or biological material.

[0135] Construction of identity barcodes (unique molecular identifiers, batch numbers, indexes). Cross-junction synthesis can be used to create unique identity barcodes in a combinatorial fashion on a surface, matrix, biomolecule, molecular library, or biological sample or material (e.g., vesicles, cells, tissues, organoids, droplets, liposomes, small molecules, beads), or to encode a unique molecular identity to a spatial position, biomolecule, or biological sample or material. This can be further combined with initial approaches such as split-and-pool synthesis or split-pool synthesis, where the addition of each guide strand is performed after the division of the target population and before pooling. See, for example, Brenner et al. (2000) Proc. Natl. Acad. Sci. USA 97:1665 and US20160251697A1, which are hereby incorporated by reference in their entirety.

[0136] Construction of spatial barcodes. Cross-junction synthesis can be used to create barcodes in a combinatorial fashion at specified positions or specified targets on a surface. Non-limiting examples of targets on a surface include biomolecules, biological materials, etc. The biological materials can be selected from tissues, tissue sections, artificial tissues, cells, patient-derived cells, primary cells, organoids, extracellular matrix, 3D biological organs, dissociated cells, live cells, fixed cells, vesicles, droplets, liposomes, etc.

[0137] Lock re-routing. Since cross-junction synthesis can be used to "re-route" the polymerase to a new backbone sequence, it can be used for gene and sequence editing involving synthetic re-routing. This can be done by ligating a strand with the new desired sequence in front of the old sequence so that the polymerase copies across the junction onto the new sequence.

[0138] In addition to the applications provided above, several types of nucleic acid synthesis can be carried out using the compositions and methods provided herein. Synthesis can include, but is not limited to, single-stranded DNA synthesis, double-stranded DNA synthesis, mutagenesis, insertion, deletion, homologous recombination, parallel / multiplex synthesis, proximity and molecular distance measurement.

[0139] Selected definition For convenience, the meanings of some terms and phrases used in this specification, the examples, and the appended claims are provided below. Unless otherwise stated or implied by the context, the following terms and phrases include the meanings provided below. Unless otherwise expressly stated or not apparent from the context, the following terms and phrases do not exclude the meanings acquired in the technical field to which the term or phrase belongs. The definitions are provided to assist in describing specific aspects of the situations provided herein and are not intended to limit the claimed invention, since the scope of the invention is limited only by the claims. Further, unless specifically required by the context, singular terms shall include the plural and plural terms shall include the singular.

[0140] Definitions of common terms in immunology and molecular biology are 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 Werner 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) can be found, and the contents of these are hereby incorporated by reference in their entirety into this specification.

[0141] As used herein, "nucleic acid" means DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. For example, nucleic acids can include double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not have the same extent (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). The term nucleic acid also includes any chemical modifications thereof, such as by methylation and / or capping. Nucleic acid modifications can include the addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interactions, and functionality to individual nucleobases or to the nucleic acid as a whole. Such modifications can include base modifications, such as 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at the exocyclic amines of cytosine, substitution of 5-bromouracil, backbone modifications, and unusual base pairing combinations such as the isobases isocytidine and isoguanidine. Nucleic acid(s) can be derived from a completely chemical synthesis process, such as solid-phase mediated chemical synthesis, from a biological source, such as isolation from any species that produces nucleic acids, or from a process involving manipulation of nucleic acids with molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or combinations of those processes.

[0142] As used herein, the terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length (either deoxyribonucleotides or ribonucleotides, or analogs thereof). A polynucleotide can have any three-dimensional structure and can perform any function, whether known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, intergenic DNA, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA) (which is a DNA representation of mRNA, usually obtained by reverse transcription or amplification of messenger RNA (mRNA)); synthetically or amplified DNA molecules, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. When present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0143] The term "statistically significant" or "significantly" refers to statistical significance and generally means a difference of two standard deviations (2SD) or more.

[0144] As used herein, the terms "comprising" or "comprises" are used in connection with a composition, method, and respective components (s) thereof, which are essential for a method or composition, but can include elements not specified, whether or not essential.

[0145] As used herein, the term "consisting essentially of" refers to the elements necessary for a given embodiment. This term allows the presence of additional elements that do not substantially affect the basic, novel, or functional features (s) of that embodiment of the invention.

[0146] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Methods and materials similar or equivalent to those provided herein can be used in the practice or testing of the present disclosure, but the preferred methods and materials are described below. The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0147] Furthermore, unless the context specifically requires otherwise, singular terms shall include the plural, and plural terms shall include the singular.

[0148] Except in the examples or unless otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about". The term "about" when used in connection with percentages can mean ± 1%.

[0149] The term "substantially identical" means that two or more nucleotide sequences have nucleotides that are at least 65%, 70%, 80%, 85%, 90%, 95%, or 97% identical. In some embodiments, "substantially identical" means that two or more nucleotide sequences have the same identical nucleotides.

[0150] As used herein, unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence to form a double-stranded structure, as understood by one of ordinary skill in the art. Such conditions can be, for example, stringent conditions, where stringent conditions can include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C, for 12 - 16 hours, followed by washing. Other conditions, such as physiologically appropriate conditions that may be encountered within an organism, can be applied. One of ordinary skill in the art will be able to determine the optimal set of conditions for testing the complementarity of two sequences according to the ultimate use of the hybridized nucleotides.

[0151] "Complementary" sequences also, as used herein, may include, or be entirely formed from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, as long as the above requirements regarding the ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogsteen base pairs.

[0152] As used herein, the terms "substrate" or "substrate surface" are used interchangeably to describe a structure to which one or more nucleic acid templates, guide strands, or portions thereof, as provided herein, can be presented or contacted for contact with additional nucleic acids and / or labels. The nucleic acid templates, guide strands, and barcode strands provided herein can be immobilized, conjugated, or linked to the substrate surface.

[0153] As used herein, the term "conjugated to" encompasses the association of a nucleic acid with a substrate surface, a phase change factor, or a member of an affinity pair by covalent bonding (including but not limited to crosslinking by a crosslinking agent) or by strong non-covalent interactions maintained under the conditions in which the conjugate is used.

[0154] As used herein, the term "hybridize" refers to the phenomenon of a single-stranded nucleic acid or a region thereof forming hydrogen-bonded base pair interactions with either another single-stranded nucleic acid or a region thereof (intermolecular hybridization) or another single-stranded region of the same nucleic acid (intramolecular hybridization). Hybridization is governed by the stability of the hybrid, which is determined by the base sequences involved (complementary nucleic acid bases forming hydrogen bonds), as well as the identity of the base pairs (e.g., G:C base pairs are stronger than A:T base pairs) and the number of consecutive base pairs, with longer stretches of complementary bases forming more stable hybrids.

[0155] As used herein, the term "spatial information" is any information, coordinates, markers within a biological tissue or matrix that can be stored in a barcode. Spatial information can provide information to one of ordinary skill in the art as to where on a substrate a particular marker, barcode, or pattern is located. For example, spatial information can be useful in creating an image or QR code with a nucleic acid barcode. Spatial information can also be useful in the detection of specific nucleic acid targets.

[0156] Some aspects of the methods and compositions described herein can be defined according to any of the following numbered paragraphs. 1) (a) A first guide strand comprising, in the 3' to 5' direction, a first synthetic region, a second synthetic region, a first junction domain, and a first blocking region; and (b) A second guide strand comprising, in the 3' to 5' direction, a second blocking region, a second junction domain, and a third synthetic region comprising a nucleic acid template, wherein the first junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the third synthetic region, the first junction domain and the second junction domain are substantially complementary to each other to form a double-stranded region, and the first blocking region and the second blocking region together form a first blocking domain that blocks the strand displacement activity of the polymerase, said nucleic acid template. 2) The nucleic acid template of paragraph 1, wherein the first and second blocking regions are covalently linked to each other. 3) The nucleic acid template of paragraph 1 or 2, wherein the first or second blocking region comprises a cross-linking segment with the other blocking region to form a first blocking domain. 4) The nucleic acid template of paragraph 3, wherein the cross-linking segment comprises 3-cyanovinylcarbazole. 5) The nucleic acid template of any one of paragraphs 1 to 4, wherein the second guide strand further comprises a fourth synthetic region at the 5' end of the third synthetic region. 6) The second guide strand further comprises, at its 5' end, in the 3' to 5' direction, a third junction domain and a third blocking region, and the nucleic acid template further comprises a third guide strand comprising a fourth blocking region, a fourth junction domain, and a fifth synthetic region in the 3' to 5' direction, wherein the third junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the fifth synthetic region, The third junction domain and the fourth junction domain are substantially complementary to each other to form a double-stranded region, and the third blocking region and the fourth blocking region together form a second blocking domain that blocks the strand displacement activity of the polymerase, a nucleic acid template according to any one of paragraphs 1 to 5. 7) A nucleic acid template according to any one of paragraphs 1 to 6, wherein the third and fourth blocking regions are covalently linked to each other. 8) A nucleic acid template according to any one of paragraphs 1 to 7, wherein the third or fourth blocking region comprises a cross-linking segment with the other blocking region to form the second blocking domain. 9) The nucleic acid template of paragraph 8, wherein the cross-linking segment comprises 3-cyanovinylcarbazole. 10) A nucleic acid template according to any one of paragraphs 6 to 9, wherein the third guide strand further comprises a sixth synthetic region at the 5'-end of the fifth synthetic region. 11) The third guide strand further comprises a fifth junction domain and a fifth blocking region at its 5'-end in the 3' to 5' direction, and the nucleic acid template further comprises a fourth guide strand comprising a sixth blocking region, a sixth junction domain and a seventh synthetic region in the 3' to 5' direction, the fifth junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the seventh synthetic region, the fourth junction domain and the fifth junction domain are substantially complementary to each other to form a double-stranded region, and the fifth blocking region and the sixth blocking region together form a third blocking domain that blocks the strand displacement activity of the polymerase, a nucleic acid template according to any one of paragraphs 6 to 10. 12) The nucleic acid template of paragraph 11, wherein the fifth and sixth blocking domains are covalently linked to each other. 13) The nucleic acid template of paragraph 11 or 12, wherein the fifth or sixth blocking region comprises a cross-linking segment with the other blocking region to form a third blocking domain. 14) The nucleic acid template of paragraph 13, wherein the cross-linking segment comprises 3-cyanovinylcarbazole. 15) The nucleic acid template of paragraphs 11 to 14, wherein the fourth guide strand further comprises an eighth synthetic region at the 5' end of the sixth synthetic region. 16) The fourth guide strand further comprises a seventh junction domain and a seventh blocking region in the 3' to 5' direction at its 5' end, and the nucleic acid template further comprises a fifth guide strand comprising an eighth blocking region, an eighth junction domain, and a ninth synthetic region in the 3' to 5' direction, wherein the seventh junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the ninth synthetic region, the seventh junction domain and the eighth junction domain are substantially complementary to each other to form a double-stranded region, and the seventh blocking region and the eighth blocking region together form a fourth blocking domain that blocks the strand displacement activity of the polymerase. The nucleic acid template of paragraphs 11 to 15. 17) The nucleic acid template according to claim 16, wherein the seventh and eighth blocking domains are covalently linked to each other. 18) The nucleic acid template of paragraph 16 or 17, wherein the seventh or eighth blocking region comprises a cross-linking segment with the other blocking region to form a fourth blocking domain. 19) The nucleic acid template of paragraph 18, wherein the cross-linking segment comprises 3-cyanovinylcarbazole. 20) The nucleic acid template of any one of paragraphs 1 to 19, wherein one or more regions utilize a three-letter code. 21) The nucleic acid template of any one of paragraphs 1 to 20, wherein at least one of the first, second, third, or fourth blocking domains comprises a double-stranded region. 22) A nucleic acid template according to any one of paragraphs 1 to 21, wherein the nucleic acid template comprises a nucleic acid modification. 23) A nucleic acid template according to any one of paragraphs 1 to 22, wherein at least one of the first, second, third or fourth blocking domains comprises a nucleic acid modification. 24) A nucleic acid template according to paragraph 22 or 23, wherein the nucleic acid modification is a modified nucleobase. 25) A nucleic acid template according to any one of paragraphs 1 to 24, wherein at least one of the first, second, third or fourth blocking domains comprises a polynomonomer stretch. 26) A nucleic acid template according to any one of paragraphs 1 to 25, wherein the nucleic acid template is conjugated to a solid support. 27) A nucleic acid template according to any one of paragraphs 1 to 26, wherein the nucleic acid template further comprises a primer annealed or hybridized to one of the guide strands. 28) A nucleic acid template according to any one of paragraphs 1 to 27, wherein the nucleic acid template further comprises a primer annealed or hybridized to the first guide strand. 29) A method for forming a nucleic acid template, comprising the step of annealing or hybridizing the first guide strand and the second guide strand, (a) the first guide strand comprises, in the 3' to 5' direction, a first synthetic region, a second synthetic region, a first junction domain and a first blocking region; and (b) the second guide strand comprises, in the 3' to 5' direction, a second blocking region, a second junction domain and a third synthetic region, the first junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the third synthetic region, the first junction domain and the second junction domain are substantially complementary to each other to form a double-stranded region, and the first blocking region and the second blocking region together form a blocking domain that blocks the strand displacement activity of the polymerase. The method. 30) The method of paragraph 29, wherein the first or second blocking region comprises a cross-linking segment with the other region to form a blocking domain, and the method further comprises a step of forming a cross-link. 31) A reaction mixture comprising any one of the nucleic acid templates of paragraphs 1-28. 32) The reaction mixture of paragraph 31, further comprising nucleotide triphosphates or deoxynucleotide triphosphates. 33) The reaction mixture of paragraph 32, wherein the reaction mixture does not contain one of adenosine, thymidine / uridine, cytosine or guanosine triphosphate or deoxynucleotide triphosphate. 34) The reaction mixture of any one of paragraphs 31-33, further comprising DNA or RNA polymerase. 35) The reaction mixture of any one of paragraphs 31-35, further comprising a buffer or salt for nucleic acid synthesis. 36) A reaction mixture for synthesizing a guide strand by chemical or enzymatic addition of a junction domain to a nucleic acid. 37) A reaction mixture for synthesizing a guide strand by chemical or enzymatic addition of a blocking region to a nucleic acid strand. 38) A kit comprising any one of the nucleic acid templates of paragraphs 1-28. 39) A kit comprising a component or reaction mixture for producing any one of the nucleic acid templates of paragraphs 1-28 from a nucleic acid sequence (partial guide strand) by enzymatic or chemical addition of a junction region and / or a blocking region onto an existing nucleic acid strand. 40) The kit of paragraph 38 or 39, further comprising nucleotide triphosphates or deoxynucleotide triphosphates. 41) The kit of any one of paragraphs 38-40, further comprising DNA or RNA polymerase. 42) The kit of any one of paragraphs 38-41, further comprising a buffer or salt for nucleic acid synthesis. (43) (a) Preparing or obtaining any one nucleic acid template of paragraphs 1 to 28; (b) If not annealed, annealing or hybridizing the primer to the nucleic acid template; and (c) Extending the nucleotide sequence from the 3′ end of the primer using a DNA polymerase having strand displacement activity A method for synthesizing a nucleic acid sequence, comprising the steps above. (44) The method of paragraph 43, further comprising the step of amplifying the nucleic acid sequence. (45) (a) If not annealed, annealing or hybridizing the primer to the first guide strand, wherein the first guide strand comprises a first synthetic region, a second synthetic region, a junction domain and a blocking region in the 3′ to 5′ direction; and (b) Extending the nucleotide sequence from the 3′ end of the primer using a DNA polymerase having strand displacement activity; (c) Adding a second guide strand to the synthesis reaction, wherein the second guide strand comprises a blocking region, a junction domain, a first synthetic region, a second synthetic region, a second junction domain and a second blocking region in the 3′ to 5′ direction, the first blocking domain of the second guide strand and the blocking domain of the first guide strand together form a blocking domain that blocks the strand displacement activity of the polymerase, the first junction domain of the second guide strand comprises a nucleotide sequence substantially complementary to the nucleic acid sequence of the junction domain of the first guide strand for forming a double-stranded region, and the first synthetic region of the second guide strand comprises a nucleotide sequence substantially identical to the nucleic acid sequence of the junction domain of the first guide strand; (d) Optionally, crosslinking the first blocking domain of the second guide strand and the blocking domain of the first guide strand; (e) Further extending the nucleotide sequence; (f) Removing the second guide strand; and (g) Optionally, removing the first guide strand. (46) The method of paragraph 45, further comprising the step of amplifying the nucleic acid sequence. (47) The method of paragraph 45, further comprising the step of ligating the amplified nucleic acid sequence to a vector. (48) The method of paragraph 45, further comprising the step of introducing the ligated vector into a host cell. (49) The method of paragraph 45, further comprising the step of expressing the nucleic acid sequence in the host cell. (50) The method of paragraph 45, further comprising the step of purifying the expressed product. (51) The method of paragraph 45, further comprising the step of analyzing the purified product. (52) The method of paragraph 45, further comprising the step of storing the purified product. (53) The method of paragraph 45, further comprising the step of using the purified product in a biological assay. (54) The method of paragraph 45, further comprising the step of using the purified product in a therapeutic application. (55) The method of paragraph 45, further comprising the step of using the purified product in a diagnostic application. (56) The method of paragraph 45, further comprising the step of using the purified product in a research application. (57) The method of paragraph 45, further comprising the step of using the purified product in a manufacturing process. (58) The method of paragraph 45, further comprising the step of using the purified product in a quality control process. (59) The method of paragraph 45, further comprising the step of using the purified product in a regulatory compliance process. (60) The method of paragraph 45, further comprising the step of using the purified product in a documentation process. (61) The method of paragraph 45, further comprising the step of using the purified product in a storage process. (62) The method of paragraph 45, further comprising the step of using the purified product in a transportation process. (63) The method of paragraph 45, further comprising the step of using the purified product in a disposal process. (64) The method of paragraph 45, further comprising the step of using the purified product in a recycling process. (65) The method of paragraph 45, further comprising the step of using the purified product in a waste management process. (66) The method of paragraph 45, further comprising the step of using the purified product in a pollution prevention process. (67) The method of paragraph 45, further comprising the step of using the purified product in a resource conservation process. (68) The method of paragraph 45, further comprising the step of using the purified product in a sustainable development process. (69) The method of paragraph 45, further comprising the step of using the purified product in a green chemistry process. (70) The method of paragraph 45, further comprising the step of using the purified product in a chemical engineering process. (71) The method of paragraph 45, further comprising the step of using the purified product in a biotechnology process. (72) The method of paragraph 45, further comprising the step of using the purified product in a pharmaceutical process. (73) The method of paragraph 45, further comprising the step of using the purified product in a medical device process. (74) The method of paragraph 45, further comprising the step of using the purified product in a food and beverage process. (75) The method of paragraph 45, further comprising the step of using the purified product in a cosmetic process. (76) The method of paragraph 45, further comprising the step of using the purified product in a textile process. (77) The method of paragraph 45, further comprising the step of using the purified product in a paper and pulp process. (78) The method of paragraph 45, further comprising the step of using the purified product in a plastics process. (79) The method of paragraph 45, further comprising the step of using the purified product in a rubber process. (80) The method of paragraph 45, further comprising the step of using the purified product in a paint and coating process. (81) The method of paragraph 45, further comprising the step of using the purified product in a adhesive process. (82) The method of paragraph 45, further comprising the step of using the purified product in a lubricant process. (83) The method of paragraph 45, further comprising the step of using the purified product in a fuel process. (84) The method of paragraph 45, further comprising the step of using the purified product in a battery process. (85) The method of paragraph 45, further comprising the step of using the purified product in a solar cell process. (86) The method of paragraph 45, further comprising the step of using the purified product in a wind turbine process. (87) The method of paragraph 45, further comprising the step of using the purified product in a hydroelectric power process. (88) The method of paragraph 45, further comprising the step of using the purified product in a nuclear power process. (89) The method of paragraph 45, further comprising the step of using the purified product in a geothermal power process. (90) The method of paragraph 45, further comprising the step of using the purified product in a biomass power process. (91) The method of paragraph 45, further comprising the step of using the purified product in a waste-to-energy process. (92) The method of paragraph 45, further comprising the step of using the purified product in a desalination process. (93) The method of paragraph 45, further comprising the step of using the purified product in a water treatment process. (94) The method of paragraph 45, further comprising the step of using the purified product in a wastewater treatment process. (95) The method of paragraph 45, further comprising the step of using the purified product in a solid waste treatment process. (96) The method of paragraph 45, further comprising the step of using the purified product in a hazardous waste treatment process. (97) The method of paragraph 45, further comprising the step of using the purified product in a radioactive waste treatment process. (98) The method of paragraph 45, further comprising the step of using the purified product in a noise pollution control process. (99) The method of paragraph 45, further comprising the step of using the purified product in a air pollution control process. (100) The method of paragraph 45, further comprising the step of using the purified product in a water pollution control process. (101) The method of paragraph 45, further comprising the step of using the purified product in a soil pollution control process. (102) The method of paragraph 45, further comprising the step of using the purified product in a climate change mitigation process. (103) The method of paragraph 45, further comprising the step of using the purified product in a climate change adaptation process. (104) The method of paragraph 45, further comprising the step of using the purified product in a biodiversity conservation process. (105) The method of paragraph 45, further comprising the step of using the purified product in a ecosystem restoration process. (106) The method of paragraph 45, further comprising the step of using the purified product in a sustainable agriculture process. (107) The method of paragraph 45, further comprising the step of using the purified product in a forestry process. (108) The method of paragraph 45, further comprising the step of using the purified product in a fisheries process. (109) The method of paragraph 45, further comprising the step of using the purified product in a wildlife conservation process. (110) The method of paragraph 45, further comprising the step of using the purified product in a cultural heritage conservation process. (111) The method of paragraph 45, further comprising the step of using the purified product in a historical building restoration process. (112) The method of paragraph 45, further comprising the step of using the purified product in a museum collection management process. (113) The method of paragraph 45, further comprising the step of using the purified product in a library collection management process. (114) The method of paragraph 45, further comprising the step of using the purified product in a archive collection management process. (115) The method of paragraph 45, further comprising the step of using the purified product in a digital preservation process. (116) The method of paragraph 45, further comprising the step of using the purified product in a software development process. (117) The method of paragraph 45, further comprising the step of using the purified product in a computer programming process. (118) The method of paragraph 45, further comprising the step of using the purified product in a database management process. (119) The method of paragraph 45, further comprising the step of using the purified product in a network security process. (120) The method of paragraph 45, further comprising the step of using the purified product in a cloud computing process. (121) The method of paragraph 45, further comprising the step of using the purified product in a artificial intelligence process. (122) The method of paragraph 45, further comprising the step of using the purified product in a machine learning process. (123) The method of paragraph 45, further comprising the step of using the purified product in a deep learning process. (124) The method of paragraph 45, further comprising the step of using the purified product in a natural language processing process. (125) The method of paragraph 45, further comprising the step of using the purified product in a computer vision process. (126) The method of paragraph 45, further comprising the step of using the purified product in a robotics process. (127) The method of paragraph 45, further comprising the step of using the purified product in a autonomous vehicle process. (128) The method of paragraph 45, further comprising the step of using the purified product in a virtual reality process. (129) The method of paragraph 45, further comprising the step of using the purified product in a augmented reality process. (130) The method of paragraph 45, further comprising the step of using the purified product in a gaming process. (131) The method of paragraph 45, further comprising the step of using the purified product in a entertainment process. (132) The method of paragraph 45, further comprising the step of using the purified product in a education process. (133) The method of paragraph 45, further comprising the step of using the purified product in a training process. (134) The method of paragraph 45, further comprising the step of using the purified product in a research and development process. (135) The method of paragraph 45, further comprising the step of using the purified product in a product design process. (136) The method of paragraph 45, further(f) Optionally, adding an additional new guide strand to the synthesis reaction and further extending the nucleotide sequence before adding each additional new guide strand, wherein each additional guide strand comprises, in the 3' to 5' direction, a blocking region, a junction domain, a first synthesis region, a second synthesis region, a second junction domain, and a second blocking region, and the first blocking region of the new guide strand and the second blocking region of the last guide strand added to the synthesis reaction together form a blocking domain that blocks the strand displacement activity of the polymerase; the first junction domain of the new guide strand comprises a nucleotide sequence substantially complementary to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction to form a double-stranded region; and the first synthesis region of the new guide strand comprises a nucleotide sequence substantially identical to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction; (g) Optionally, crosslinking the first blocking region of the new guide strand and the second blocking region of the last guide strand added to the synthesis reaction before further extending the nucleotide sequence A method for synthesizing a nucleic acid sequence, comprising: 46) (h) Adding a terminal guide strand to the synthesis reaction, wherein the terminal guide strand comprises, in the 3' to 5' direction, a blocking region, a junction domain, a first synthesis region, a second synthesis region, a second junction domain, and a second blocking region, and the first blocking region of the terminal guide strand and the second blocking region of the last guide strand added to the synthesis reaction together form a blocking domain that blocks the strand displacement activity of the polymerase; the first junction domain of the terminal guide strand comprises a nucleotide sequence substantially complementary to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction to form a double-stranded region; and the first synthesis region of the terminal guide strand comprises a nucleotide sequence substantially identical to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction; (i) Optionally, crosslinking the first blocking domain of the terminal guide strand and the blocking domain of the last guide strand added to the reaction; and (j) A step of further extending the nucleotide sequence The method according to claim 45, further comprising . 47) The method according to claim 45 or 46, further comprising a step of amplifying a nucleic acid sequence. 48) Use of any one of the nucleic acid templates of paragraphs 1 to 28 for generating a combinatorial barcode for assigning an identity specific to a target population, wherein the target population is a surface position / partition, matrix, biomolecule, molecular library or biological material (vesicle, cell, tissue, organoid, droplet, liposome, small molecule, bead, etc.), and optionally, the use comprises a step of dividing the target population before addition of the guide strand, and a step of pooling the divided target populations after addition of the guide strand. 49) Use of the method of paragraph 29 or 30 for generating a combinatorial barcode for assigning an identity specific to a target population, wherein the target population is a surface position / partition, matrix, biomolecule, molecular library or biological material (vesicle, cell, tissue, organoid, droplet, liposome, small molecule, bead, etc.), and optionally, the use comprises a step of dividing the target population before addition of the guide strand, and a step of pooling the divided target populations after addition of the guide strand. 50) Use of any one of the reaction mixtures of paragraphs 31 to 42 for generating a combinatorial barcode for assigning an identity specific to a target population, wherein the target population is a surface position / partition, matrix, biomolecule, molecular library or biological material (vesicle, cell, tissue, organoid, droplet, liposome, small molecule, bead, etc.), and optionally, the use comprises a step of dividing the target population before addition of the guide strand, and a step of pooling the divided target populations after addition of the guide strand. 51) Use of any one of the methods of paragraphs 42 to 47 for generating a combinatorial barcode to assign a target population-specific identity, wherein the target population is a surface position / compartment, matrix, biomolecule, molecular library, or biological material (vesicles, cells, tissues, organoids, droplets, liposomes, small molecules, beads, etc.), and optionally, the use includes, prior to addition of the guide strand, a step of dividing the target population, and after addition of the guide strand, a step of pooling the divided target populations. 52) Use of any one of the nucleic acid templates of paragraphs 1 to 28 for generating a combinatorial barcode to assign a spatial position identifier to a target, wherein the target is a surface, biomolecule, or biological material (including, but not limited to, biomolecules, cells, tissues, organs, organoids, vesicles, liposomes, and droplets). 53) Use of the method of paragraph 29 or 30 for generating a combinatorial barcode to assign a spatial position identifier to a target, wherein the target is a surface, biomolecule, or biological material (including, but not limited to, biomolecules, cells, tissues, organs, organoids, vesicles, liposomes, and droplets). 54) Use of any one of the reaction mixtures of paragraphs 31 to 37 for generating a combinatorial barcode to assign a spatial position identifier to a target, wherein the target is a surface, biomolecule, or biological material (including, but not limited to, biomolecules, cells, tissues, organs, organoids, vesicles, liposomes, and droplets). 55) Use of any one of the kits of paragraphs 38 to 42 for generating a combinatorial barcode to assign a spatial position identifier to a target, wherein the target is a surface, biomolecule, or biological material (including, but not limited to, biomolecules, cells, tissues, organs, organoids, vesicles, liposomes, and droplets). Use of any one of the methods of paragraphs 43-47 to generate a combinatorial barcode for assigning a spatial location identifier to a target, wherein the target is a surface, biomolecule or biological material (including, but not limited to, biomolecules, cells, tissues, organs, organoids, vesicles, liposomes and droplets).

[0157] It should be understood that the present disclosure is not limited to the specific methodologies, protocols, reagents, etc. provided herein and can therefore vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present disclosure, which is defined only by the claims. The invention is further illustrated by the following examples, which should not be construed as further limitations.

Examples

[0158] Example 1: Method for synthesizing a strand across a nucleic acid junction The ability to synthesize any sequence of nucleic acids, particularly DNA, has revolutionized the ways in which scientists can study and manipulate biology. Currently, whole genomes can be efficiently sequenced (Schwarze et al., 2018), but the technology for synthesizing genomes lags behind. Synthetic single-stranded sequences (“oligos”) are typically chemically synthesized through cyclic coupling steps (Caruthers et al., 1987). However, oligos are typically not chemically synthesized beyond 200 bases due to the limitations of chemical coupling efficiency. Thus, synthetic methods for assembling larger fragments of single- and double-stranded sequences have been developed, including enzymatic assembly using sequential or simultaneous combinations of multiple enzyme activities (e.g., restriction enzyme digestion and ligation, or isothermal Gibson assembly (Gibson, 2011; Gibson et al., 2009) that combines 5’ exonuclease, 3’ extension activity of DNA polymerase, and DNA ligase activity), non-enzymatic twin primer assembly (Liang et al., 2017), and chemical assembly (e.g., click DNA assembly (Kukwikila et al., 2017)). These can be effective for generating longer sequences of single- and double-stranded DNA, but sequences of hundreds of thousands of bases must be further assembled from shorter fragments in vivo using yeast vectors (Hutchison et al., 2016). A simple and robust new synthetic method that can generate long DNA sequences could have important and achievable applications in molecular biology, genome engineering, nanotechnology, and polymer-based data storage. Provided herein is a method for synthesizing nucleic acid strands across hybridized different nucleic acid backbones using strand displacement polymerases. The method for synthesizing strands across nucleic acid junctions can effectively decouple the synthetic step from the assembly step of template oligos. In this way, it is possible to sequentially perform robust annealing of template oligos that can hybridize in a predictable manner close to thermodynamically optimal conditions, followed by polymerization to create new transcripts.This method has the potential to scale up to arbitrarily long arrays. Furthermore, the entire assembly and synthesis process can occur isothermally, including at room temperature, which enables a generalized workflow for the synthesis of arbitrarily long arrays under relatively mild conditions.

[0159] The basic strategy for cross-junction synthesis is depicted in FIGS. 1A-1B. Shown are two template oligos hybridized together to form a junction, and a primer attached upstream of that junction on the first template strand (left). A strand-displacing polymerase is used to copy the x domain until it reaches a stopper (shown in black). The new and old x domains then compete in a random-walk branch-point migration process (Lee et al., 1970). Eventually, the new x domain binds to the exposed x* domain on the second template strand (right), thus enabling successful crossing of the junction. Polymerization then continues on the second template strand and can copy along the new backbone.

[0160] As depicted in FIG. 2, the cross-junction synthesis reaction can be cascaded to form longer arrays by hybridizing multiple template oligos together. Each junction between template strands exhibits the same domain motif, such that the strand domains (b, c, d, e) copied onto the 3’ end of the growing strand before reaching the stopper can reach and bind across the junction to the exposed complementary sequences (b*, c*, d*, e*) on the next template strand. Arbitrary sequences can be added into the template regions between motif domains (shown in gray) to enable the assembly of longer arrays.

[0161] This method can ligate any given sequence and requires only the basic domain constraints depicted in FIGS. 1A-1B.

[0162] It is also suitable for synthesizing a specific number of repetitive sequences by using a programmable unique hybridization domain that fixes the length of the template concatenator, which has been a long-standing problem. By utilizing the specificity of DNA self-organization, unique binding sequences promote the assembly of a predetermined length, even if the synthesized domains are identical (Figure 3).

[0163] The strategy for assembling cross-junctions with light uses photo-reactive interstrand cross-linking bases, which can serve the dual purpose of linking two cross-junction strands together and acting as a stopper for polymerase synthesis (Figures 4A - 4C). Cross-junctions can be repeatedly assembled through cycles of hybridization, cross-linking, and washing. Cross-junctions can also be assembled and spatially addressed on a surface, and subsequent synthesis steps can still be performed on the surface.

[0164] Assembling a library of orthogonal cross-junction arrays on a surface can enable the multiplex synthesis of orthogonal DNA of a predetermined sequence and arbitrary length.

[0165] In this implementation, 3-cyanovinylcarbazole (CNVK) (Vieregg et al., 2013) base modification and a UV light source were used to cross-link DNA junctions together. Strand design can utilize a set of two or more hybridization domains (1 and 1*, 2 and 2* in this implementation), internal barcode sequences (b1, b2, etc.) that serve as growing sequence strands, and CNVK base cross-linkers (Figure 4A, cyan circles).

[0166] Experimental Verification The stepwise assembly of template concatenators on a surface using cascaded cross-junction synthesis, as well as photo-reactive cross-linking chemistry (see above), followed by cross-junction synthesis, was experimentally verified.

[0167] The synthetic cascades in the range of 1-8-junction synthesis as depicted in FIGS. 5-12 were designed using random three-letter (A, T, and C) 7nt “flap” primers (sequences b, d, f, h, j, l, and n), a 3-consecutive G as a stopper (dGTP removed from the dNTP mix), random four-letter 9nt second hybridization domains (r0, r1, ..., r7), and 5nt three-letter (A, T, and C) intercalating domains (c, e, g, i, k, m, o, q). Template concatemers were formed by combining the strands at a concentration of 1 μM in 1x PBS and annealing from 80 °C to 20 °C over 1 hour (lowering 1 degree per minute). The concatemers were then introduced into the synthesis reaction at a final concentration of 50 nM in 0.95x PBS containing 10 mM MgSO4 together with 40 nM a primer, 1 μM dGTP clean-up hairpin, 600 μM dATP / dCTP / dTTP mix, and 800 units / ml of Bst Large Fragment polymerase. The reaction was incubated at room temperature for 15 minutes, then the primer was added and the incubation was continued for an additional 30 minutes. 2 μL of 6 mM dGTP was added to the 20 μL reaction and incubated for an additional 5 minutes to complete the copy of the For primer sequence. The reaction was then heat inactivated at 80 °C for 20 minutes and then 1000x (final) diluted into Taq 1X PCR containing 200 nM For and rev primers. The qPCR instrument was used to track the amplification in real time using the Sybr Green I intercalating time (FIG. 13A), and the PCR products were run on a PAGE denaturing gel (FIG. 13B). The longest 8-junction synthesis product was further verified using Sanger sequencing (FIG. 13C).

[0168] Optical directed concatenation formation (see above) was also experimentally verified. The reactions performed are depicted in FIGS. 14-16, and the results are shown in FIG. 17. After binding the probe array to the glass slide, the template strand was hybridized at a concentration of 1 μM in 1x PBS for 5 minutes, the bound strand was crosslinked with UV light for about 3 seconds, and then several washing steps were performed with 1x PBS to remove the unbound strands before the next hybridization step. All of these steps were performed at room temperature. After template formation, cross-junction synthesis was performed at room temperature using 40 nM primers, the product was heat inactivated at 80 °C for 20 minutes, and then diluted into the PCR solution and amplified to form double-stranded products. A PAGE denaturing gel was run to show the products for three different template sizes (FIG. 17).

[0169] Discussion The new ability to program synthesis across nucleic acid junctions enables a new paradigm of synthesis that is no longer limited to copying along a single backbone. The formation of template concatemers (assembly steps) can be performed under favorable conditions, such as by annealing, which can help improve the specificity of the formed hybridization interactions. The primer strands may be included in the formation step or hybridized later, and cross-junction synthesis proceeds with a strand-displacing polymerase to generate the sequence record of the template concatemer. As shown above, any programmable sequence can be assembled in this way to generate a long single-stranded sequence. The single-stranded sequence can be used directly or amplified (e.g., by PCR) to form single-stranded or double-stranded products. This ability opens up a number of exciting applications ranging from the generation of combinatorial libraries of sequences to in situ synthesis.

[0170] References TIFF0007692355000001.tif140141

[0171] Example 2: Nucleic Acid Synthesis across Multiple Junctions In addition to the method described above, synthesis beyond 8-junctions was performed using fluorophore-labeled primers according to the process outlined in Figure 19A. The template strand (also referred to as the guide strand) was annealed (1 μM of each strand in 1× PBS, from 80 °C to 20 °C over 1 hour) and diluted into various reaction buffer conditions at two time points (10 and 20 minutes). To block polymerization, the template sequence utilized G-C pairs and the reactants were pre-incubated with the hairpin for 5 minutes. The hairpin was able to sequester the extra dGTP in the mix of dATP, dTTP, and dCTP, after which the fluorophore-labeled primer could be added (using 100 nM of each template strand, at a final 40 nM). The reactants were then incubated at the indicated temperature for 10 or 20 minutes for 2 minutes, after which dGTP was added. Following this step, heat inactivation was performed at 80 °C for 20 minutes. To determine the effect on synthesis efficiency, the reactants were run on a 15% TBE-Urea PAGE denaturing gel (Figure 19B). The two time points (10 and 20 minutes) allowed for monitoring of the reaction process over time. More bands appearing on the gel indicated that synthesis occurred beyond higher numbered junctions. Lanes 1 and 7 were found to have the highest efficiency for nucleic acid synthesis using the method shown in Figure 19A (Figure 19B).

[0172] Several salt conditions were also tested in the 5-junction cross-synthesis reaction. In this aspect of the method described herein, longer binding domains between the template strands were used. The primer and template strands were annealed together at increasing concentrations in 1× PBS from 80 °C to 20 °C over a 1-hour incubation period. A total of 48 reaction conditions (8 salt conditions × 3 polymerase concentrations × 2 time points) were tested with incubation at 37 °C, followed by incubation with dGTP and heat inactivation at 80 °C for 20 minutes. Efficiency was evaluated based on the Cq value from amplification of the complete reaction product (Figures 20A - 20B).

[0173] In another example, instead of pre-assembling the template strands as described above, cross-junction synthesis was performed by successively adding one template strand at a time (see the schematic in FIG. 21A). In continuous nucleic acid synthesis, first, the complete reaction conditions for the 5-junction cross-synthesis reaction excluding the oligo were pre-incubated, and then a primer was introduced. After incubation at 37° C. every 2 minutes, new template strands were added in the order of synthesis. To ensure that nucleic acid synthesis occurred while successively adding each template strand, a separate strand (the terminal barcode strand) was added. The terminal barcode strand used in this example included (i) one or more binding domains substantially identical to the third template strand; and (ii) different barcodes. The barcodes were not complementary to the template strands. The terminal barcode strand was added at the end of the synthesis process. Finally, the reaction mixture was incubated with dGTP and heat inactivated. The finished product was amplified by PCR and subjected to Sanger sequencing (FIG. 21B). The sequencing results showed a nucleic acid sequence that was not interfered with by the barcode from the last terminal barcode strand (FIG. 21B, bottom).

[0174] All patents and other publications identified are hereby expressly incorporated by reference herein for the purpose of describing and disclosing, for example, the methods described in such publications that may be used in connection with the present disclosure. These publications are provided only for the purpose of their disclosure prior to the filing date of the present application. No admission is made that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or other reason. All statements as to the date or content of these documents are based on the information available to the applicant, and no admission is made as to the accuracy of the date or content of these documents.

Claims

1. (a) A first guide strand comprising, in the 3’ to 5’ direction, a first synthesis region, a second synthesis region, a first junction domain, and a first blocking region; and (b) A second guide strand comprising, in the 3’ to 5’ direction, a second blocking region, a second junction domain, and a third synthesis region comprising a nucleic acid template, wherein the first junction domain comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of the third synthesis region, the first junction domain and the second junction domain are complementary to each other to form a double-stranded region, and the first blocking region and the second blocking region together form a first blocking domain that blocks the strand displacement activity of polymerase, said nucleic acid template.

2. (a) The first and second blocking regions are covalently linked to each other; and / or (b) The first or second blocking region comprises a cross-linking segment with the other blocking region to form the first blocking domain, the nucleic acid template according to Claim 1.

3. The cross-linking segment comprises 3-cyanovinylcarbazole, the nucleic acid template according to Claim 2.

4. The second guide strand further comprises a fourth synthesis region at the 5’ end of the third synthesis region, the nucleic acid template according to Claim 1.

5. The second guide strand further comprises, at its 5’ end, in the 3’ to 5’ direction, a third junction domain and a third blocking region, and the nucleic acid template further comprises a third guide strand comprising, in the 3’ to 5’ direction, a fourth blocking region, a fourth junction domain, and a fifth synthesis region, the third junction domain comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of the fifth synthesis region, the third junction domain and the fourth junction domain are complementary to each other to form a double-stranded region, and the third blocking region and the fourth blocking region together form a second blocking domain that blocks the strand displacement activity of polymerase, the nucleic acid template according to Claim 4.

6. (a) The third and fourth blocking regions are covalently linked to each other; and / or (b) The third or fourth blocking region includes a cross-linking segment with the other blocking region to form a second blocking domain. The nucleic acid template according to claim 5. **Claim 7** The cross-linking segment includes 3-cyanovinylcarbazole. The nucleic acid template according to claim 6. **Claim 8** The third guide strand further includes a sixth synthetic region at the 5'-end of the fifth synthetic region. The nucleic acid template according to claim 5. **Claim 9** The third guide strand further includes a fifth junction domain and a fifth blocking region in the 3' to 5' direction at its 5'-end, and the nucleic acid template further includes a fourth guide strand including a sixth blocking region, a sixth junction domain, and a seventh synthetic region in the 3' to 5' direction. The fifth junction domain includes a nucleotide sequence that is at least 80% identical to the nucleotide sequence of the seventh synthetic region. The fifth junction domain and the sixth junction domain are complementary to each other to form a double-stranded region, and The fifth blocking region and the sixth blocking region together form a third blocking domain that blocks the strand displacement activity of the polymerase. The nucleic acid template according to claim 8. **Claim 10** (a) The fifth and sixth blocking regions are covalently linked to each other, and / or (b) The fifth or sixth blocking region includes a cross-linking segment with the other blocking region to form a third blocking domain. The nucleic acid template according to claim 9. **Claim 11** The cross-linking segment includes 3-cyanovinylcarbazole. The nucleic acid template according to claim 10. **Claim 12** The fourth guide strand further includes an eighth synthetic region at the 5'-end of the seventh synthetic region. The nucleic acid template according to claim 9. **Claim 13** The fourth guide strand further includes a seventh junction domain and a seventh blocking region in the 3' to 5' direction at its 5'-end, and the nucleic acid template further includes a fifth guide strand including an eighth blocking region, an eighth junction domain, and a ninth synthetic region in the 3' to 5' direction. The seventh junction domain includes a nucleotide sequence that is at least 80% identical to the nucleotide sequence of the ninth synthetic region. The seventh junction domain and the eighth junction domain are complementary to each other to form a double-stranded region, and The seventh and eighth blocking regions together form a fourth blocking domain that blocks the strand displacement activity of the polymerase. The nucleic acid template according to claim 12.

14. (a) The seventh and eighth blocking regions are covalently linked to each other, and / or (b) The seventh or eighth blocking region includes a cross-linking segment with the other blocking region to form the fourth blocking domain. The nucleic acid template according to claim 13.

15. The cross-linking segment includes 3-cyanovinylcarbazole. The nucleic acid template according to claim 14.

16. (a) One or more regions utilize a three-letter code; or (b) At least one of the first, second, third, or fourth blocking domains includes a double-stranded region or a polymonomer stretch. The nucleic acid template according to any one of claims 1 to 15.

17. The nucleic acid template or at least one of the first, second, third, or fourth blocking domains includes a nucleic acid modification. The nucleic acid template according to claim 1.

18. The nucleic acid modification is a modified nucleobase or a fluorophore at the 3' and / or 5' ends. The nucleic acid template according to claim 17.

19. (a) The nucleic acid template is conjugated to a solid support; (b) The nucleic acid template further includes a primer annealed or hybridized to one of the guide strands; or (c) The nucleic acid template further includes a primer annealed or hybridized to the first guide strand. The nucleic acid template according to claim 1.

20. The step of annealing or hybridizing the first guide strand and the second guide strand A method for forming a nucleic acid template, including: (a) The first guide strand includes a first synthetic region, a second synthetic region, a first junction domain, and a first blocking region in the 3' to 5' direction; and (b) The second guide strand includes a second blocking region, a second junction domain, and a third synthetic region in the 3' to 5' direction, The first junction domain includes a nucleotide sequence that is at least 80% identical to the nucleotide sequence of the third synthetic region, the first junction domain and the second junction domain are complementary to each other to form a double-stranded region, and The first blocking region and the second blocking region together form a blocking domain that blocks the strand displacement activity of the polymerase. The method.

21. (a) A step of preparing or obtaining a nucleic acid template according to any one of claims 1 to 19; (b) If not annealed, a step of annealing or hybridizing a primer to the nucleic acid template; and (c) A step of extending the nucleotide sequence from the 3′ end of the primer using a DNA polymerase having strand displacement activity A method for synthesizing a nucleic acid sequence, comprising:

22. The method according to claim 21, further comprising a step of amplifying the nucleic acid sequence or a step of sequencing the synthesized nucleic acid sequence. The method according to claim 21.

23. (a) A step of annealing or hybridizing a primer to a first guide strand, wherein the first guide strand comprises a first synthesis region, a second synthesis region, a junction domain, and a blocking region in the 3′ to 5′ direction; and (b) A step of extending the nucleotide sequence from the 3′ end of the primer using a DNA polymerase having strand displacement activity; (c) A step of adding a second guide strand to the synthesis reaction, wherein the second guide strand comprises a blocking region, a junction domain, a first synthesis region, a second synthesis region, a second junction domain, and a second blocking region in the 3′ to 5′ direction, the first blocking domain of the second guide strand and the blocking domain of the first guide strand together form a blocking domain that blocks the strand displacement activity of the polymerase, the first junction domain of the second guide strand comprises a nucleotide sequence complementary to the nucleic acid sequence of the junction domain of the first guide strand for forming a double-stranded region, and the first synthesis region of the second guide strand comprises a nucleotide sequence that is at least 80% identical to the nucleic acid sequence of the junction domain of the first guide strand; and (d) A step of further extending the nucleotide sequence; A method for synthesizing a nucleic acid sequence, comprising:

24. The method according to claim 23, further comprising a step of cross-linking the first blocking domain of the second guide strand and the blocking domain of the first guide strand before extending the nucleotide sequence. The method according to claim 23.

25. (e) A step of adding an additional new guide strand to the synthesis reaction and further extending the nucleotide sequence before adding each additional new guide strand, wherein each additional guide strand comprises, in the 3' to 5' direction, a blocking region, a junction domain, a first synthesis region, a second synthesis region, a second junction domain, and a second blocking region, and the first blocking region of the new guide strand and the second blocking region of the last guide strand added to the synthesis reaction together form a blocking domain that blocks the strand displacement activity of the polymerase; the first junction domain of the new guide strand comprises a nucleotide sequence complementary to the nucleotide sequence of the second junction domain of the last guide strand added to the synthesis reaction to form a double-stranded region; and the first synthesis region of the new guide strand comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of the second junction domain of the last guide strand added to the synthesis reaction; The method according to claim 23, further comprising. **Claim 26** (f) A step of cross-linking the first blocking region of the new guide strand and the second blocking region of the last guide strand added to the synthesis reaction before further extending the nucleotide sequence The method according to claim 25, further comprising. **Claim 27** (g) A step of adding a terminal guide strand to the synthesis reaction, wherein the terminal guide strand comprises, in the 3' to 5' direction, a blocking region, a junction domain, a first synthesis region, a second synthesis region, a second junction domain, and a second blocking region, and the first blocking region of the terminal guide strand and the second blocking region of the last guide strand added to the synthesis reaction together form a blocking domain that blocks the strand displacement activity of the polymerase; the first junction domain of the terminal guide strand comprises a nucleotide sequence complementary to the nucleotide sequence of the second junction domain of the last guide strand added to the synthesis reaction to form a double-stranded region; and the first synthesis region of the terminal guide strand comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of the second junction domain of the last guide strand added to the synthesis reaction; and (h) A step of further extending the nucleotide sequence The method according to claim 26, further comprising. **Claim 28** Before extending the nucleotide sequence, the step of cross-linking the first blocking domain of the terminal guide strand and the blocking domain of the last guide strand added to the reaction The method according to claim 27, further comprising.

29. The step of amplifying a nucleic acid sequence or sequencing a synthesized nucleic acid sequence The method according to claim 27, further comprising.

30. Use of the nucleic acid template according to any one of claims 1 to 19 for the following: (a) Preparation of a combinatorial barcode for assigning an identity specific to a target population, wherein the target population is a surface position / partition, matrix, biomolecule, molecular library or biological material; (b) Preparation of a combinatorial barcode for assigning a spatial position identifier to a target, wherein the target is a surface, biomolecule or biological material; (c) Determination of the proximity of biomolecules and preparation of a record indicating distance information between labeled biomolecules; or (d) Preparation of a nucleic acid sequence library.

31. The use according to claim 30, wherein the biological material is a vesicle, cell, tissue, organoid, droplet, liposome, small molecule, or bead.

32. The use according to claim 30, wherein the use further comprises the step of dividing the target population before adding the guide strand and the step of pooling the divided target population after adding the guide strand The use according to claim 30, further comprising.

33. The nucleic acid template according to claim 1, wherein the first junction domain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of the third synthesis region.

34. The nucleic acid template according to claim 1, wherein the first junction domain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of the third synthesis region.

35. The nucleic acid template according to claim 1, wherein the first junction domain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of the third synthesis region.

36. The nucleic acid template according to claim 5, wherein the third junction domain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of the fifth synthesis region.

37. The nucleic acid template according to claim 5, wherein the third junction domain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of the fifth synthesis region.

38. The nucleic acid template according to claim 5, wherein the third junction domain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of the fifth synthetic region.

39. The nucleic acid template according to claim 9, wherein the fifth junction domain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of the seventh synthetic region.

40. The nucleic acid template according to claim 9, wherein the fifth junction domain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of the seventh synthetic region.

41. The nucleic acid template according to claim 9, wherein the fifth junction domain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of the seventh synthetic region.

42. The nucleic acid template according to claim 13, wherein the seventh junction domain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of the ninth synthetic region.

43. The nucleic acid template according to claim 13, wherein the seventh junction domain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of the ninth synthetic region.

44. The nucleic acid template according to claim 13, wherein the seventh junction domain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of the ninth synthetic region.

45. The method according to claim 20, wherein the first junction domain comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of the third synthetic region.

46. The method according to claim 20, wherein the first junction domain comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of the third synthetic region.

47. The method according to claim 20, wherein the first junction domain comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of the third synthetic region.

48. The method according to claim 23, wherein the first synthetic region of the second guide strand comprises a nucleotide sequence that is at least 85% identical to the nucleic acid sequence of the junction domain of the first guide strand.

49. The method according to claim 23, wherein the first synthetic region of the second guide strand comprises a nucleotide sequence that is at least 90% identical to the nucleic acid sequence of the junction domain of the first guide strand.

50. The method according to claim 23, wherein the first synthetic region of the second guide strand comprises a nucleotide sequence that is at least 95% identical to the nucleic acid sequence of the junction domain of the first guide strand.

51. The method according to claim 25, wherein the first synthetic region of the new guide strand comprises a nucleotide sequence that is at least 85% identical to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction.

52. The method according to claim 25, wherein the first synthetic region of the new guide strand comprises a nucleotide sequence that is at least 90% identical to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction.

53. The method according to claim 25, wherein the first synthetic region of the new guide strand comprises a nucleotide sequence that is at least 95% identical to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction.

54. The method according to claim 27, wherein the first synthetic region of the terminal guide strand comprises a nucleotide sequence that is at least 85% identical to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction.

55. The method according to claim 27, wherein the first synthetic region of the terminal guide strand comprises a nucleotide sequence that is at least 90% identical to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction.

56. The method according to claim 27, wherein the first synthetic region of the terminal guide strand comprises a nucleotide sequence that is at least 95% identical to the nucleic acid sequence of the second junction domain of the last guide strand added to the synthesis reaction.

Images