Compositions and methods for synthesizing nucleic acids

Abstract

Provided herein are compositions, kits, and methods for nucleic acid synthesis. Also provided herein are compositions and methods for synthesizing a strand of nucleic acid between different nucleic acid backbones that are hybridized together using a strand-displacing polymerase.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Pursuant to 35 U.S.C. §119(e), this application claims the benefit of U.S. Provisional Application No. 62 / 796,982, filed on January 25, 2019, and U.S. Provisional Application No. 62 / 845,539, filed on May 9, 2019, the contents of both of which are incorporated herein by reference in their entireties.

[0003] Government support

[0004] This invention was made with government support under Grants 1317291 and 1729397 from the National Science Foundation, GM133052 from the National Institutes of Health, and N00014-16-1-2410 and N00014-18-1-2549 from the Office of Naval Research of the Department of Defense. The U.S. Government has certain rights in this invention. Technical Field

[0005] The present disclosure relates to compositions and methods for synthesizing nucleic acids using strand-displacing polymerases. Background Art

[0006] The ability to synthesize any nucleic acid sequence (especially DNA) has revolutionized the way scientists can study and design biology. Now it is possible to sequence the entire genome efficiently, but the technology for synthesizing genomes lags behind. Synthetic single-stranded sequences ("oligos") are usually synthesized chemically by cyclic coupling steps. However, due to the limitations of chemical coupling efficiency, oligonucleotides of more than 200 bases cannot usually be synthesized chemically. Therefore, synthetic methods for assembling larger single-stranded and double-stranded sequence fragments have been developed, including enzyme assembly (such as restriction enzyme digestion and ligation, or isothermal Gibson assembly combining 5' exonuclease, DNA polymerase 3' extension activity and DNA ligase activity), non-enzymatic double primer assembly, and chemical assembly (such as click DNA assembly). Although these methods can effectively produce longer single-stranded and double-stranded DNA sequences, yeast vectors must be used in vivo to further assemble sequences of hundreds of thousands of bases from shorter fragments. A simple and robust new synthetic method capable of generating long DNA sequences could have important enabling applications in molecular biology, genome engineering, nanotechnology, and polymer-based data storage.

[0007] Therefore, there remains a need in the art for compositions and methods for synthesizing nucleic acids having a specified sequence of arbitrary length, a polymer sequence of a specified length, or a sequence comprising a specified number of repeats. The present disclosure addresses some of these needs. Summary of the invention

[0008] Provided herein are compositions and methods for synthesizing nucleic acid chains using strand displacement polymerases across different nucleic acid backbones that are hybridized together. Compositions and methods for synthesizing chains across nucleic acid junctions as described herein can effectively separate the assembly step of nucleic acid templates from the synthesis step. This provides, among other aspects, the robust annealing of sequentially executed guide chains (they can hybridize in a predictable manner close to thermodynamic optimum) and the ability to then polymerize to produce new transcripts. Compositions and methods as described herein can allow expansion to very long arbitrary sequences. In addition, the entire assembly and synthesis process can be carried out isothermally (including at room temperature), which makes it possible to synthesize a general workflow of long arbitrary sequences under relatively mild conditions.

[0009] The methods and compositions provided herein are based, in part, on the discovery that by using specific nucleic acid template secondary structures ( Figure 1A ), any desired DNA sequence can be synthesized using DNA polymerase displacement. Generally, and without limitation, the nucleic acid template may include oligonucleotides that hybridize together to form one or more junctions that prevent the DNA polymerase from using complementary nucleotides to synthesize nucleic acid sequences along the guide strand, but allow the DNA polymerase to displace and cross the junction. Polymerization can then continue on the second guide strand and replicate along the new backbone.

[0010] By hybridizing multiple oligonucleotides together (called guide strands), cross-junction synthesis can be cascaded to form longer sequences ( Figure 2 ). Multiple guide strands can be hybridized to form a nucleic acid template of any length. The nucleic acid template can be used to synthesize a nucleic acid sequence of any desired length.

[0011] Therefore, in one aspect, nucleic acid templates are provided herein. For example, nucleic acid templates for synthesizing nucleic acid sequences by the methods described herein. Typically, the nucleic acid templates include a region that can inhibit the advancement of a strand displacement polymerase, such as a double-stranded region. The double-stranded region together with the sequences at the 3'-end and the 5'-end may also be referred to herein as a synthetic domain or synthetic region.

[0012] like Figure 1BAs shown in, an exemplary cross-link-junction (100) comprises a synthesis region (101), a double-stranded region (102), and another synthesis region (103) in a 3' to 5' direction. The 3'-end (104) of the double-stranded region (102) has a sequence substantially similar to the nucleic acid sequence of the synthesis region (103) at the 5'-end of the duplex. The double-stranded region (102) further comprises a blocking domain (105) located at the 5'-end of the region (104), wherein the blocking domain (105) is capable of blocking the strand displacement activity of a polymerase. The cross-link-junction can be a continuous polynucleotide, or the cross-link-junction can be prepared by annealing two separate guide strands (106 and 107).

[0013] The nucleic acid template may comprise any number of cross-linking-junctions. For example, the nucleic acid template may comprise more than two (e.g., three, four, five, six, seven, eight, nine, ten or more) cross-linking-junctions. When the nucleic acid template comprises more than two cross-linking-junctions, they may be connected together by a nucleic acid connector. For example, the 5'-end of the first cross-linking-junction may be connected to the 3'-end of the second cross-linking-junction. Without limitation, the connector may be any desired length and / or nucleotide sequence. For example, the connector may simply be a nucleic acid backbone connection (e.g., a phosphodiester bond). In addition, the nucleic acid connectors may be all the same, all different, or some the same and some different.

[0014] In addition, when the nucleic acid template comprises more than two cross-linking junctions, the two or more cross-linking junctions may be all the same, some the same, some different, and / or all different. For example, the synthesis region (101) of the two or more cross-linking junctions may have substantially the same nucleotide sequence; the synthesis region (103) of the two or more cross-linking junctions may have substantially the same nucleotide sequence; and / or the synthesis region (101) of the first cross-linking junction may have substantially the same nucleotide sequence as the synthesis region (103) of the second cross-linking junction.

[0015] Cross-linking-junctions can be prepared from two separate nucleic acid chains (e.g., guide chains). Therefore, in some embodiments, the nucleic acid template comprises a first guide chain and a second guide chain, the first guide chain comprising a first synthesis zone, a second synthesis zone, a first junction domain and a first blocking zone in a 3' to 5' direction; the second guide chain comprises a second blocking zone, a second junction domain and a third synthesis zone in a 3' to 5' direction. The first junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the third synthesis zone, 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 zone and the second blocking zone together form a first blocking domain that blocks the strand displacement activity of the polymerase. In a non-limiting manner, the above-mentioned regions and domains can independently comprise any nucleotide sequence, provided that the nucleotide sequences of the first junction domain of the first guide chain and the third synthesis zone of the second guide chain are substantially identical, and the nucleotide sequences of the first junction domain of the first guide chain and the second junction domain of the second guide chain are substantially complementary to each other and form a double-stranded region. In a non-limiting manner, the nucleic acid template can comprise any number of guide chains.

[0016] On the other hand, the present invention provides a method for forming a nucleic acid template. The method includes annealing a first guide strand and a second guide strand. The first guide strand comprises a first synthesis zone, a second synthesis zone, a first junction domain and a first blocking zone in a 3' to 5' direction. The second guide strand comprises a second blocking zone, a second junction domain and a third synthesis zone in a 3' to 5' direction, wherein the first junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the third synthesis zone, 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 zone and the second blocking zone together form a blocking domain that blocks the strand displacement activity of a polymerase.

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

[0018] In another aspect, provided herein is a reaction mixture that can be used to create a guide strand by enzymatically or chemically adding a junction region to an existing nucleic acid strand. For example, a reaction mixture can be used to create a guide strand by enzymatically or chemically adding a blocking region to an existing nucleic acid strand.

[0019] In another aspect, provided herein are kits comprising the nucleic acid templates provided herein.

[0020] In yet another aspect, provided herein are kits comprising components or reaction mixtures for creating a 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 to an existing nucleic acid strand.

[0021] Also provided herein is a method for synthesizing a nucleic acid sequence. Typically, the method comprises extending a nucleotide sequence from the 3'-end of a primer annealed to a nucleic acid template using a DNA polymerase having a strand displacement activity. If the primer has not yet annealed to the nucleic acid template, the method may comprise a step of annealing or hybridizing the primer to the nucleic acid template.

[0022] In some embodiments, a method for synthesizing a nucleic acid sequence comprises: 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 continuously adding additional guide strands in the synthesis reaction, wherein the nucleotide sequence is extended before adding each additional guide strand.

[0023] In some embodiments, provided herein is the use of nucleic acid templates for creating combinatorial barcodes to assign unique identities to target populations, wherein the target populations are surface locations / partitions, matrices, biomolecules, molecular libraries, or biomaterials (e.g., vesicles, cells, tissues, organoids, droplets, liposomes, small molecules, beads), and optionally the use comprises splitting the target population prior to adding a guide strand and pooling the split target population after adding a guide strand. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A Schematic diagram of an exemplary cross-link-junction synthesis mechanism. First, a strand-displacing polymerase replicates along a template strand until it reaches a stopper (step 1). As shown, the first template strand (left) is designed to have an x* domain so that a complementary x sequence is attached to the 3' end of the growing strand. The second template strand (right) that pairs with the first template strand is designed to have an exposed x* binding domain and an x ​​domain that binds to the x* domain on the first template strand. When the x domain on the second template strand displaces the newly synthesized x domain at the 3' end of the growing strand by branch migration (Lee et al., 1970), the strand is then free to pair that domain with the complementary exposed x* domain on the second template strand. At this point, the strand has now successfully "jumped" over the junction, hybridizing with the backbone of the new strand, and synthesis can continue.

[0025] Figure 1B is a schematic diagram of an exemplary cross-link-junction point.

[0026] Figure 2 It is a schematic diagram of exemplary cascade cross-linking-junction synthesis. Many template chains can be hybridized to enable cascade cross-linking-junction synthesis reaction. Template chains with corresponding motif domains can be hybridized together, and strand displacement polymerase can be used to replicate across each junction. In the above example, the primer with domain a replicates the base at the end of domain b until the first terminator is reached, which is then displaced and crosses the junction to reach b* (from the left) on the second template chain. Until the sequence of c, d and e is also similarly replicated (with optional extra bases inserted in the gray domain). This method can link any specified sequence, and only requires the basic domain constraints depicted in Figure 1.

[0027] Figure 3 is a schematic diagram showing the synthesis of a fixed number of repeat sequences. By programming each template strand with a unique hybridization domain (1, 2, 3, and 4), a fixed length template concatemer can be formed for cross-link-junction synthesis. However, if desired, each of these template strands can be used to append the same sequence as the previous junction (e.g., xy sequence). This allows the synthesis of a fixed number of repeat sequence domains. For example, Figure 3 As shown in , the sequence xy is copied exactly 4 times in the sequence.

[0028] Figure 4A-4C is a schematic representation of the light-directed cross-linking junction (blocking domain) assembly. Figure 4A It is shown that interstrand cross-linking reactions between complementary sequences can be achieved using cross-linking base modifications (solid cyan circles). Figure 4B The iterative assembly of crosslink-junctions is shown: (1) an initial docking strand with a crosslink base modification and hybridization domain 1 is hybridized to a strand with complementary site 1*, a first barcode sequence b1, the next hybridization domain 2, and a crosslink base; (2) a UV crosslinking light source is used to covalently link the two sequences together, wash away the uncrosslinked sequences, and introduce the next iterative strand carrying the b2 sequence; (3) UV crosslinking and growth sequence chain extension on a glass surface. Figure 4C Cross-links showing photo-cross-linking will only be produced within the irradiated areas.

[0029] Figure 5-Figure 12 For the following reaction scheme: template nucleic acid synthesis and the resulting 8-junction or blocking domain ( Figure 5 )、7-junction( Figure 6 )、6-junction( Figure 7 )、5-junction( Figure 8 )、4-junction( Fig. 9 ), 3-junction( Fig.10 ), 2-junction( Fig.11 ) and 1-junction ( Fig.12 ) is synthesized by annealing the template strands together. Then, the cross-linking-junction synthesis reaction is performed with primers as described in the figure. Finally, PCR is performed to amplify the synthesized product into a double-stranded amplicon.

[0030] Fig.13A Shows Figure 5-Figure 12 Real-time qPCR amplification of the depicted synthesis. The corresponding reactions 1-8 ( Figure 5-Figure 12 ) PCR reaction. All 8 cross-link-junction synthesis reactions were diluted 1000X (final) into PCR solution and showed amplification at approximately the same cycle. As a control, 8 reactions with all components except dNTPs were also mixed with PCR master mix to check that the amplicons were not generated by the PCR step and template strand alone.

[0031] Fig. 13B is a denaturing PAGE gel showing eight synthetic products of expected length.

[0032] Fig. 13C Shows the results for the 8-junction template ( Figure 5 ) synthetic Sanger sequencing results matched the planned sequence in both directions.

[0033] Figure 14-16 It is about guiding light through 2-junctions ( Fig.14 )、4-junction( Fig.15 ) and 6-junction ( Fig.16 ) concatemer formation and then synthesis to synthesize the reaction scheme of template nucleic acid. The biotinylated probe sequence is attached to the glass slide by biotin-streptavidin interaction. The template strands are iteratively introduced to form a template containing three strands, and then cross-linked-junction synthesis is performed with rev a primer. After dilution and PCR, a double-stranded product is formed.

[0034] Fig.17 It is shown Figure 5-Figure 12 Depicted is a denaturing PAGE gel readout of the synthesized PCR products.

[0035] Fig.18 is a schematic diagram of an exemplary combinatorial cross-link-junction synthesis. The above example shows a combinatorial library formed by including three possible options for the second template strand from the left and two options for the second template strand from the right. By including all possibilities in the annealing reaction, a concatemer library is formed, thereby forming a library of synthetic sequences.

[0036] Fig.19Ais a schematic diagram of an embodiment of synthesizing a template nucleic acid and synthesizing a nucleic acid strand therefrom, wherein a full-length template is assembled and added to a synthesis reaction. Fig.19B Shows the different conditions detailed under each lane Fig.19A Results of the synthetic scheme shown. Each band represents an extended step. It can be seen that lanes 1 and 7 have the highest efficiency.

[0037] Figure 20A-20B is a bar graph showing the amplification under different salt and polymerase conditions at 10 min ( Fig. 20A ) or 20 minutes ( Fig. 20B ) Cq value of the qPCR reaction of the synthesis product spanning 5-junctions performed by incubation.

[0038] Fig.21A Schematic diagram showing an exemplary embodiment of a method of nucleic acid synthesis in which guide strands are continuously added to a reaction mixture during synthesis to assemble a full-length template nucleic acid strand. Fig.21B Expected sequencing results (top) are shown, compared to Sanger sequencing results of PCR amplification products synthesized across 5-junctions using an embodiment of the exemplary method depicted in FIG. 21 . DETAILED DESCRIPTION

[0039] The basic strategy of cross-linking-junction synthesis is described in Figure 1. Two guide strands (also referred to as template strands) that hybridize together to form a junction are shown, as well as a primer (left) in front of the junction that is bound to the first template strand. Strand displacement polymerase is used to replicate the x domain (also referred to as the junction domain in this article) until it reaches the terminator (shown in black and also referred to as the blocking domain in this article). Afterwards, the new and old x domains compete in the random walk branch migration process (Lee et al., 1970). Ultimately, the new x domain can be bound to the x* domain (right, also referred to as the synthesis zone) exposed on the second template strand, thereby successfully crossing the junction. Then, polymerization can continue on the second template strand and replicate along the new main chain.

[0040] Therefore, 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-linking-junction (100). Without limitation, the nucleic acid template can comprise any number of cross-linking-junctions (100), that is, the nucleic acid template can comprise any number of guide strands for forming a desired number of cross-linking-junctions. Multiple guide strands can hybridize to form a complete nucleic acid template that promotes the synthesis of a desired nucleic acid sequence. Figure 1A-Figure 2Guidance for exemplary nucleic acid template structures is provided based on the secondary structure and hybridization of the first guide strand and the second guide strand. The nucleic acid template can be designed based on the desired nucleotide sequence to be synthesized.

[0041] As disclosed herein, the nucleic acid template comprises one or cross-linking-junctions. Typically, each cross-linking-junction comprises a first guide strand and a second guide strand in a 3' to 5' direction. The first guide strand comprises an optional first blocking region, an optional first junction domain, a first synthesis region, a second synthesis region, a second junction domain, and a second blocking region in a 3' to 5' direction. The second guide strand comprises a first blocking region, a first junction domain, a first synthesis region, a second synthesis region, an optional second junction domain, and an optional second blocking region in a 3' to 5' direction. 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 comprises a nucleotide sequence that is substantially complementary to the nucleic acid 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 comprises a nucleotide sequence that is substantially identical to the nucleotide sequence of the first synthesis region of the second guide strand.

[0042] When the nucleic acid template comprises more than two cross-linking junctions, the first guide strand of the first cross-linking junction can be the second guide strand of the second cross-linking junction. Similarly, the second guide strand of the first cross-linking junction can be the first guide strand of the second cross-linking junction.

[0043] It should be noted that the guide strands described herein may include a barcode domain. For example, the second guide strand of the junction may include a barcode domain at the 5'-end. In some embodiments, one of the first synthesis region or the second synthesis region of the guide strand includes a barcode domain.

[0044] As used herein, "barcode domain" refers to a portion of a strand (e.g., a guide strand) that contains a nucleic acid sequence representing information or data. Non-limiting examples include spatial information, sequence, molecular sequence, experimental / batch number, binary encoding, random barcodes for creating unique molecular identifiers (UMIs), or any combination thereof. The barcode domain sequence may be predetermined by a barcode library. The barcode domain may be any suitable sequence. A bit value may be assigned to the barcode domain. For example, a bit value may be independently assigned to each barcode domain. Note that bit values ​​are not limited to 0 and 1. In some embodiments, the length of the barcode domain sequence may be 0 nucleotides. For example, the absence of a barcode sequence may provide information (e.g., a 0 bit value is assigned to a situation where a barcode is not present, and a 1 bit value may be represented as a barcode domain having a length of 1 or more nucleotides).

[0045] 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 represents spatial information.

[0046] The guide strands described herein may also include a primer sequence. For example, the first guide strand of the junction may include a primer sequence at the 3'-end. In another non-limiting example, the second guide strand of the junction may include a primer sequence at the 5'-end.

[0047] In some embodiments, the nucleic acid template comprises a first guide strand and a second guide strand. The first guide strand comprises a first synthesis region, a second synthesis region, a first junction domain, and a first blocking region in a 3' to 5' direction. The second guide strand comprises a second blocking region, a second junction domain, and a third synthesis region in a 3' to 5' direction. The first junction domain comprises a nucleotide sequence 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 a polymerase.

[0048] In some embodiments, the second guide strand further comprises a fourth synthesis region located at the 5' end of the third synthesis region.

[0049] As used herein, the terms "guide strand" or "template strand" are used interchangeably and refer to a portion of a nucleic acid template and serve as a basic building block of a nucleic acid template. Several guide strands can be combined to form a nucleic acid template for synthesizing a desired output nucleic acid sequence. For example, the guide strand can include a first synthesis region, a second synthesis region, a first junction domain, and a first blocking region in the 3' to 5' direction. The guide strand can also include a second blocking region, a second junction domain, and a third synthesis region in the 3' to 5' direction. For reference, Figure 2 Several exemplary guide strands are depicted in . A set of guide strands (template strands) can be used to assemble a full-length template nucleic acid for nucleic acid synthesis.

[0050] As used herein, "synthesis zone" refers to the portion of the template strand (guide strand) that will be copied to the nucleic acid sequence being synthesized. Without limitation, each synthesis zone can independently contain any desired nucleotide sequence. In other words, each synthesis zone can independently be any length. For example, the length of each synthesis zone can be one nucleotide to several thousand nucleotides. In some embodiments, the length of each synthesis zone 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, or 50 nucleotides or less. Without limitation, a synthesis zone can have only one nucleotide.

[0051] Without limitation, the double-stranded regions of the cross-link-junctions may independently comprise any desired nucleotide sequence or number of base pairs. In other words, the double-stranded regions may independently be of any length. For example, the length of each double-stranded region may be from one base pair to several dozen base pairs. In some embodiments, the length of the double-stranded regions may independently be at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten nucleotides or base pairs.

[0052] As used herein, "junction domain" refers to a portion of a guide strand that can hybridize with a portion of a second guide strand to form a duplex, and the nucleic acid sequence generated by a 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 by a nucleic acid template comprising the guide strand comprises a sequence synthesized from the junction domain sequence or the reverse complement of the junction domain sequence.

[0053] Without limitation, each junction domain can independently comprise any desired nucleotide sequence or number of nucleotides. In other words, each junction domain can independently be of any length. For example, the length of each junction domain can be from one nucleotide to tens of nucleotides. In some embodiments, the length of each junction domain can independently be one, two, three, four, five, six, seven, eight, nine, or ten nucleotides.

[0054] As described above, the junction domain of the first guide strand can hybridize with the junction domain of the second guide strand to form a double-stranded structure. Without limitation, each duplex region can independently contain any desired number of base pairs. In other words, each duplex region can independently be of any length. For example, the length of each duplex region can be from one base pair to tens of base pairs. In some embodiments, the length of each duplex region can independently be one, two, three, four, five, six, seven, eight, nine or ten nucleotides or base pairs.

[0055] As used herein, a "blocking domain" refers to a portion of a nucleic acid template that can prevent or stop a polymerase from continuing to move along the backbone of the template strand and / or to replicate the template strand. For example, the blocking domain can be a double-stranded region with high stability (e.g., a hyperstable double-stranded region) such that the polymerase cannot separate the two strands. Methods for forming hyperstable double-stranded nucleic acids are well known in the art and are suitable for use in the present invention. For example, the blocking domain can include a known method for increasing the T of a double-stranded nucleic acid. m One or more modified nucleotides. Exemplary such nucleotides include, but are not limited to, locked nucleic acids (LNA), 2'-O-methoxy-ethyl (2'-MOE) nucleotides, 2,6-diaminopurine (diamopurine), G-clamp (C analog with 4 hydrogen bonds), and guanidinyl G-clamp nucleotides, etc.

[0056] In some embodiments, the blocking domain may include covalent crosslinking of two guide strands. It is worth noting that covalent crosslinking may include a situation where the nucleotide at the 5'-end of the first guide strand is crosslinked with the nucleotide at the 3'-end of the second template strand, for example, connected by an oligonucleotide or a single nucleic acid backbone (e.g., a phosphodiester bond). Typically, the covalent crosslinking of the two guide strands excludes the kind of strand that can produce a continuous sequence from the two guide strands. Therefore, in some embodiments, at least one (or two) of the nucleotides involved in the covalent crosslinking is not a terminal nucleotide. In other words, at least one (or two) of the nucleotides involved in the covalent crosslinking is not the first or last nucleotide of the template strand.

[0057] In a non-limiting manner, each blocking domain can independently contain any desired nucleotide sequence or number of base pairs. In other words, each blocking domain can independently be of any length. For example, the length of each blocking domain can be from one base pair to tens of base pairs. In some embodiments, the length of each blocking domain can independently be one, two, three, four, five, six, seven, eight, nine or ten nucleotides or base pairs.

[0058] In some embodiments, at least one of the blocking domains comprises a multi-monomer stretch. For example, the blocking domain comprises a stretch of polyA, polyT, polyC, or polyG.

[0059] As used herein, "blocking region" refers to a portion of a template strand that forms a blocking domain together with a second template strand. In a non-limiting manner, the blocking region may independently comprise any desired nucleotide sequence or number of nucleotides. In other words, each blocking region may independently be of any length. For example, the length of each blocking region may be from one nucleotide to several tens of nucleotides. In some embodiments, the length of each blocking region may independently be one, two, three, four, five, six, seven, eight, nine or ten nucleotides. It should be noted that the blocking region may be a single nucleotide.

[0060] In some embodiments, at least one blocking region within a blocking domain comprises modified nucleotides that include modifications for cross-linking to another blocking region in the blocking domain.

[0061] As used herein, the term "cross-linked segment" refers to a portion of a nucleic acid template, which comprises modified nucleotides that cross-link with another oligonucleotide upon exposure to a phase change stimulus (such as ultraviolet light, temperature change, pH change, etc.) and allow assembly of the complete nucleic acid template sequence. Figure 4A Examples of cross-linked segments of the nucleic acid templates are provided.

[0062] Monomers and modified nucleotides for oligonucleotide crosslinking are well known in the art. For example, photoreactive nucleotides for crosslinking oligonucleotides are well known in the art. Therefore, in some embodiments, the blocking region may include photoreactive nucleotides, i.e., nucleotides with photoreactive groups. Exemplary photoreactive nucleotides include, but are not limited to: 3-cyanovinylcarbazole (CNVK) nucleotides; 5-bromodeoxycytosine; 5-iododeoxycytosine; 5-bromodeoxyuridine; 5-iododeoxyuridine; and nucleotides comprising aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluorinated aryl azide (FAB-dUMP) or diazomethane (diazirine) (DB-dUMP). In some embodiments, the blocking region includes CNVK as a photoreactive nucleotide.

[0063] In some embodiments of any aspect, the crosslinking segment comprises 3-cyanovinylcarbazole.

[0064] For example, the first blocking region and the second blocking region can be covalently linked to each other. Thus, in some embodiments, the first blocking region or the second blocking region comprises a crosslinking segment, which together with the other blocking region forms a first blocking domain. For example, the crosslinking segment can comprise 3-cyanovinylcarbazole for crosslinking.

[0065] The nucleic acid template and the exemplary secondary structure of the guide strand constituting the nucleic acid template are Figure 1A-Figure 2 The secondary structure allows the 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 obtained. Different structure prediction models can produce different predicted structures, and even the same model may produce different predicted structures if different baseline parameters (such as temperature, ionic strength, etc.) are used.

[0066] That is, when the change of one nucleotide in the base pairing structure of the guide strand in the nucleic acid template is accompanied by a compensatory change of the complementary nucleotide that maintains the ability of base pairing, the structure of the guide strand and the function derived therefrom can be maintained. In other words, some guide strands can tolerate a certain degree of sequence variation and still retain the DNA polymerase displacement activity. In addition, the truncated or partial sequence of the nucleic acid template or guide strand as described herein can also retain the displacement activity, provided that the truncation does not change the necessary intramolecular base pairing of the secondary structure of the guide strand or nucleic acid template. The embodiments herein provide a working demonstration of a nucleic acid template for a specific displacement DNA polymerase.

[0067] It is expected that the reverse sequence, complement sequence, reverse complement sequence or truncated sequence of the nucleic acid template described herein can also maintain the secondary structure of the template. The secondary structure model can be used to predict the stability of the guide strand in the nucleic acid template. The maintenance or improvement of the modified guide strand to produce the nucleic acid output sequence (e.g., compensatory changes or non-compensatory changes) can be performed on the basis of the predicted structure and should be experimentally tested.

[0068] In some embodiments of any aspect, one or more regions of the nucleic acid template use a 3-letter code. As used herein, a "3-letter code" means that the region contains only three of the four nucleobases, i.e., only three of adenine, thymine / uracil, guanine and cytosine, or modified forms thereof. For example, the 3-letter code may contain or consist of a nucleobase selected from one of the following:

[0069] (i) adenine, thymine / uracil and guanine;

[0070] (ii) adenine, thymine / uracil, and cytosine;

[0071] (iii) adenine, guanine and cytosine; or

[0072] (iv) Thymine / uracil, guanine and cytosine.

[0073] 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 synthesis region, junction domain, and / or blocking domain may comprise a nucleic acid modification. Exemplary nucleic acid modifications include, but are not limited to, nucleobase modifications, sugar modifications, inter-sugar linkage modifications, conjugates (e.g., ligands), and any combination thereof.

[0074] Exemplary modified nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine, and uracil, such as 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, uracil, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyl uracil, 8-halogen, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenine and guanine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methyl Guanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines (including 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-pyridone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-hydroxyacetic 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 O-alkylated bases. Further, purines and pyrimidines include those disclosed in: U.S. Pat. No. 3,687,808; the Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, JI ed., John Wiley & Sons, 1990; and Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

[0075] In some embodiments, the modified nucleobase can be selected from the group consisting of inosine, xanthine, hypoxanthine, nubularine, isoguanosine, tuberculocidin, 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-(halogenated)adenine, 8-(hydroxylated)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-(hydroxy)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 )uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidinyl)uracil, 5-(1,3-oxadiazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halogenated)uracil, 5-(methoxy)uracil, uracil-5-hydroxyacetic 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 pyrimidine, 1-(aminocarbonylvinyl)-pseudouracil, 1-(aminocarbonylvinyl)-2(thio)-pseudouracil, 1-(aminocarbonylvinyl)-4(thio)pseudouracil, 1-(aminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-pseudouracil, 1-(aminoalkylamino-carbonylvinyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-4(thio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl , 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tuberculocidin, isoguanosine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyrazinyl, i socarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, stilbene, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridone, 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 purines, substituted 1,2,4-triazoles, and any O-alkylated or N-alkylated derivatives thereof.

[0076] Exemplary sugar modifications include, but are not limited to, 2'-fluoro, 3'-fluoro, 2'-OMe, 3'-OMe, 2'-deoxy modifications, and acyclic nucleotides, such as peptide nucleic acids (PNA), unlocked nucleic acids (UNA), or glycol nucleic acids (GNA).

[0077] In some embodiments, nucleic acid modifications may include replacement or modification of intersugar linkages. Exemplary intersugar linkage modifications include, but are not limited to, phosphotriesters, methylphosphonates, phosphoramidates, phosphorothioates, methylenemethylimino, thiodiesters, thiocarbamates, siloxanes, N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5'), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate, thioformal (3'-S-CH2-O- 5'), acetal (3'-O-CH2-O-5'), oxime, methyleneimino, methylenecarbonylamino, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazine, methylenedimethylhydrazine, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-C5'), C3'-OP(O)-O-SS-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5'.

[0078] In some embodiments, nucleic acid modifications can include peptide nucleic acids (PNA), bridged nucleic acids (BNA), morpholinos, locked nucleic acids (LNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), or any other xenonucleic acids (XNA) described in the art.

[0079] In some embodiments of various aspects, the nucleic acid template, guide strand and / or synthetic nucleic acid sequence can be modified at the 3'-end and / or the 5'-end. For example, a marker, fluorophore, tag or cap can be added to the 3'-end and / or the 5'-end of the nucleic acid template, guide strand and / or synthetic nucleic acid sequence.

[0080] In some embodiments of various aspects, the nucleic acid template, guide strand and / or synthesized nucleic acid sequence can be modified with a linker or spacer, for example, at an internal position, at the 3'-end and / or the 5'-end. Without wishing to be bound by theory, the linker or spacer can be used to connect the nucleic acid template, guide strand and / or synthesized nucleic acid sequence to a portion (e.g., a solid support or a label). In some embodiments, the linker or spacer can be selected from the group consisting of a photocleavable linker, a hydrolyzable linker, a redox cleavable linker, a phosphate / ester-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, and any combination thereof. In some embodiments, the cleavable linker can include a disulfide bond, a tetrazine-trans-cyclooctene group, a thiol group, a nitrobenzyl group, a nitoindoline group, a bromohydroxycoumarin group, a bromohydroxyquinoline group, a hydroxyphenacyl group, a dimethozybenzoin group, or any combination thereof.

[0081] Any art-recognized photocleavable linker can be used. In some embodiments, the cleavable linker may include a photocleavable linker. Typically, a photocleavable linker includes a photolabile functional group that is cleavable when exposed to a light source (e.g., UV light) or a specific wavelength. Non-limiting examples of photocleavable spacers can be found in, for example, U.S. Patent Nos. 6,589,736B1, 7,622,279B2, 9,371,348B2, 7,547,530B2, and 7,057,031B2, and PCT Publication No. WO2014200767, all of which are incorporated herein by reference in their entirety.

[0082] In some embodiments of various aspects, the nucleic acid template, guide strand and / or synthetic nucleic acid sequence can be modified with a detectable label, for example, at an internal position, at the 3'-end and / or the 5'-end. Without wishing to be bound by theory, such detectable labels can assist in detection. As used herein, the term "detectable label" refers to a component that can produce a detectable signal indicating the presence of a target. Detectable labels include any component that can be detected by spectral, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, bioluminescent moieties, etc. Similarly, labels are any components that can be detected by spectral, photochemical, biochemical, immunochemical, electrical, optical or chemical means.

[0083] A wide variety of fluorescent reporter dyes are known in the art. Typically, the fluorophore is an aromatic or heteroaromatic compound and may be pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine, or other similar compounds.

[0084] Exemplary fluorophores include, but are not limited to, 1,5IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein (5-Carboxynapthofluorescein) (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; acriflavin; acriflavin Feulgen SITSA; aequorin (photoprotein); Alexa Fluor 350 TM Alexa Fluor 430 TM ;Alexa Fluor 488 TM Alexa Fluor 532 TM ; Alexa Fluor 546 TM ;AlexaFluor 568 TM Alexa Fluor 594 TM ;Alexa Fluor 633 TM ;Alexa Fluor 647 TM Alexa Fluor 660 TM Alexa Fluor 680 TM; Alizarin complex indicator; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (aminomethylcoumarin); AMCA-X; Aminactinomycin D; Aminocoumarin; Aniline Blue; Anthrocylstearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; AstrazonRed 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG TM CBQCA; ATTO-TAG TM FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); berberine sulfate; β-lactamase; BFP blue-shifted GFP (Y66H); BG-647; Bimane; bisbenzamide; Blancophor FFG; Blancophor SV; BOBO TM -1;BOBO TM -3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-ceramide; Bodipy R6GSE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO TM -1; BO-PRO TM -3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson TM ; Calcium Green; Calcium Green-1Ca2+ dye; Calcium Green-2Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcium Fluorescent White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue TM); 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 TM ;Cy3.1 8;Cy3.5 TM ;Cy3 TM ;Cy5.1 8;Cy5.5 TM ;Cy5 TM ;Cy7 TM ; cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2; Dabcyl; Dansyl; Dansylamide; 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; Erythrosine; Erythrosine ITC; Ethidium homodimer-1 (EthD-1); Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue (Fast Blue); FDA; Feulgen (pararosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4; Fluorescein diacetate; Fluoro-Emerald; Fluoro-Gold (hydroxystilbene); Fluoro-Ruby; FluorX; FM 1-43 TM ;FM 4-46; Fura Red TM(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); red-shifted GFP (rsGFP); wild-type GFP, non-UV excited (wtGFP); wild-type GFP, UV excited (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; hydroxycoumarin; hydroxystilbene (fluorescent gold); hydroxytryptamine; indole dicarbocyanine (DiD); indole tricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine rhodamine; Lissamine rhodamine B; LOLO-1; LO-PRO-1; Lucifer Yellow; Mag Green; Sudan Red (Magdala Red) (Root Bark Red B); Magnesium Green; Magnesium Orange; Malachite Green; MarinaBlue; Maxilon Brilliant Flavin 10GFF; Maxilon Brilliant Flavin 8GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD amine; Nile Red; Nitrobenzoxadiazole; Norepinephrine; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Iavin E8G; Oregon Green TM ; Oregon Green 488-X; Oregon Green TM 488; Oregon Green TM 500; Oregon Green TM514; Pacific Blue; Feulgen; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Root Bark Red B (Sudan Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Photoresist; Phycoerythrin B [PE]; Phycoerythrin R [ΡΕ]; PKH26; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard); resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; 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 TM ;sgBFP TM (superluminescent BFP); sgGFP TM ;sgGFP TM (superluminescent GFP); SITS; SITS (primrose yellow); SITS (stilbene isothiosulfonate); SPQ (6-methoxy-N-(3-sulfopropyl)-quinolinium); stilbene; sulforhodamine B can C; sulforhodamine G Extra; tetracycline; tetramethylrhodamine; Texas Red TM ;Texas Red-X TMConjugates; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (calcium fluorescent white); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC (tetramethylrhodamine isothiocyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. A variety of suitable forms of these fluorescent compounds are available and can be used.

[0085] Other exemplary detectable labels include luminescent and bioluminescent markers (e.g., biotin, luciferase (e.g., bacteria, fireflies, click beetles, etc.), luciferin, and aequorin); radioactive labels (e.g., 3 H. 125 I. 35 S. 14 C or 32 P); enzymes (e.g., β-galactosidases, glucorinidases, phosphatases (e.g., alkaline phosphatase), peroxidases (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 that teach the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference.

[0086] Methods for detecting such labels are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme labels can be detected by providing an enzyme and an enzyme substrate and detecting the reaction product generated by the action of the enzyme on the enzyme substrate; and calorimetric labels can be detected by visualizing a colored label.

[0087] In some embodiments, the detectable label is a fluorophore or quantum dot. Without wishing to be bound by theory, the use of fluorescent agents can reduce the signal-to-noise ratio in imaging / readout, thereby maintaining sensitivity.

[0088] In some embodiments, labels can be configured to include "smart labels" that are undetectable when conjugated to the nucleic acid template, guide strand, and / or synthetic nucleic acid sequence, but produce a color change when released.

[0089] The nucleic acid template, guide strand and / or synthetic nucleic acid sequence may also be modified with acrylamide. Acrylamide modification may allow oligonucleotides to be used in reactions with nucleophilic substances (such as thiols) (e.g., microarrays) or to be incorporated into gels (e.g., polyacrylamide). Therefore, in some embodiments, the nucleic acid template, guide strand and / or synthetic nucleic acid sequence comprises one or more acrylamide nucleosides. The acrylamide nucleosides may be located at the 3'-end, 5-end and / or internal position of the nucleic acid template, guide strand and / or synthetic nucleic acid sequence.

[0090] Any modification may be made to the nucleic acid templates, guide strands and / or synthesized nucleic acid sequences provided herein to allow for purification, extraction, expression quantification, binding, electrophoresis, and the like.

[0091] As disclosed herein, the nucleic acid template may comprise any desired number of cross-links-junctions, i.e., the nucleic acid template may comprise more than two guide strands. For example, the nucleic acid template comprises two cross-links-junctions, e.g., the nucleic acid template comprises three guide strands. Thus, in some embodiments, the second guide strand further comprises a third junction domain and a third blocking region at its 5' end in a 3' to 5' direction, and the nucleic acid template further comprises a third guide strand. The third guide strand comprises a fourth blocking region, a fourth junction domain, and a fifth synthesis region in a 3' to 5' direction. The third junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the fifth synthesis region, wherein the third junction domain and the fourth junction domain are substantially complementary to each other and form a double-stranded region, wherein the third blocking region and the fourth blocking region together form a second blocking domain that blocks the strand displacement activity of a polymerase.

[0092] In some embodiments of any of the aspects, the third guide strand further comprises a sixth synthesis region located at the 5' end of the fifth synthesis region.

[0093] As disclosed herein, the third blocking region or the fourth blocking region may be covalently linked to each other. Thus, one of the third blocking region or the fourth blocking region may comprise a crosslinking segment that together with the other blocking region forms a second blocking domain. In some embodiments, the crosslinking segment comprises 3-cyanovinylcarbazole for crosslinking.

[0094] In some embodiments of any aspect, the nucleic acid template comprises three cross-linking-junctions, for example, the nucleic acid template comprises four guide strands. Therefore, in some embodiments, the third guide strand further comprises a fifth junction domain and a fifth blocking region at its 5' end in a 3' to 5' direction, and the nucleic acid template further comprises a fourth guide strand. The fourth guide strand comprises a sixth blocking region, a sixth junction domain, and a seventh synthesis region in a 3' to 5' direction. The fifth junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the seventh synthesis region, and the fourth junction domain and the fifth junction domain are substantially complementary to each other and form a double-stranded region, and wherein the fifth blocking region and the sixth blocking region together form a third blocking domain that blocks the strand displacement activity of the polymerase.

[0095] In some embodiments, the fourth guide strand further comprises an eighth synthesis region located at the 5' end of the sixth synthesis region.

[0096] As described herein, the fifth and sixth blocking regions may be covalently linked to each other. Thus, one of the fifth or sixth blocking regions may include a crosslinking segment that together with the other blocking region forms a third blocking domain. In some embodiments, the crosslinking segment includes 3-cyanovinylcarbazole for crosslinking.

[0097] In some embodiments of any aspect, the nucleic acid template comprises four cross-linking-junctions, for example, the nucleic acid template comprises five guide strands. Thus, in some embodiments, the fourth guide strand further comprises a seventh junction domain and a seventh blocking region at its 5' end in a 3' to 5' direction, and the nucleic acid template further comprises a fifth guide strand. The fifth guide strand comprises an eighth blocking region, an eighth junction domain, and a ninth synthesis region in a 3' to 5' direction. The seventh junction domain comprises a nucleotide sequence 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 and form a double-stranded region, and wherein the seventh blocking region and the eighth blocking region together form a fourth blocking domain that blocks the strand displacement activity of the polymerase.

[0098] As described herein, the seventh and eighth blocking regions may be covalently linked to each other. Thus, one of the seventh or eighth blocking regions may comprise a crosslinking segment that together with the other blocking region forms a fourth blocking domain. In some embodiments, the crosslinking segment comprises 3-cyanovinylcarbazole.

[0099] In some embodiments, all guide strands in the nucleic acid template may comprise substantially the same nucleotide sequence in the synthesis region on the 5' side of the guide strand. In some embodiments, all guide strands in the nucleic acid template may comprise substantially the same nucleotide sequence in the synthesis region on the 3' side of the guide strand.

[0100] In some embodiments of any aspect, the nucleic acid template, guide strand, or portion thereof is fixed, conjugated, or connected to a solid support or substrate. In some embodiments, the nucleic acid template, guide strand, or portion thereof is fixed on a substrate surface. In some embodiments, the nucleic acid template, guide strand, or portion thereof represents information, date, or spatial information. Note that the nucleic acid template, guide strand, or portion thereof can be covalently or non-covalently fixed, conjugated, or connected to a solid support or substrate.

[0101] In some embodiments of any aspect, the nucleic acid template, guide strand, or portion thereof is fixed in a predetermined pattern. In some embodiments of any aspect, the predetermined pattern is a geometric shape, a square, a circle, or a triangle. In some embodiments of any aspect, the predetermined pattern includes repeating elements. In some embodiments of any aspect, the predetermined pattern is asymmetric or symmetric. In some embodiments of any aspect, the predetermined pattern includes spatial information and / or special information.

[0102] In a non-limiting manner, the solid support or substrate can be present in the form of a platform, a column, a filter or sheet, a disk, a microfluidic capture device, a capillary, an electrochemical response platform, a bracket, a box, a resin, a matrix, a bead, or other solid supports known in the art. The substrate may also include a biomaterial. Biomaterials are known in the art. Non-limiting examples of biomaterials include tissues, tissue sections, engineered tissues, cells, patient-derived cells, primary cells, organoids, extracellular matrices, 3D biological organs, isolated cells, living cells, fixed cells, vesicles, droplets, liposomes, etc.

[0103] In some embodiments, the material of the solid support or substrate includes, but is not limited to, polymers, metals, ceramics, gels, paper, or glass. As non-limiting examples, the material of the solid support may further include polystyrene, agarose, gelatin, alginate, iron oxide, stainless steel, gold nanobeads or particles, copper, silver chloride, polycarbonate, polydimethylsiloxane, polyethylene, acrylonitrile butadiene styrene, cycloolefin polymers or cycloolefin copolymers, or Sepharose. TM Resin.

[0104] 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 may be any water-swellable, cross-linked polymeric material produced by the reaction of one or more monomers. The hydrogel may be a polymeric material that is capable of swelling to retain a substantial portion of water within its structure without dissolving into an aqueous solution. The hydrogel may also be any shrinkable material, such as heat shrinkable plastics, viscoelastic foams, memory foams.

[0105] In some embodiments, the solid support or substrate may also include a magnetically responsive element, such as a magnetically responsive bead. In some embodiments, the magnetically responsive element or bead is in the form of a sphere, cube, rectangle, cylinder, cone, or any other shape described in the art.

[0106] In some embodiments, the magnetically responsive element comprises magnetite, iron (III) oxide, samarium-cobalt, terfenol-D, or any other magnetic element described in the art.

[0107] Synthesis of nucleic acid templates

[0108] On the other hand, the present invention provides a method for forming a nucleic acid template as described herein. Generally, the method includes annealing or hybridizing a first guide strand and a second guide strand. The first guide strand comprises a first synthesis region, a second synthesis region, a first junction domain and a first blocking region in a 3' to 5' direction. The second guide strand comprises a second blocking region, a second junction domain and a third synthesis region in a 3' to 5' direction. The first junction domain comprises a nucleotide sequence 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, wherein the first blocking region and the second blocking region together form a blocking domain that blocks the strand displacement activity of the polymerase.

[0109] In some embodiments, the first blocking region or the second blocking region comprises a crosslinking segment that together with another region forms a blocking domain, and the method further comprises forming a crosslink.

[0110] The compositions and methods described herein can be used to synthesize a given sequence of any length. Figure 2 As shown in Figure 2As shown, by hybridizing multiple guide strands together, cross-link-junction synthesis reactions can be cascaded to form longer sequences. Each junction between guide strands displays the same domain motif, so before reaching the terminator (b, c, d, e), the chain domain replicated on the 3' end of the growing strand is able to cross the junction and bind to the exposed complementary sequence (b*, c*, d*, e*) on the next guide strand. Arbitrary sequences can be added to the template region between the motif domains (shown in gray) to enable assembly of longer sequences.

[0111] The compositions and methods described herein can be used to synthesize polymer sequences of specified lengths. For example, the compositions and methods described herein can be used to fix the length of the template concatemer to synthesize a specific number of repeat sequences by using programmable unique hybridization domains, which has been a long-standing challenge. Figure 3 As shown in Figure 3 As shown, even though the synthesized domains are identical, unique binding sequences promote assembly of a specified length by exploiting the specificity of DNA self-assembly.

[0112] In some embodiments, cross-linking-junctions can be assembled by utilizing photoreactive interchain cross-linking bases, which can serve the dual purpose of connecting two cross-linking-junction chains together and acting as terminators for polymerase synthesis (Fig. 4). Cross-linking-junctions can be assembled iteratively through hybridization, cross-linking, and washing cycles. Cross-linking-junctions can also be assembled and spatially addressed on a surface, and subsequent synthesis steps can still be performed on the surface.

[0113] Assembling libraries of orthogonal crosslink-junction arrays on a surface can enable multiplexed synthesis of orthogonal and arbitrary-length DNA of specified sequence.

[0114] In one exemplary embodiment, 3-cyanovinylcarbazole (CNVK) (Vieregg et al., 2013) base modification and a UV light source are used to crosslink DNA junctions together. The design of the strand can utilize: a set of two or more hybridization domains (1 and 1*, 2 and 2* in the current implementation), an internal barcode sequence (b1, b2, etc.) that will be used as a growing sequence strand, and a CNVK base crosslinker ( Figure 4A , cyan circle).

[0115] Nucleic acid synthesis

[0116] In another aspect, provided herein is a method for synthesizing a nucleic acid using a nucleic acid template as described herein. Generally, the method comprises extending a nucleotide sequence from the 3'-end of a primer annealed or hybridized to the nucleic acid template using a DNA polymerase having strand displacement activity.

[0117] In some embodiments, the method further comprises 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 may be annealed or hybridized to a guide strand of the nucleic acid template prior to assembling the full-length nucleic acid.

[0118] The nucleic acid templates used in the methods for synthesizing nucleic acids described herein can be fully assembled prior to synthesis. Figure 5-Figure 12 and Figure 14-19A As shown in , all 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 a nucleic acid includes annealing or hybridizing the 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 guide strands can be added to the initial reaction mixture and allowed to anneal or hybridize prior to the extension step.

[0119] Alternatively, synthesis can be continuous. For example, guide strands can be added continuously to the synthesis reaction over a period of time under conditions that allow the synthesis of the desired nucleic acid sequence across the junction. Typically, synthesis begins with a 3'-end extension nucleotide sequence of the primer that anneals or hybridizes with the guide strands. The reaction is substantially free of other guide strands of the full-length template. After a period of time (e.g., 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes or longer), the next guide strand is added. This can continue until the desired full-length template is assembled.

[0120] like Fig.21A As shown in the exemplary embodiment of , synthesis begins by annealing or hybridizing the first guide strand to the primer. The first guide strand comprises a synthesis region, a junction domain, and a blocking region in a 3' to 5' direction. After the primer is annealed or hybridized to the first guide strand, the primer is extended from its 3' end. For example, after a period of time (such as 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 longer), additional new guide strands can be continuously added to the synthesis reaction.

[0121] Note that after adding a new guide strand to the synthesis reaction, the new guide strand is allowed to anneal or otherwise hybridize to the guide strands already in the synthesis reaction. Optionally, the complementary blocking regions are covalently cross-linked (e.g., by photocross-linking). Synthesis is allowed to continue for a period of time (e.g., 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 longer) before the next guide strand is added to the reaction.

[0122] Typically, each additional guide strand comprises, in a 3' to 5' direction, 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 comprises 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 comprises a nucleotide sequence that is substantially identical to the nucleotide sequence of the junction domain of the first guide strand.

[0123] Each additional new guide chain added after the first new guide chain has the following characteristics: the first blocking region of the new guide chain and the second blocking region of the last guide chain 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 chain contains a nucleotide sequence that is substantially complementary to the nucleic acid sequence of the second junction domain of the last guide chain added to the synthesis reaction to form a double-stranded region; and the first synthesis region of the new guide chain contains a nucleotide sequence that is substantially identical to the nucleic acid sequence of the second junction domain of the last guide chain added to the synthesis reaction.

[0124] There is no limit to the number of additional guide strands that can be added. 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.

[0125] In some embodiments, the method further comprises adding a terminal guide strand to the reaction synthesis. The terminal guide strand comprises a blocking region, a junction domain, a first synthesis region, an optional second synthesis region and an optional primer sequence in a 3' to 5' direction.

[0126] If one or more additional guide chains are added to the synthesis reaction, the terminal guide chain has the following characteristics: the blocking region of the terminal guide chain and the second blocking region of the last guide chain 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 chain contains a nucleotide sequence that is substantially complementary to the nucleic acid sequence of the second junction domain of the last guide chain added to the synthesis reaction to form a double-stranded region; and the first synthesis region of the terminal guide chain contains a nucleotide sequence that is substantially identical to the nucleic acid sequence of the second junction domain of the last guide chain added to the synthesis reaction.

[0127] If no additional guide strands are added to the synthesis, the terminal guide strand 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.

[0128] In some embodiments, the terminal guide strand comprises a primer sequence at its 5 '-end.

[0129] As used herein, the term "primer" is used to describe a DNA (or RNA) sequence that pairs with a DNA chain and provides a free 3'-OH, at which DNA polymerase begins to synthesize a deoxyribonucleotide chain. Preferably, the primer is composed of an oligonucleotide. The exact length of the primer will depend on a variety of factors, including temperature and primer source. For example, depending on the complexity of the target sequence, an oligonucleotide primer typically contains 15-25 or more nucleotides, although it can contain fewer nucleotides. Short primer molecules typically require lower temperatures to form sufficiently stable hybrid complexes with templates.

[0130] "Polymerase" refers to an enzyme that performs template-directed synthesis of polynucleotides (e.g., DNA and / or RNA). The 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 versions thereof. Other examples of commercially available polymerases include, but are not limited to, Klenow fragment (New England Biolabs, Inc., New England Biolabs, NE, USA). Inc.), Taq DNA polymerase (QIAGEN), 9°NTM DNA polymerase (New England Biolabs Inc.), Deep Vent TM 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 DNA-dependent polymerases and RNA-dependent polymerases (e.g., reverse transcriptases). At least five DNA-dependent DNA polymerase families are known, although most belong to the A family, the B family, and the C family. There is little or no sequence similarity between the various families. Most A-family polymerases are single-chain proteins that can contain multiple enzymatic functions including polymerase, 3' to 5' exonuclease activity, and 5' to 3' exonuclease activity. B-family polymerases typically have a single catalytic domain and an auxiliary factor, and the single catalytic domain has polymerase and 3' to 5' exonuclease activity. C-family polymerases are typically multi-subunit proteins with polymerization and 3' to 5' exonuclease activity. In Escherichia coli (E. coli), three types of DNA polymerases have been found: DNA polymerase I (A family), DNA polymerase II (B family), and DNA polymerase III (C family). In eukaryotic cells, three different B-family polymerases, DNA polymerases α, δ, and ε, are involved in nuclear replication, and the A-family polymerase, polymerase γ, is used for mitochondrial DNA replication. Other types of DNA polymerases include bacteriophage polymerases. Similarly, RNA polymerases generally include eukaryotic RNA polymerases I, II, and III, as well as bacterial RNA polymerases, and bacteriophage and viral polymerases. RNA polymerases can be DNA-dependent and RNA-dependent.

[0131] It should be noted that reagents (e.g., strand displacement DNA or RNA polymerases), and methods for synthesizing nucleic acid sequences from nucleic acid templates are well known in the art and are applicable to the present invention. See, for example, US20050277146A1, US20100035303A1, and WO2006030455A1, all of which are incorporated herein by reference in their entirety.

[0132] In some embodiments of any aspect, the method further includes amplifying nucleic acid sequences. As used herein, the term "amplification" refers to a step in which the nucleic acid sequence is placed under conditions sufficient to allow polynucleotide amplification if all components of the reaction are complete. The components of the amplification reaction include, for example, primers, polynucleotide templates, polymerases, nucleotides, etc. The term "amplification" generally refers to an "exponential" increase in a target nucleic acid. However, "amplification" as used herein may also refer to a linear increase in the quantity of a selected target sequence of a nucleic acid, such as obtained with cycle sequencing. Amplification and methods for synthesizing nucleic acid sequences are known in the art. For example, referring to U.S. Patent Nos. 7,906,282, 8,367,328, 5,518,900, 7,378,262, 5,476,774, and 6,638,722, all of which are incorporated herein by reference in their entirety.

[0133] In some embodiments, amplifying a nucleic acid sequence comprises a polymerase chain reaction (PCR). PCR is well known to those skilled 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., 1990, which is incorporated herein by reference in its entirety. Exemplary PCR reaction conditions typically include two or three-step cycles. 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 a separate extension step.

[0134] In some embodiments, the amplification step includes additional polynucleotide sequences or templates having hairpins orthogonal to the amplification step. Without wishing to be bound by theory, such additional DNA hairpins can reduce or correct off-target reactions. For example, when using a 3-letter code, these sequences or templates comprising additional hairpins can be used to absorb trace amounts of unwanted nucleotides that may be present in some samples.

[0135] In some embodiments of any aspect described herein, the guide strands are added to the initial reaction mixture. In some embodiments of any aspect described herein, the synthesis is continuous. In continuous synthesis, the guide strands are each added continuously under conditions that allow synthesis of the desired nucleic acid across the junction. See, for example, Fig.21A , which provides a schematic diagram of one embodiment of the method described herein.

[0136] In order to complete a round of continuous nucleic acid synthesis as described herein, a terminal barcode chain can be used. As used herein, a "terminal barcode chain" comprises (i) one or more binding domains substantially identical to a template chain; optionally (ii) different barcodes; and optionally (iii) a PCR primer sequence on the 5' end so that the complete synthetic product can be amplified exponentially. Specifically, the optional barcode of the terminal barcode chain is not complementary to the template chain. In a non-limiting manner, the terminal barcode chain can be added to the 3' end or the 5' end of the template nucleic acid. The terminal barcode chain may also include a label or a fluorophore.

[0137] In another aspect, provided herein is a reaction mixture comprising the nucleic acid template.

[0138] In some embodiments of any aspect, the nucleic acid template further comprises a primer that anneals or otherwise hybridizes to one of the guide strands. In another embodiment, the nucleic acid template further comprises a primer that anneals or hybridizes to the first guide strand, the second guide strand, the third guide strand, the fourth guide strand, the fifth guide strand, the sixth guide strand, the seventh guide strand, etc.

[0139] In some embodiments of any aspect, the reaction mixture further comprises nucleoside triphosphates or deoxynucleoside triphosphates. In some embodiments, the reaction mixture does not include one of adenosine, thymidine / uridine, cytosine or guanosine triphosphates or deoxynucleoside triphosphates.

[0140] In some embodiments, the reaction mixture further comprises a DNA or RNA polymerase.

[0141] In some embodiments, the reaction mixture further comprises a buffer or salt for nucleic acid synthesis. It is contemplated that the buffer selected for use in the reaction mixture is to allow the stability of the nucleic acid template and required nucleotide sequence. The method for selecting this type of buffer is known in the art, and can also be selected with regard to their properties under various conditions (comprising the temperature or pH of the reaction carried out).

[0142] In some embodiments, the reaction mixture comprises components that can be used to create a guide strand by enzymatically or chemically adding a junction domain to an existing nucleic acid strand. For example, the reaction mixture can be used to create a guide strand by enzymatically or chemically adding a blocking region to an existing nucleic acid strand.

[0143] In one aspect, provided herein are kits comprising nucleic acid templates as provided herein. In certain embodiments, such kits are intended for use in therapeutic applications. In certain embodiments, such kits are intended for use in research applications.

[0144] In one embodiment of any aspect, the kit further comprises nucleoside triphosphates or deoxynucleoside triphosphates.

[0145] In another embodiment of any aspect, the kit further comprises a DNA polymerase or an RNA polymerase.

[0146] In one embodiment of any aspect, the kit further comprises a buffer or salt for nucleic acid synthesis.

[0147] In one embodiment of any aspect, the kit further comprises a solid support as provided herein to isolate specific sequences.

[0148] In yet another aspect, provided herein are kits comprising components or reaction mixtures for creating a nucleic acid template described herein from a nucleic acid sequence (partial guide strand) by enzymatically or chemically adding a junction domain and / or a blocking region to an existing nucleic acid strand.

[0149] In some embodiments of the various aspects described herein, the kit further comprises instructions for use.

[0150] Methods for purification and analysis of nucleic acids are known to those skilled 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 nucleotide sequence, secondary structure, chemical composition, expression, thermodynamics, binding or function.

[0151] Example Applications

[0152] The nucleic acid templates and methods provided herein can be used in a number of different applications in principle. Non-limiting examples include, but are not limited to, the applications described below.

[0153] Proximity and molecular distance measurements. Since the synthesis steps occur isothermally and the hybridization kinetics can also be tuned to occur under mild (non-denaturing) conditions, the proximity of biomolecules can be determined and a record can be created indicating the distance information between the labeled biomolecules. This applies to distance / proximity measurements of DNA / RNA / proteins and other biomolecules of interest, and structural determination of DNA, RNA, proteins and RNA-protein complexes. If the synthesis reaction is programmed to produce a record pattern that is relatively different from other targets, then this can be further used as a fingerprint recognition method for proteins and other biomolecules.

[0154] Long-chain synthesis and DNA assembly. Because the assembly and synthesis steps can be separated, it is possible to perform assembly of larger sequences than would otherwise be possible.

[0155] Combinatorial synthesis for library generation. Template concatenation can be designed to generate programmable combinatorial sequence sets ( Fig.18 ). Multiple options for inserting domains may be desired in a sequence library, which can be achieved by simply including a relevant guide strand with each of the options (included at the desired relative frequency). For example, having 10 options for each of 5 different domains, instead of the five normally used for fixed domains, would require 10*5=50 guide strands, but would allow the creation of 10^5=100,000 possible sequences. Typically, for positions p1, p2, ..., pn, each position has multiple possibilities x1, x3, ..., xn, and the total number of strands required for these positions would be x1+x2+...+xn, which would generate a library of size x1*x3*...*xn.

[0156] Parallel synthesis of multiple orthogonal sequences. Multiple orthogonal cross-link-junction synthesis reactions can be performed together in the same reaction. These may or may not use the same primers or insert sequences.

[0157] In situ synthesis. The synthesis steps are carried out in an aqueous isothermal environment, allowing the in situ synthesis of arbitrary programmable chains in biological samples or biomaterials.

[0158] Construct identification barcodes (unique molecular identifiers, batch numbers, indexes). Cross-linking-junction synthesis can be used to create unique identification barcodes on surfaces, matrices, biomolecules, molecular libraries, or biological samples or biomaterials (e.g., vesicles, cells, tissues, organoids, droplets, liposomes, small molecules, beads) in a combinatorial manner, or encode unique molecular identifiers to spatial locations, biomolecules, or biological samples or biomaterials. This can be further combined with early methods, such as split-and-pool synthesis or split-pool synthesis, in which the addition of each guide chain is performed after the target population is split and before pooling. See, for example, Brenner et al., (2000) Proc. Natl. Acad. Sci. USA 97: 1665 and US20160251697A1, which are incorporated herein by reference in their entirety.

[0159] Construction of spatial barcodes. Cross-linking-junction synthesis can be used to create barcodes in a combinatorial manner at a defined location or defined target on a surface. Non-limiting examples of targets on a surface include biomolecules, biomaterials, and the like. Biomaterials can be selected from tissues, tissue sections, engineered tissues, cells, patient-derived cells, primary cells, organoids, extracellular matrices, 3D bio-organs, isolated cells, living cells, fixed cells, vesicles, droplets, liposomes, and the like.

[0160] Strand re-routing. Since cross-link-junction synthesis can be used to "re-route" polymerases to new backbone sequences, it can be used for gene and sequence editing with synthetic rerouting. This can be done by joining a strand with a new desired sequence in front of the old sequence so that the polymerase crosses the junction to the new sequence for replication.

[0161] In addition to the applications provided above, several types of nucleic acid synthesis can be employed 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 / multiplexed synthesis, proximity and molecular distance measurement.

[0162] Selected Definitions

[0163] For convenience, the meanings of some terms and phrases used in the specification, examples and appended claims are provided below. Unless otherwise specified or implied by the context, the following terms and phrases include the meanings provided below. Unless otherwise explicitly stated, or obvious from the context, the following terms and phrases do not exclude the meanings that the term or phrase has acquired in the art to which it belongs. These definitions are provided to help describe the specific embodiments of the aspects provided herein and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. In addition, unless the context requires otherwise, singular terms shall include pluralities, and plural terms shall include the singular.

[0164] Definitions of commonly used terms in immunology and molecular biology can be found in: The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Werner Luttmann, Immunology, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (editor), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN044460149X); Laboratory Methods in Enzymolog: DNA, Jon Lorsch (editor), Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (editor), 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 (editor), John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the entire contents of which are incorporated herein by reference in their entirety.

[0165] As used herein, "nucleic acid" refers to DNA, RNA, single-stranded, double-stranded or more highly aggregated hybridization motifs, and any chemically modified forms thereof. For example, nucleic acid can encompass double-stranded nucleic acids or triple-stranded nucleic acids, as well as single-stranded molecules. In double-stranded nucleic acids or triple-stranded nucleic acids, the nucleic acid chains do not need to be coextensive (i.e., double-stranded nucleic acids do not need to be double-stranded along the entire length of the two chains). The term nucleic acid also encompasses any chemically modified forms thereof, such as by methylation and / or by capping. Nucleic acid modifications may include the addition of chemical groups that incorporate additional charge, polarity, hydrogen bonds, electrostatic interactions, and functionality into a single nucleic acid base or the entire nucleic acid. Such modifications may include base modifications (e.g., 2'-sugar modifications, 5-pyrimidine modifications, 8-purine modifications, modifications at cytosine outer ring amines, substitutions of 5-bromouracil), backbone modifications, unusual base pairing combinations (e.g., isobases isocytidine and isoguanidine), etc. Nucleic acids can be derived from completely chemical synthetic processes (e.g., solid phase-mediated chemical synthesis), from biological sources (e.g., by isolation from any species that produces nucleic acids), or from processes involving the manipulation of nucleic acids by molecular biology tools (e.g., DNA replication, PCR amplification, reverse transcription), or a combination of these processes.

[0166] As used herein, the terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides, or analogs thereof) of any length. A polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci / locus defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), small RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA) (which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification); DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component.

[0167] The term "statistically significant" or "significantly" refers to statistical significance and generally means a difference of two standard deviations (2SD) or greater.

[0168] As used herein, the terms "comprising or comprises" are used to refer to compositions, methods, and their respective components that are essential to the method or composition, but both remain open to the inclusion of unspecified elements, whether essential or not.

[0169] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or operating characteristics of the embodiments of the invention.

[0170] Unless the context clearly indicates otherwise, the singular terms "a / an" and "the" include plural referents. Similarly, the word "or" is intended to include "and / as well as" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to the methods and materials provided herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Therefore, the abbreviation "eg" is synonymous with the term "for example".

[0171] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0172] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” When used in conjunction with a percentage, the term “about” may mean ±1%.

[0173] The term "substantially identical" refers to two or more nucleotide sequences having at least 65%, 70%, 80%, 85%, 90%, 95% or 97% identical nucleotides. In some embodiments, "substantially identical" refers to two or more nucleotide sequences having the same identical nucleotides.

[0174] As used herein, unless otherwise indicated, when used to describe a first nucleotide sequence associated with a second nucleotide sequence, the term "complementary" will be understood by the skilled person as referring to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. For example, such conditions may be stringent conditions, wherein stringent conditions may 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 may be applied, such as physiologically relevant conditions that may be encountered in an organism. The skilled person will be able to determine a set of conditions that are most suitable for testing the complementarity of two sequences based on the ultimate application of the hybridizing nucleotides.

[0175] As used herein, "complementary" sequences may also include non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleotides, or may be formed entirely by non-Watson-Crick base pairs and / or base pairs formed by non-natural and modified nucleotides, satisfying the above requirements with respect to their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogsteen base pairing.

[0176] As used herein, the terms "substrate" or "substrate surface" are used interchangeably to describe a structure on which one or more nucleic acid templates, guide strands, or portions thereof as provided herein can be displayed or contacted for contact with other nucleic acids and / or labels. Nucleic acid templates, guide strands, and barcode strands as provided herein can be immobilized, conjugated, or attached to a substrate surface.

[0177] As used herein, the term "conjugated to" includes association of the nucleic acid with a substrate surface, phase change agent, or affinity pair member by covalent bonds, including but not limited to cross-linking by a cross-linking agent, or by strong non-covalent interactions that are maintained under the conditions in which the conjugate is used.

[0178] As used herein, the term "hybridization" refers to the following phenomenon: a single-stranded nucleic acid or a region thereof forms a hydrogen bond base pair interaction with another single-stranded nucleic acid or a region thereof (intermolecular hybridization), or a single-stranded nucleic acid or a region thereof forms a hydrogen bond base pair interaction with another single-stranded region of the same nucleic acid (intramolecular hybridization). Hybridization is dominated by the base sequence involved and the complementary nucleobases that form hydrogen bonds, and the stability of any hybridization depends on the type of base pair (e.g., G:C base pairs are stronger than A:T base pairs) and the number of continuous base pairs, and longer complementary base stretches form more stable hybrids.

[0179] As used herein, the term "spatial information" is any information, coordinates, or markers in a biological tissue or matrix that can be stored in a barcode. The spatial information can inform a person skilled in the art where a particular marker, barcode, or pattern is located on a substrate. 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 detecting a specific nucleic acid target.

[0180] Some embodiments of the methods and compositions described herein may be defined according to any of the following numbered paragraphs:

[0181] 1) A nucleic acid template, comprising:

[0182] (a) a first guide strand comprising, in a 3' to 5' direction, a first synthesis region, a second synthesis region, a first junction domain, and a first blocking region; and

[0183] (b) a second guide strand comprising, in a 3' to 5' direction, a second blocking region, a second junction domain, and a third synthesis region;

[0184] wherein the first junction domain comprises a nucleotide sequence substantially identical to a 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, and

[0185] The first blocking region and the second blocking region together form a first blocking domain, and the first blocking domain blocks the strand displacement activity of the polymerase.

[0186] 2) The nucleic acid template as described in paragraph 1, wherein the first blocking region and the second blocking region are covalently linked to each other.

[0187] 3) The nucleic acid template as described in any of paragraphs 1-2, wherein the first blocking region or the second blocking region comprises a cross-linking segment, and the cross-linking segment forms the first blocking domain with the other blocking region.

[0188] 4) The nucleic acid template as described in paragraph 3, wherein the cross-linking segment comprises 3-cyanovinylcarbazole.

[0189] 5) The nucleic acid template as described in any of paragraphs 1 to 4, wherein the second guide strand further comprises a fourth synthesis region located at the 5' end of the third synthesis region.

[0190] 6) The nucleic acid template of any of paragraphs 1-5, wherein the second guide strand further comprises a third junction domain and a third blocking region at its 5' end in a 3' to 5' direction, and the nucleic acid template further comprises a third guide strand, wherein the third guide strand comprises a fourth blocking region, a fourth junction domain, and a fifth synthesis region in a 3' to 5' direction,

[0191] wherein the third junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the fifth synthetic region,

[0192] wherein the third junction domain and the fourth junction domain are substantially complementary to each other and form a double-stranded region, and

[0193] The third blocking region and the fourth blocking region together form a second blocking domain, and the second blocking domain blocks the strand displacement activity of the polymerase.

[0194] 7) The nucleic acid template as described in any of paragraphs 1-6, wherein the third blocking region and the fourth blocking region are covalently linked to each other.

[0195] 8) The nucleic acid template as described in any of paragraphs 1-7, wherein the third blocking region or the fourth blocking region comprises a cross-linking segment, and the cross-linking segment forms the second blocking domain with the other blocking region.

[0196] 9) The nucleic acid template as described in paragraph 8, wherein the cross-linking segment comprises 3-cyanovinylcarbazole.

[0197] 10) The nucleic acid template as described in any of paragraphs 6 to 9, wherein the third guide strand further comprises a sixth synthesis region located at the 5' end of the fifth synthesis region.

[0198] 11) The nucleic acid template of any of paragraphs 6-10, wherein the third guide strand further comprises a fifth junction domain and a fifth blocking region at its 5' end in a 3' to 5' direction, and the nucleic acid template further comprises a fourth guide strand, wherein the fourth guide strand comprises a sixth blocking region, a sixth junction domain and a seventh synthesis region in a 3' to 5' direction,

[0199] wherein the fifth junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the seventh synthetic region, and the fourth junction domain and the fifth junction domain are substantially complementary to each other and form a double-stranded region, and

[0200] The fifth blocking region and the sixth blocking region together form a third blocking domain, and the third blocking domain blocks the strand displacement activity of the polymerase.

[0201] 12) The nucleic acid template of paragraph 11, wherein the fifth blocking domain and the sixth blocking domain are covalently linked to each other.

[0202] 13) The nucleic acid template as described in paragraph 11 or 12, wherein the fifth blocking region or the sixth blocking region comprises a cross-linking segment, and the cross-linking segment forms the third blocking domain with the other blocking region.

[0203] 14) The nucleic acid template as described in paragraph 13, wherein the cross-linking segment comprises 3-cyanovinylcarbazole.

[0204] 15) The nucleic acid template as described in any of paragraphs 11-14, wherein the fourth guide strand further comprises an eighth synthesis region located at the 5' end of the sixth synthesis region.

[0205] 16) The nucleic acid template of any of paragraphs 11-15, wherein the fourth guide strand further comprises a seventh junction domain and a seventh blocking region at its 5' end in a 3' to 5' direction, and the nucleic acid template further comprises a fifth guide strand, wherein the fifth guide strand comprises an eighth blocking region, an eighth junction domain and a ninth synthesis region in a 3' to 5' direction,

[0206] wherein the seventh junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the ninth synthetic region, and the seventh junction domain and the eighth junction domain are substantially complementary to each other and form a double-stranded region, and

[0207] The seventh blocking region and the eighth blocking region together form a fourth blocking domain, and the fourth blocking domain blocks the strand displacement activity of the polymerase.

[0208] 17) The nucleic acid template of claim 16, wherein the seventh blocking domain and the eighth blocking domain are covalently linked to each other.

[0209] 18) The nucleic acid template as described in paragraph 16 or 17, wherein the seventh blocking region or the eighth blocking region comprises a cross-linking segment, and the cross-linking segment forms the fourth blocking domain with the other blocking region.

[0210] 19) The nucleic acid template as described in paragraph 18, wherein the cross-linking segment comprises 3-cyanovinylcarbazole.

[0211] 20) The nucleic acid template of any of paragraphs 1-19, wherein one or more regions use a 3-letter code.

[0212] 21) The nucleic acid template of any of paragraphs 1-20, wherein at least one of the first blocking domain, the second blocking domain, the third blocking domain or the fourth blocking domain comprises a double-stranded region.

[0213] 22) The nucleic acid template as described in any of paragraphs 1-21, wherein the nucleic acid template comprises a nucleic acid modification.

[0214] 23) The nucleic acid template of any of paragraphs 1-22, wherein at least one of the first blocking domain, the second blocking domain, the third blocking domain or the fourth blocking domain comprises a nucleic acid modification.

[0215] 24) The nucleic acid template of paragraph 22 or 23, wherein the nucleic acid is modified to a modified nucleobase.

[0216] 25) The nucleic acid template of any of paragraphs 1-24, wherein at least one of the first blocking domain, the second blocking domain, the third blocking domain or the fourth blocking domain comprises a multimer extension.

[0217] 26) The nucleic acid template as described in any of paragraphs 1-25, wherein the nucleic acid template is conjugated to a solid support.

[0218] 27) The nucleic acid template as described in any of paragraphs 1-26, wherein the nucleic acid template further comprises a primer that anneals or hybridizes to one of the guide strands.

[0219] 28) The nucleic acid template as described in any of paragraphs 1-27, wherein the nucleic acid template further comprises a primer that anneals or hybridizes to the first guide strand.

[0220] 29) A method for forming a nucleic acid template, the method comprising annealing or hybridizing a first guide strand and a second guide strand, wherein:

[0221] (a) the first guide strand comprises, in a 3' to 5' direction, a first synthesis region, a second synthesis region, a first junction domain and a first blocking region; and

[0222] (b) the second guide strand comprises, in 3' to 5' direction, a second blocking region, a second junction domain, and a third synthesis region;

[0223] wherein the first junction domain comprises a nucleotide sequence substantially identical to a 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, and

[0224] The first blocking region and the second blocking region together form a blocking domain, and the blocking domain blocks the strand displacement activity of the polymerase.

[0225] 30) The method of paragraph 29, wherein the first blocking region or the second blocking region comprises a cross-linking segment, the cross-linking segment forms the blocking domain with the other region, and the method further comprises forming a cross-link.

[0226] 31) A reaction mixture comprising the nucleic acid template described in any of paragraphs 1-28.

[0227] 32) The reaction mixture as described in paragraph 31, further comprising nucleoside triphosphates or deoxynucleoside triphosphates.

[0228] 33) The reaction mixture of paragraph 32, wherein the reaction mixture does not contain one of the following: adenosine, thymidine / uridine, cytosine or guanosine triphosphate or deoxynucleotide triphosphate.

[0229] 34) The reaction mixture as described in any of paragraphs 31-33, further comprising a DNA polymerase or an RNA polymerase.

[0230] 35) The reaction mixture as described in any of paragraphs 31-35, further comprising a buffer or salt for nucleic acid synthesis.

[0231] 36) A reaction mixture for synthesizing a guide strand by chemically or enzymatically adding a junction domain to a nucleic acid.

[0232] 37) A reaction mixture for synthesizing a guide strand by chemically or enzymatically adding a blocking region to a nucleic acid strand.

[0233] 38) A kit comprising the nucleic acid template described in any of paragraphs 1-28.

[0234] 39) A kit comprising components or a reaction mixture for creating a nucleic acid template as described in any of paragraphs 1-28 from a nucleic acid sequence (partial guide strand) by enzymatically or chemically adding a joining region and / or a blocking region to an existing nucleic acid strand.

[0235] 40) The kit of claim 38 or 39, further comprising nucleoside triphosphates or deoxynucleoside triphosphates.

[0236] 41) The kit as described in any of paragraphs 38-40, further comprising a DNA polymerase or an RNA polymerase.

[0237] 42) The kit as described in any of paragraphs 38-41, further comprising a buffer or salt for nucleic acid synthesis.

[0238] 43) A method for synthesizing a nucleic acid sequence, the method comprising:

[0239] (a) providing or obtaining the nucleic acid template described in any of paragraphs 1-28;

[0240] (b) annealing or hybridizing a primer to the nucleic acid template, if not already annealed; and

[0241] (c) The nucleotide sequence is extended from the 3'-terminus of the primer using a DNA polymerase having strand displacement activity.

[0242] 44) The method as described in paragraph 43 further comprises amplifying the nucleic acid sequence.

[0243] 45) A method for synthesizing a nucleic acid sequence, the method comprising:

[0244] (a) annealing or hybridizing a primer to a first guide strand, if not already annealed, wherein the first guide strand comprises, in a 3' to 5' direction, a first synthesis region, a second synthesis region, a junction domain, and a blocking region; and

[0245] (b) extending the nucleotide sequence from the 3′-end of the primer using a DNA polymerase having a strand displacement activity;

[0246] (c) adding a second guide strand to the synthesis reaction, wherein the second guide strand comprises, in a 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, wherein 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 a polymerase, the first junction domain of the second 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 second guide strand comprises a nucleotide sequence that is substantially identical to the nucleic acid sequence of the junction domain of the first guide strand;

[0247] (d) optionally, cross-linking the first blocking domain of the second guide strand and the blocking domain of the first guide strand;

[0248] (e) further extending the nucleotide sequence;

[0249] (f) optionally, adding additional new guide strands to the synthesis reaction and further extending the nucleotide sequence before adding each additional new guide strand, wherein each additional guide strand comprises, in a 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, wherein the first blocking region of the new guide strand and the second blocking region of a previous guide strand added to the synthesis reaction together form a blocking domain that blocks the strand displacement activity of a polymerase; the first junction domain of the new guide strand comprises a nucleotide sequence that is substantially complementary to the nucleic acid sequence of the second junction domain of the previous 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 substantially identical to the nucleic acid sequence of the second junction domain of the previous guide strand added to the synthesis reaction;

[0250] (g) Optionally, prior to further extending the nucleotide sequence, 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.

[0251] 46) The method of claim 45, further comprising:

[0252] (h) adding a terminal guide strand to the synthesis reaction, wherein the terminal guide strand comprises, in a 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, wherein the first blocking region of the terminal guide strand and the second blocking region of the previous guide strand added to the synthesis reaction together form a blocking domain that blocks the strand displacement activity of a polymerase; the first 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 previous 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 previous guide strand added to the synthesis reaction;

[0253] (i) optionally, cross-linking the first blocking domain of the terminal guide strand and the blocking domain of the previous guide strand added to the reaction; and

[0254] (j) further extending the nucleotide sequence.

[0255] 47) The method described in paragraph 45 or 46 further comprises amplifying the nucleic acid sequence.

[0256] 48) Use of the nucleic acid template described in any of paragraphs 1-28 for creating a combinatorial barcode to assign a unique identifier to a target population, wherein the target population is a surface location / partition, a matrix, a biomolecule, a molecular library or a biological material (such as a vesicle, a cell, a tissue, an organoid, a droplet, a liposome, a small molecule, a bead), and optionally the use includes splitting the target population before adding the guide strand and pooling the split target population after adding the guide strand.

[0257] 49) Use of the method described in any of paragraphs 29-30 for creating a combinatorial barcode to assign a unique identifier to a target population, wherein the target population is a surface location / partition, a matrix, a biomolecule, a molecular library or a biological material (such as a vesicle, a cell, a tissue, an organoid, a droplet, a liposome, a small molecule, a bead), and optionally the use includes splitting the target population before adding the guide strand and pooling the split target population after adding the guide strand.

[0258] 50) Use of the reaction mixture described in any of paragraphs 31-42 for creating a combinatorial barcode to assign a unique identifier to a target population, wherein the target population is a surface location / partition, a matrix, a biomolecule, a molecular library or a biological material (such as a vesicle, a cell, a tissue, an organoid, a droplet, a liposome, a small molecule, a bead), and optionally the use includes splitting the target population before adding the guide strand and pooling the split target population after adding the guide strand.

[0259] 51) Use of the method described in any of paragraphs 42-47 for creating a combinatorial barcode to assign a unique identifier to a target population, wherein the target population is a surface location / partition, a matrix, a biomolecule, a molecular library or a biological material (such as a vesicle, a cell, a tissue, an organoid, a droplet, a liposome, a small molecule, a bead), and optionally the use includes splitting the target population before adding the guide strand and pooling the split target population after adding the guide strand.

[0260] 52) Use of the nucleic acid template described in any of paragraphs 1-28 for creating a combinatorial barcode to assign a spatial location identifier to a target, wherein the target is a surface, a biomolecule or a biomaterial (including but not limited to biomolecules, cells, tissues, organs, organoids, vesicles, liposomes and droplets).

[0261] 53) Use of the method described in any of paragraphs 29-30 for creating a combinatorial barcode to assign a spatial location identifier to a target, wherein the target is a surface, a biomolecule or a biomaterial (including but not limited to biomolecules, cells, tissues, organs, organoids, vesicles, liposomes and droplets).

[0262] 54) Use of the reaction mixture described in any of paragraphs 31-37 for creating a combinatorial barcode to assign a spatial location identifier to a target, wherein the target is a surface, a biomolecule or a biomaterial (including but not limited to biomolecules, cells, tissues, organs, organoids, vesicles, liposomes and droplets).

[0263] 55) Use of the kit described in any of paragraphs 38-42 for creating a combinatorial barcode to assign a spatial location identifier to a target, wherein the target is a surface, a biomolecule or a biomaterial (including but not limited to biomolecules, cells, tissues, organs, organoids, vesicles, liposomes and droplets).

[0264] 56) Use of the method described in any of paragraphs 43-47 for creating a combinatorial barcode to assign a spatial location identifier to a target, wherein the target is a surface, a biomolecule or a biomaterial (including but not limited to biomolecules, cells, tissues, organs, organoids, vesicles, liposomes and droplets).

[0265] It should be understood that the present disclosure is not limited to the specific methodology, protocols and reagents provided herein, and may vary. The terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the claims. The present invention is further illustrated by the following examples, which should not be construed as further limiting.

[0266] Example

[0267] Example 1: Method for synthesizing strands across nucleic acid junctions

[0268] The ability to synthesize arbitrary nucleic acid sequences, particularly DNA, has revolutionized the way scientists can study and engineer biology. Whole genomes can now be sequenced efficiently (Schwarze et al., 2018), but technologies for synthesizing genomes have lagged behind. Synthetic single-stranded sequences ("oligos") are typically synthesized chemically via cyclic coupling steps (Caruthers et al., 1987). However, oligos longer than 200 bases are typically not synthesized chemically due to limitations in chemical coupling efficiency. As a result, synthetic methods have been developed to assemble larger single-stranded and double-stranded sequence fragments, including enzymatic assembly using sequential or simultaneous combinations of multiple enzyme activities (e.g., restriction enzyme digestion and ligation, or isothermal Gibson assembly that combines 5' exonuclease, DNA polymerase 3' extension activity, and DNA ligase activity (Gibson, 2011; Gibson et al., 2009)), non-enzymatic two-primer assembly (Liang et al., 2017), and chemical assembly (e.g., click DNA assembly (Kukwikila et al., 2017)). While these methods can effectively produce longer single-stranded and double-stranded DNA sequences, yeast vectors must be used to further assemble hundreds of thousands of bases from shorter fragments in vivo (Hutchison et al., 2016). A simple and robust new synthesis method capable of generating long DNA sequences may have important enabling applications in molecular biology, genome engineering, nanotechnology, and polymer-based data storage. This article provides a method for synthesizing nucleic acid chains across different nucleic acid backbones that are hybridized together using a strand displacement polymerase. This method of synthesizing chains across nucleic acid junctions can effectively separate the assembly step of template oligos from the synthesis step. In this way, template oligos can be robustly annealed sequentially, hybridized in a predictable manner close to the thermodynamic optimum, and then polymerized to create new transcripts. This method has the potential to be extended to very long arbitrary sequences. In addition, the entire assembly and synthesis process can be performed isothermally (including at room temperature), which makes a general workflow for synthesizing long arbitrary sequences under relatively mild conditions possible.

[0269] The basic strategy for cross-linking junction synthesis is outlined in Figure 1A-1B Figure 2. Two template oligos that have hybridized together to form a junction are shown, along with a primer that binds in front of the junction on the first template strand (left). A strand-displacing polymerase is used to replicate the x domain until it reaches the terminator (shown in black). Afterwards, the old and new x domains compete in a random walk branch migration process (Lee et al., 1970). Eventually, the new x domain is able to bind to the exposed x* domain on the second template strand (right), successfully crossing the junction. Polymerization can then continue on the second template strand, replicating along the new backbone.

[0270] like Figure 2 As depicted, by hybridizing multiple template oligos together, cross-link-junction synthesis reactions can be cascaded to form longer sequences. Each junction between template strands displays the same domain motif, so before reaching the terminator (b, c, d, e), the chain domain replicated on the 3' end of the growing strand can cross the junction and bind to the exposed complementary sequence (b*, c*, d*, e*) on the next template strand. Arbitrary sequences can be added in the template regions between the motif domains (shown in gray), enabling the assembly of longer sequences.

[0271] This method can connect any specified sequence and only requires Figure 1A-1B Basic domain constraints depicted.

[0272] This approach is also applicable to fixing the template concatemer length to synthesize a specific number of repeats by using programmable unique hybridization domains, which has been a long-standing challenge. Figure 3 ), by exploiting the specificity of DNA self-assembly, unique binding sequences promote assembly of specified lengths.

[0273] A strategy to assemble cross-link junctions with light would exploit photoreactive interstrand cross-link bases that could serve the dual purpose of linking the two cross-linked strands together and acting as terminators for polymerase synthesis ( Figure 4A-4C ). Cross-link junctions can be assembled iteratively through hybridization, cross-linking, and washing cycles. Cross-link junctions can also be assembled and spatially addressed on surfaces, and subsequent synthetic steps can still be performed on the surface.

[0274] Assembly of orthogonal crosslink-junction array libraries on surfaces enables multiplexed synthesis of orthogonal and arbitrary-length DNA of specified sequence.

[0275] In current practice, 3-cyanovinylcarbazole (CNVK) (Vieregg et al., 2013) base modification and a UV light source are used to crosslink DNA junctions together. The design of the strand can utilize: a set of two or more hybridization domains (1 and 1*, 2 and 2* in the current implementation), an internal barcode sequence (b1, b2, etc.) that will be used as the growing sequence strand, and the CNVK base crosslinker ( Figure 4A , cyan circle).

[0276] Experimental verification

[0277] Cascaded cross-link-junction synthesis and stepwise assembly of template concatemers on surfaces using photoreactive cross-linking chemistry (see above) followed by cross-link-junction synthesis were demonstrated experimentally.

[0278] like Figure 5-Figure 12 As depicted, the synthetic cascade from 1-junction synthesis to 8-junction synthesis was designed using a random 3-letter (A, T, and C) 7nt 'flap' primer (sequences b, d, f, h, j, l, and n), three consecutive Gs as terminators (dGTP was excluded from the dNTP mix), a random 4-letter 9nt secondary hybridization domain (r0, r1, ..., r7), and a 5nt 3-letter (A, T, and C) insertion domain (c, e, g, i, k, m, o, q). Template concatemers were formed by mixing the strands together at a concentration of 1 μM in 1× PBS and annealing from 80° C. to 20° C. for one hour (1 degree decrease per minute). The concatemers were then introduced into a synthesis reaction at a final concentration of 50 nM in 0.95× PBS with 10 mM MgSO4, with 40 nM primers, 1 μM dGTP cleanup hairpin, 600 μM dATP / dCTP / dTTP mix, and 800 units / mL Bst large fragment polymerase. The reactions were incubated at room temperature for 15 minutes, then primers were added and incubated 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 replication of the For primer sequence. The reaction was then heat inactivated at 80°C for 20 minutes and then diluted 1000X (final) into a Taq 1× PCR with 200 nM For and rev primers. A qPCR machine was used to set the time for real-time tracking amplification using Sybr Green I ( 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 ).

[0279] Light-directed concatemer formation (see above) was also experimentally demonstrated. The reactions performed are depicted in Figure 14-16 The results are shown in Fig.17 After the probe sequence is bound to the glass slide, the template strands are hybridized at 1 μM concentration in 1× PBS for 5 minutes, the bound strands are cross-linked with UV light for ~3 seconds, and then several washing steps are performed before the next hybridization step, and unbound strands are removed with 1× PBS. All these steps are performed at room temperature. After template formation, cross-link-junction synthesis is performed with 40nM primers at room temperature, and the results are heat-inactivated at 80°C for 20 minutes before being diluted into PCR solution and amplified to form double-stranded products. PAGE denaturing gel electrophoresis is run to show the products of three different template sizes ( Fig.17 ).

[0280] discuss

[0281] The new ability to program synthesis across nucleic acid junctions enables a new paradigm for synthesis that is no longer limited to replication along a single backbone. The formation of template concatemers (assembly step) can be carried out under favorable conditions (e.g., by annealing), which helps to improve the specificity of the hybridization interactions formed. The primer chain can be included in the formation step or in subsequent hybridization, and the cross-linking-junction synthesis is carried out with a chain displacement polymerase to produce a sequence record of the template concatemer. As shown above, any programmable sequence can be assembled in this way to produce a long single-stranded sequence. Single-stranded sequences can be used directly or amplified (e.g., using PCR) to form single-stranded or double-stranded products. This ability opens up many exciting applications, from the generation of sequence combinatorial libraries to in situ synthesis.

[0282] References

[0283] 1.Caruthers, MH, Barone, AD, Beaucage, SL, Dodds, DR, Fisher, EF, McBride, LJ, Matteucci, M., Stabinsky, Z., and Tang, J.-Y. (1987).

[15] Chemicalsynthesis of deoxyoligonucleotides by the phosphoramidite method. In Methods in Enzymology, (Academic Press), pp.287-313.

[0284] 2.Gibson,D.G.(2011).Chapter fifteen-Enzymatic Assembly of OverlappingDNA Fragments.In Methods in Enzymology,C.Voigt,ed.(Academic Press),pp.349-361.

[0285] 3.Gibson,D.G.,Young,L.,Chuang,R.-Y.,Venter,J.C.,Hutchison,C.A.,3rd,and Smith,H.O.(2009)Enzymatic assembly of DNA molecules up to several hundredkilobases.Nat.Methods 6,343-345.

[0286] 4.Hutchison,C.A.,3rd,Chuang,R.-Y.,Noskov,V.N.,Assad-Garcia,N.,Deerinck,T.J.,Ellisman,M.H.,Gill,J.,Kannan,K.,Karas,B.J.,Ma,L.,et al.(2016).Design and synthesis of a minimal bacterial genome.Science 351,aad6253.

[0287] 5.Kukwikila,M.,Gale,N.,El-Sagheer,A.H.,Brown,T.,and Tavassoli,A.(2017).Assembly of a biocompatible triazole-linked gene by one-pot click-DNAligation.Nat.Chem.9,1089-1098.

[0288] 6. Lee, C.S., Davis, R.W., and Davidson, N. (1970). A physical study by electron microscopy of the terminally repetitious, circularly permuted DNA from the coliphage particles of Escherichia coli 15. J. Mol. Biol. 48, 1 - 22.

[0289] 7. Liang, J., Liu, Z., Low, X.Z., Ang, E.L., and Zhao, H. (2017). Twin - primer non - enzymatic DNA assembly: an efficient and accurate multi - part DNA assembly method. Nucleic Acids Res. 45, e94.

[0290] 8. Schwarze, K., Buchanan, J., Taylor, J.C., and Wordsworth, S. (2018). Are whole - exome and whole - genome sequencing approaches cost - effective? A systematic review of the literature. Genet. Med. 20, 1122 - 1130.

[0291] 9. Vieregg, J.R., Nelson, H.M., Stoltz, B.M., and Pierce, N.A. (2013). Selective nucleic acid capture with shielded covalent probes. J. Am. Chem. Soc. 13, 9691 - 9699.

[0292] Example 2: Nucleic Acid Synthesis across Multiple Junctions

[0293] In addition to the above methods, in Fig.19AFollowing the process outlined in , synthesis across the 8-junction was performed using fluorophore-labeled primers. The template strands (also called guide strands) were annealed (1 μM per 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 minutes and 20 minutes). The template sequence was polymerized using a GC pair and the reaction was pre-incubated with a hairpin for 5 minutes. The hairpin can sequester any extraneous dGTP in the mixture of dATP, dTTP, and dCTP before the fluorophore-labeled primers can be added (final 40 nM, 100 nM per template strand). The reaction was then incubated at the temperature for 2 minutes before the addition of dGTP, about 10 minutes or 20 minutes. This step was followed by heat inactivation at 80°C for 20 minutes. The reactions were run on a 15% TBE-urea PAGE denaturing gel to determine the effect on synthesis efficiency ( Fig.19B ). Two time points (10 min and 20 min) allow monitoring of the course of the reaction over time. More bands appearing on the gel indicate that synthesis across a higher number of junctions has occurred. Fig.19A ( Fig.19B ), it was found that lanes 1 and 7 had the highest nucleic acid synthesis efficiency.

[0294] Several salt conditions were also tested with the 5-junction cross-linking-synthesis reaction. In this embodiment of the method described herein, a longer binding domain between the template strands was used. The primers and template strands were annealed in 1×PBS at increasing concentrations from 80°C to 20°C during a 1-hour incubation. A total of 48 reaction conditions (8 salt conditions × 3 polymerase concentrations × 2 time points) were tested, incubated at 37°C, then incubated with dGTP and heat inactivated at 80°C for 20 minutes. Efficiency was assessed based on Cq values ​​from all reaction product amplifications ( Figure 20A-20B ).

[0295] In another example, cross-linking-junction synthesis is performed by sequentially adding template strands one at a time rather than preassembling the template strands as described above (see Fig.21A Schematic diagram in ). In continuous nucleic acid synthesis, the complete reaction conditions for the 5-junction cross-linking-synthesis reaction without any oligos are initially pre-incubated before the introduction of primers. After each 2-minute incubation at 37°C, a new template strand is added in the order of synthesis. To ensure that nucleic acid synthesis is performed while each template strand is added continuously, a separate strand (the terminal barcode strand) is added. The terminal barcode strand used in this example comprises (i) one or more binding domains that are substantially identical to the third template strand; and (ii) a different barcode. The barcode is not complementary to the template strand. The terminal barcode strand is added at the end of the synthesis process. Finally, the reaction mixture is incubated with dGTP and heat inactivated. The complete product is amplified by PCR and subjected to Sanger sequencing ( Fig.21B). The sequencing results show the nucleic acid sequence without interference from the barcode from the last terminal barcode strand ( Fig.21B ,bottom).

[0296] All patents and other public documents indicated are expressly incorporated herein by reference for the purpose of describing or disclosing, for example, the methodology that can be used in combination with the present disclosure described in these public documents. These public documents are provided only because they are disclosed earlier than the filing date of the present application. In this respect, it should not be regarded as an admission that the inventor has no right to advance the disclosed content by means of previous inventions or for any other reason. All statements about the dates of these documents or the representation of the contents of these documents are based on the information available to the applicant and do not constitute any admission about the correctness of the dates of these documents or the contents of these documents.

Claims

1. A nucleic acid template, comprising: (a) a first guide strand comprising, in a 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 a 3' to 5' direction, a second blocking region, a second junction domain, and a third synthesis region; in, The first junction domain comprises a nucleotide sequence substantially identical to a 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, and The first blocking region and the second blocking region together form a first blocking domain, and the first blocking domain blocks the strand displacement activity of the polymerase.

2. The nucleic acid template according to claim 1, wherein The first blocking region and the second blocking region are covalently linked to each other.

3. The nucleic acid template according to claim 1 or 2, wherein The first blocking region or the second blocking region comprises a cross-linking segment, and the cross-linking segment forms the first blocking domain with the other blocking region.

4. The nucleic acid template according to claim 3, wherein The crosslinking segment comprises 3-cyanovinylcarbazole.

5. The nucleic acid template according to claim 1, wherein The second guide strand further comprises a fourth synthesis zone located at the 5' end of the third synthesis zone.

6. The nucleic acid template according to claim 5, wherein the second guide strand further comprises a third junction domain and a third blocking region at its 5' end in a 3' to 5' direction, and the nucleic acid template further comprises a third guide strand comprising a fourth blocking region, a fourth junction domain, and a fifth synthesis region in a 3' to 5' direction, wherein the third junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the fifth synthetic region, wherein the third junction domain and the fourth junction domain are substantially complementary to each other and form a double-stranded region, and The third blocking region and the fourth blocking region together form a second blocking domain, and the second blocking domain blocks the strand displacement activity of the polymerase.

7. The nucleic acid template according to claim 6, wherein The third blocking region and the fourth blocking region are covalently linked to each other.

8. The nucleic acid template according to claim 6, wherein The third blocking zone or the fourth blocking zone comprises a cross-linking segment, and the cross-linking segment forms the second blocking domain with the other blocking zone.

9. The nucleic acid template according to claim 8, wherein The crosslinking segment comprises 3-cyanovinylcarbazole.

10. The nucleic acid template according to claim 6, wherein The third guide strand further comprises a sixth synthesis zone located at the 5' end of the fifth synthesis zone.

11. The nucleic acid template according to claim 6, wherein the third guide strand further comprises a fifth junction domain and a fifth blocking region at its 5' end in a 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 synthesis region in a 3' to 5' direction, wherein the fifth junction domain comprises a nucleotide sequence substantially identical to a nucleotide sequence of the seventh synthetic region, wherein the fifth junction domain and the sixth junction domain are substantially complementary to each other and form a double-stranded region, and The fifth blocking region and the sixth blocking region together form a third blocking domain, and the third blocking domain blocks the strand displacement activity of the polymerase.

12. The nucleic acid template of claim 11, wherein The fifth blocking region and the sixth blocking region are covalently linked to each other.

13. The nucleic acid template according to claim 11, wherein The fifth blocking zone or the sixth blocking zone comprises a cross-linking segment, and the cross-linking segment forms the third blocking domain with the other blocking zone.

14. The nucleic acid template according to claim 13, wherein The crosslinking segment comprises 3-cyanovinylcarbazole.

15. The nucleic acid template according to claim 11, wherein The fourth guide strand further comprises an eighth synthesis zone located at the 5' end of the seventh synthesis zone.

16. The nucleic acid template of claim 11, wherein: the fourth guide strand further comprises a seventh junction domain and a seventh blocking region at its 5' end in a 3' to 5' direction, and the nucleic acid template further comprises a fifth guide strand comprising an eighth blocking region, an eighth junction domain, and a ninth synthesis region in a 3' to 5' direction, wherein the seventh junction domain comprises a nucleotide sequence substantially identical to the nucleotide sequence of the ninth synthetic region, and the seventh junction domain and the eighth junction domain are substantially complementary to each other and form a double-stranded region, and The seventh blocking region and the eighth blocking region together form a fourth blocking domain, and the fourth blocking domain blocks the strand displacement activity of the polymerase.

17. The nucleic acid template of claim 16, wherein: The seventh blocking region and the eighth blocking region are covalently linked to each other.

18. The nucleic acid template of claim 16, wherein The seventh blocking zone or the eighth blocking zone comprises a cross-linking segment, and the cross-linking segment forms the fourth blocking domain with the other blocking zone.

19. The nucleic acid template of claim 18, wherein The crosslinking segment comprises 3-cyanovinylcarbazole.

20. The nucleic acid template of claim 1, wherein One or more regions use a 3-letter code, wherein the 3-letter code comprises three of adenine, thymine / uracil, guanine and cytosine, or modified forms thereof.

21. The nucleic acid template of claim 1, wherein At least one of the first blocking domain, the second blocking domain, the third blocking domain or the fourth blocking domain comprises a double-stranded region.

22. The nucleic acid template of claim 1, wherein The nucleic acid template comprises a nucleic acid modification.

23. The nucleic acid template of claim 1, wherein: At least one of the first blocking domain, the second blocking domain, the third blocking domain, or the fourth blocking domain comprises a nucleic acid modification.

24. The nucleic acid template of claim 22, wherein The nucleic acid modifications are modified nucleobases.

25. The nucleic acid template of claim 1, wherein: At least one of the first blocking domain, the second blocking domain, the third blocking domain or the fourth blocking domain comprises a multimer stretch.

26. The nucleic acid template of claim 1, wherein The nucleic acid template comprises a fluorophore at the 3' end and / or the 5' end.

27. The nucleic acid template of claim 1, wherein: The nucleic acid template is conjugated to a solid support.

28. The nucleic acid template of claim 1, wherein The nucleic acid template further comprises a primer that anneals or hybridizes to one of the guide strands.

29. The nucleic acid template of claim 1, wherein: The nucleic acid template further comprises a primer that anneals or hybridizes to the first guide strand.

30. A method for forming a nucleic acid template, the method comprising annealing or hybridizing a first guide strand and a second guide strand, wherein: (a) the first guide strand comprises, in a 3' to 5' direction, a first synthesis region, a second synthesis region, a first junction domain and a first blocking region; and (b) the second guide strand comprises, in a 3' to 5' direction, a second blocking region, a second junction domain, and a third synthesis region; wherein the first junction domain comprises a nucleotide sequence substantially identical to a 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, and The first blocking region and the second blocking region together form a blocking domain, and the blocking domain blocks the strand displacement activity of the polymerase.

31. The method of claim 30, wherein: The first blocking region or the second blocking region comprises a cross-linking segment, the cross-linking segment forms the blocking domain with the other blocking region, and the method further comprises forming a cross-link.

32. A reaction mixture comprising the nucleic acid template of any one of claims 1-29.

33. The reaction mixture of claim 32, further comprising nucleoside triphosphates or deoxynucleoside triphosphates.

34. The reaction mixture of claim 33, wherein The reaction mixture does not contain one of the following: adenosine, thymidine / uridine, cytidine or guanosine triphosphates or deoxynucleoside triphosphates.

35. The reaction mixture of claim 32, further comprising a DNA polymerase or an RNA polymerase.

36. The reaction mixture of claim 32, further comprising a buffer or salt for nucleic acid synthesis.

37. A kit comprising the nucleic acid template of any one of claims 1-29.

38. The kit of claim 37, further comprising nucleoside triphosphates or deoxynucleoside triphosphates.

39. The kit of claim 37 or 38, further comprising a DNA polymerase or an RNA polymerase.

40. The kit of claim 37 or 38, further comprising a buffer or salt for nucleic acid synthesis.

41. A method for synthesizing a nucleic acid sequence, the method comprising: (a) providing or obtaining the nucleic acid template according to any one of claims 1 to 29; (b) annealing or hybridizing a primer to the nucleic acid template, if not already annealed; as well as (c) Extending a nucleotide sequence from the 3'-terminus of the primer using a DNA polymerase having a strand displacement activity.

42. The method of claim 41, further comprising amplifying the nucleic acid sequence.

43. The method of claim 41 or 42, further comprising sequencing the synthesized nucleic acid sequence.

44. A method for synthesizing a nucleic acid sequence, the method comprising: (a) annealing or hybridizing a primer to a first guide strand, if not already annealed, wherein the first guide strand comprises, in a 3' to 5' direction, a first synthesis region, a second synthesis region, a junction domain, and a blocking region; and (b) extending a nucleotide sequence from the 3'-end of the primer using a DNA polymerase having a strand displacement activity; (c) adding a second guide strand to the synthesis reaction, wherein the second guide strand comprises, in a 3' to 5' direction, 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, wherein the first blocking region of the second guide strand and the blocking region of the first guide strand together form a blocking domain that blocks the strand displacement activity of a polymerase, the first junction domain of the second 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 second guide strand comprises a nucleotide sequence that is substantially identical to the nucleic acid sequence of the junction domain of the first guide strand; and (d) further extending the nucleotide sequence.

45. The method of claim 44, further comprising: (e) cross-linking the first blocking region of the second guide strand and the blocking region of the first guide strand before further extending the nucleotide sequence.

46. ​​The method of claim 44, further comprising: (f) adding additional new guide strands to the synthesis reaction and further extending the nucleotide sequence before adding each additional new guide strand, wherein each additional guide strand comprises, in a 3' to 5' direction, 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, wherein 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 a polymerase; the first junction domain of the new 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 new 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.

47. The method of claim 46, further comprising: (g) prior to further extending the nucleotide sequence, cross-linking the first blocking region of the new guide strand and the second blocking region of the previous guide strand added to the synthesis reaction.

48. The method of claim 46, further comprising: (h) adding a terminal guide strand to a synthesis reaction, wherein the terminal guide strand comprises, in a 3' to 5' direction, 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, wherein the first blocking region of the terminal guide strand forms a blocking domain together with the second blocking region of the previous guide strand added to the synthesis reaction, and the blocking domain blocks the strand displacement activity of a polymerase; the first 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 previous 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 previous guide strand added to the synthesis reaction; and (i) further extending the nucleotide sequence.

49. The method of claim 48, further comprising: (j) prior to further extending the nucleotide sequence, cross-linking the first blocking region of the terminal guide strand and the second blocking region of the previous guide strand added to the reaction.

50. The method of any one of claims 44-49, further comprising amplifying the nucleic acid sequence.

51. The method of any one of claims 44-49, further comprising sequencing the synthesized nucleic acid sequence.

52. Use of the nucleic acid template of any one of claims 1-29 for creating a combinatorial barcode to assign a unique identifier to a target population, wherein: The target population is a surface location / partition, a matrix, a biomolecule, a molecular library or a biological material.

53. The use of claim 52, further comprising splitting the target population before adding the guide strand and pooling the split target population after adding the guide strand.

54. The use according to claim 52 or 53, wherein The biological material is a cell, a tissue, an organoid, a liposome, a small molecule or a bead.

55. The use according to claim 52 or 53, wherein The biological material is a vesicle.

56. The use according to claim 52 or 53, wherein The biological material is in the form of droplets.

57. Use of the method of any one of claims 30-31 for creating a combined barcode to assign a unique identifier to a target group, wherein: The target population is a surface location / partition, a matrix, a biomolecule, a molecular library or a biological material.

58. The use of claim 57, further comprising splitting the target population before adding the guide strand and pooling the split target population after adding the guide strand.

59. The use according to claim 57 or 58, wherein The biological material is a cell, a tissue, an organoid, a liposome, a small molecule or a bead.

60. The use according to claim 57 or 58, wherein The biological material is a vesicle.

61. The use according to claim 57 or 58, wherein The biological material is in the form of droplets.

62. Use of the reaction mixture of any one of claims 32 to 36 for creating a combined barcode to assign a unique identity to a target population, wherein The target population is a surface location / partition, a matrix, a biomolecule, a molecular library or a biological material.

63. The use of claim 62, further comprising splitting the target population before adding the guide strand and pooling the split target population after adding the guide strand.

64. The use according to claim 62 or 63, wherein The biological material is a cell, a tissue, an organoid, a liposome, a small molecule or a bead.

65. The use according to claim 62 or 63, wherein The biological material is a vesicle.

66. The use according to claim 62 or 63, wherein The biological material is in the form of droplets.

67. Use of the kit of any one of claims 37-40 for creating a combined barcode to assign a unique identifier to a target population, wherein: The target population is a surface location / partition, a matrix, a biomolecule, a molecular library or a biological material.

68. The use of claim 67, further comprising splitting the target population before adding the guide strand and pooling the split target population after adding the guide strand.

69. The use according to claim 67 or 68, wherein The biological material is a cell, a tissue, an organoid, a liposome, a small molecule or a bead.

70. The use according to claim 67 or 68, wherein The biological material is a vesicle.

71. The use of claim 67 or 68, wherein The biological material is in the form of droplets.

72. Use of the method of any one of claims 41 to 51 for creating a combined barcode to assign a unique identifier to a target group, wherein: The target population is a surface location / partition, a matrix, a biomolecule, a molecular library or a biological material.

73. The use of claim 72, further comprising splitting the target population before adding the guide strand and pooling the split target population after adding the guide strand.

74. The use of claim 72 or 73, wherein The biological material is a cell, a tissue, an organoid, a liposome, a small molecule or a bead.

75. The use of claim 72 or 73, wherein The biological material is a vesicle.

76. The use of claim 72 or 73, wherein The biological material is in the form of droplets.

77. Use of the nucleic acid template of any one of claims 1-29 for creating a combinatorial barcode to assign a spatial location identifier to a target, wherein: The target is a surface, a biomolecule or a biomaterial.

78. The use according to claim 77, wherein The biological material is a biological molecule, a cell, a tissue, an organ, an organoid or a liposome.

79. The use according to claim 77, wherein The biological material is a vesicle.

80. The use according to claim 77, wherein The biological material is in the form of droplets.

81. Use of the method of any one of claims 30-31 for creating a combined barcode for assigning a spatial location identifier to an object, wherein: The target is a surface, a biomolecule or a biomaterial.

82. The use of claim 81, wherein The biological material is a biological molecule, a cell, a tissue, an organ, an organoid or a liposome.

83. The use of claim 81, wherein The biological material is a vesicle.

84. The use of claim 81, wherein The biological material is in the form of droplets.

85. Use of the reaction mixture of any one of claims 32-36 for creating a combined barcode to assign a spatial location identifier to a target, wherein The target is a surface, a biomolecule or a biomaterial.

86. The use of claim 85, wherein The biological material is a biological molecule, a cell, a tissue, an organ, an organoid or a liposome.

87. The use of claim 85, wherein The biological material is a vesicle.

88. The use of claim 85, wherein The biological material is in the form of droplets.

89. Use of the kit of any one of claims 37-40 for creating a combined barcode to assign a spatial location identifier to a target, wherein: The target is a surface, a biomolecule or a biomaterial.

90. The use of claim 89, wherein The biological material is a biological molecule, a cell, a tissue, an organ, an organoid or a liposome.

91. The use of claim 89, wherein The biological material is a vesicle.

92. The use of claim 89, wherein The biological material is in the form of droplets.

93. Use of the method of any one of claims 41-51 for creating a combined barcode to assign a spatial location identifier to an object, wherein: The target is a surface, a biomolecule or a biomaterial.

94. The use of claim 93, wherein The biological material is a biological molecule, a cell, a tissue, an organ, an organoid or a liposome.

95. The use of claim 93, wherein The biological material is a vesicle.

96. The use of claim 93, wherein The biological material is in the form of droplets.

97. Use of the method of any one of claims 44-51 for determining the proximity of biomolecules and creating a record indicative of distance information between tagged biomolecules.

98. Use of the nucleic acid template of any one of claims 1-29 for generating a nucleic acid sequence library.

99. Use of the method of any one of claims 30-31 for generating a nucleic acid sequence library.

100. Use of the reaction mixture of any one of claims 32 to 36 for generating a nucleic acid sequence library.

101. Use of the kit of any one of claims 37-40 for generating a nucleic acid sequence library.

102. Use of the method of any one of claims 41-51 for generating a nucleic acid sequence library.

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