Assembly of long nucleic acids by ligation using indexed splint oligos

Abstract

The present disclosure relates to a method for assembling long nucleic acids by enzymatic ligation of oligonucleotide molecules hybridized to indexed splint oligonucleotide molecules. Oligonucleotide structures comprising indexed splint oligonucleotides useful in carrying out the disclosed methods are also disclosed. Kits of the invention include nucleic acid libraries comprising the disclosed oligonucleotide structures. The oligonucleotides are ligated in a highly specific order to construct optimal desired ligation products.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 228,448, filed August 2, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the field of molecular biology. In particular, the present disclosure relates to methods for generating long nucleic acids, particularly long DNA libraries. [Background technology]

[0003] DNA libraries are used in a wide range of applications in biological research and drug discovery. Currently, DNA libraries are most often generated by chemical synthesis of oligos, either in a massively parallel manner on solid surfaces or by pooling individually chemically synthesized oligonucleotides ("oligos"). One of the major limitations of these approaches is the length of the DNA molecules that can be made by single-stranded chemical synthesis. The longest commercially available oligo libraries currently have a maximum length of 350 nucleotides. However, many research applications, such as gene synthesis, protein engineering, genome engineering and mutation saturation scanning, would benefit from longer DNA libraries.

[0004] One approach to generate libraries of longer length involves enzymatic assembly of library members by the action of DNA ligase, which joins two or more chemically synthesized oligos into longer constructs. Methods known in the art rely on compatible single-stranded overhangs in specific double-stranded sequences to achieve specific annealing of sequences to join the sequences in a predefined order. See, for example, U.S. Pat. No. 6,495,318. The '318 patent also discloses two specific sequences with terminal single-stranded portions hybridized to a single splint oligo with a complementary region in each piece. Under ideal conditions, the oligo will hybridize properly to the splint and a ligase will join the two pieces to form a longer construct. Similar methods are currently being used by various research groups, but with limited success. The main weakness of this method is the hybridization specificity, which can result in libraries of poor quality.

[0005] An alternative method for enzymatic assembly of long oligos involves hybridization of library pieces to solid surface DNA arrays to spatially isolate the constructs prior to ligation, see, e.g., U.S. Patent No. 10,538,796. Summary of the Invention

[0006] In some aspects, the present invention addresses the need for methods that have high specificity for sequences of interest and that can be performed in solution. [Brief description of the drawings]

[0007] [Figure 1]Figure 1 depicts one type of oligonucleotide structure for assembling oligonucleotides into a multicomponent DNA construct. The oligonucleotide structure consists of two oligonucleotide pieces (1 is the upstream assembly component, assembly component 1, and 2 is the downstream assembly component, assembly component 2) hybridized to a single splint oligonucleotide (sprint) that has adjacent sequence regions complementary to each oligonucleotide piece. The arrow indicates the 3' end of the oligonucleotide; the flat end is the 5' end of the oligonucleotide. The intended ligation point is the junction of assembly components 1 and 2. [Diagram 2] FIG. 2 depicts another type of oligonucleotide structure for assembling oligonucleotides into a multicomponent DNA construct. The oligonucleotide structure consists of two oligonucleotide pieces (assembly component 1 and assembly component 2) hybridized to an indexed splint structure. The indexed splint structure has a pair of splint oligonucleotide members. Each member of the pair has an index sequence that hybridizes to the other index sequence of the other member to form a double-stranded index element (index), thus serving to bring the two assembly components in close proximity to each other. One splint oligonucleotide member has a splint sequence region (L-sprint) that is complementary to the assembly component 1 oligonucleotide. The other splint oligonucleotide member has a splint sequence region (R-sprint) that is complementary to the 3'-terminal portion of assembly component 1 and the 5'-terminal portion of assembly component 2 oligonucleotide. The arrowhead indicates the 3'-end of the oligonucleotide; the flat end is the 5'-end of the oligonucleotide. The intended ligation points are the junctions of assembly components. [Diagram 3]FIG. 3 depicts another type of oligonucleotide structure for assembling oligonucleotides into a multicomponent DNA construct. The oligonucleotide structure consists of two oligonucleotide pieces (assembly component 1 and assembly component 2) hybridized to an indexed splint structure, similar to the structure in FIG. 2. The indexed splint structure has a pair of splint oligonucleotide members. Each member of the pair has an index sequence that hybridizes to the other index sequence of the other member to form a double-stranded index element. In this structure, the splint oligonucleotide member has a splint sequence region (L-sprint) that is complementary to a sequence in the assembly component 1 oligonucleotide that is not proximal to the 3' end of the assembly component 1 oligonucleotide, resulting in a looped portion of assembly component 1. The other splint oligonucleotide member has a splint sequence region (R-sprint) that is complementary to the 3' end portion of assembly component 1 and the 5' end portion of the assembly component 2 oligonucleotide. The arrow indicates the 3' end of the oligonucleotide; the flat end is the 5' end of the oligonucleotide. The intended ligation points are the junctions of assembly components. [Figure 4]FIG. 4 depicts another type of oligonucleotide structure for assembling oligonucleotides into a multicomponent DNA construct. The oligonucleotide structure consists of two oligonucleotide pieces (assembly component 1 and assembly component 2) hybridized to a single indexed splint molecule. In this structure, the assembly component 1 oligonucleotide has a 5' index sequence that is 5' to the assembly sequence. The index sequence hybridizes to the index sequence of the indexed splint molecule to form a double-stranded index element. The splint oligonucleotide member has a sequence region (R-sprint) that is complementary to a sequence in the assembly component 1 oligonucleotide at the 3' end of the assembly component 1 oligonucleotide and to the 5' end portion of the assembly component 2 oligonucleotide, resulting in a loop portion of assembly component 1 (somewhat similar to FIG. 3). The arrow indicates the 3' end of the oligonucleotide; the flat end is the 5' end of the oligonucleotide. The intended ligation point is the junction of the assembly components. [Diagram 5]5A-5C depict another type of oligonucleotide structure for assembling oligonucleotides into a multicomponent DNA construct. The oligonucleotide structure includes an indexed splint molecule (depicted in FIG. 5A) that is hybridized to two oligonucleotide pieces (assembly component 1 and assembly component 2). The arrow indicates the 3' end of the oligonucleotide; the flat end is the 5' end of the oligonucleotide. The dotted line represents an optional spacer sequence. As shown in FIG. 5A, in this type of oligonucleotide structure, the indexed splint molecule includes a splint sequence (sprint) and two index sequences A and B (index sequence B and index sequence A). The two index sequences of the indexed splint molecule flank the splint sequence with intervening spacer sequences spacer 1 and spacer 2 (dotted lines). The splint sequence has a 5' element and a 3' element, as depicted in the enlargement of the splint. The 5' element of the splint sequence hybridizes to the 5' end of the assembly component 2 oligonucleotide. The 3' element of the splint sequence hybridizes to the 3' end of the assembly component 1 oligonucleotide. One index sequence of the indexed splint molecule hybridizes to the index sequence at the end of one assembly component oligonucleotide. The other index sequence of the indexed splint molecule hybridizes to the index sequence at the end of the other assembly component oligonucleotide. Figures 5A and 5B illustrate two exemplary orientations for indexed splint molecules binding to two assembly components in an oligonucleotide structure. Figure 5B depicts an orientation in which a double-stranded index element (index 2) is formed by hybridization of index sequence A (index sequence at the 5' end of the indexed splint molecule) to the index sequence at the 3' end of assembly component 2.The double-stranded index element (index 1) is formed by index sequence B (index sequence at the 3' end of the indexed splint molecule) hybridized to the index sequence at the 5' end of assembly component 1. The 5' element of the splint sequence hybridizes to the 5' end of the assembly component 2 oligonucleotide. The 3' element of the splint sequence hybridizes to the 3' end of the assembly component 1 oligonucleotide. The intended ligation point is the junction of the assembly components. Figure 5C depicts a second orientation in which the double-stranded index element (index 1) is formed by hybridization of index sequence A (index sequence at the 5' end of the indexed splint molecule) to the index sequence at the 5' end of assembly component 1 (segment C). The double-stranded index element (index 2) is formed by index sequence B (index sequence at the 3' end of the indexed splint molecule) hybridized to the index sequence at the 3' end of assembly component 1 (segment F). The 5' element of the splint sequence hybridizes to the 5' end of the assembly component 2 oligonucleotide. The 3' element of the splint sequence hybridizes to the 3' end of the assembly component 1 oligonucleotide. The intended ligation point is the junction of the assembly components. [Figure 6]Figure 6 illustrates a variation of Figure 2, in which the oligonucleotide structure includes at least one blocking oligonucleotide (e.g., Blocker 1) that can hybridize to a region of the assembly sequence that does not hybridize to the splint. Two blocking oligonucleotides (Blocker 1 and Blocker 2) are depicted in Figure 6. The arrow indicates the 3' end of the oligonucleotide; the flat end is the 5' end of the oligonucleotide. The intended ligation point is the junction of the assembly components. One or more blocking nucleotides can be utilized in the oligonucleotide structures of Figures 5B and 5C. [Figure 7] FIG. 7 depicts the two oligonucleotide structures to be ligated in the first ligation step of the four-part assembly process. (See also FIG. 8 and FIG. 9). The first ligation step assembles assembly components 1+2 (upper structure) and assembly components 3+4 (lower structure) in separate reactions with a mechanism similar to the two-part assembly. The arrows indicate the 3' ends of the oligonucleotides; the flat ends are the 5' ends of the oligonucleotides. The intended ligation points in each oligonucleotide structure are indicated. In addition to the first splint structure (sprint 1), the indexed splint of the upper oligonucleotide structure includes a second splint sequence (sprint 2) in the spacer section, as well as another index sequence (index 3) between the 5' index sequence (index 2) and the first splint structure (sprint 1). The indexed splint of the lower oligonucleotide structure comprises a complementary sequence of index 3 (index 3 complement) between a first splint structure (sprint 3) and a 5' index sequence (index 4). [Figure 8]FIG. 8 depicts the next step of the four-part assembly process. The ligation products of the assembly step depicted in FIG. 7 are illustrated in FIG. 8. The ligation produces two multi-component ligation products, assembled product (1-2) (depicted in (a)) and assembled product (3-4) (depicted in (c)). Also depicted are type IIS restriction enzyme cleavage sites (RE cut sites) located between the index sequences and the assembly components for assembled product (1-2) and assembled product (3-4). Each of the ligation products of the assembly step depicted in FIG. 7 is subjected to digestion with a type IIS restriction enzyme to cleave at the RE cut site (not shown in FIG. 7). In the ligation product in (a), cleavage removes index 2, leaving only the assembly component sequence at the 3' end in assembled product (1-2), as illustrated in (b). In the ligation product in (c), cleavage removes index 4, as shown in (d), leaving only the assembly component sequence at the 5' end in the assembled product (3-4). The resulting product is purified, for example, by SPRI purification. [Figure 9] Figure 9 depicts the second ligation step of the four-part assembly process. The purified products of the restriction digest (see (b) and (d) in Figure 8) are then hybridized together (see index 3) and ligated as depicted in the schematic to produce the assembled product (1-2-3-4). This product can be purified, for example by SPRI, subjected to additional ligation or amplified by PCR. For example, primer sequences may be present between index 1 and the assembly sequence of assembly component 1 (forward primer) and between index 5 and the assembly sequence of assembly component 4 (reverse primer). [Figure 10]Figure 10 illustrates a schematic of a variant embodiment of the disclosed method. Figure 10 illustrates an oligonucleotide structure in which the index sequences of two assembly components are hybridized to a single splint ("index splint") that hybridizes to the two index sequences to form a double-stranded Index 1 and Index 2. [Figure 11] Figure 11 illustrates a schematic of a variant embodiment of the disclosed method. Figure 11 depicts the ligation product of the oligonucleotide structures of Figure 10. Figure 11 also illustrates an optional embodiment in which the index splint is biotinylated for attachment to a solid support or indexed array. [Figure 12] FIG. 12 illustrates a schematic of a variant embodiment of the disclosed method. FIG. 12A and FIG. 12B depict an oligonucleotide structure in which a second splint molecule ("construct splint") is hybridized to the 3' and 5' ends of an assembly component. FIG. 12A depicts an oligonucleotide structure in which an indexed splint is linked to a construct splint via a spacer ("optional tether"). This represents another embodiment of the claimed index splint molecule, which includes, in a 5' to 3' orientation, a first index sequence (binding to an index at the 5' end of oligo 1 to form index 1); a second index sequence (binding to an index at the 3' end of oligo 2 to form index 2); a spacer (optional tether); a splint in which the 5' element hybridizes to the 5' end of oligo 2 and the 3' element hybridizes to the 3' end of oligo strand 1. 12B depicts an oligonucleotide structure in which a second splint molecule ("construct splint") that is not linked to the index splint is hybridized to the 3' and 5' ends of the assembly component. Also depicted is an optional embodiment in which the index splint is biotinylated for attachment to a solid support or indexed array. [Figure 13]Figure 13 illustrates a schematic of a variant embodiment of the disclosed method. Figure 13 shows the ligation product of Figure 12B. Figure 13 also shows optional additional structural features for the assembly components of the ligation product. An optional restriction enzyme site is depicted at the 5' end of the index splint. PBS1 and BPS1 represent (optional) primer binding sites useful for amplification of the assembly product. [Figure 14] FIG. 14 depicts a graph of data for an experiment evaluating solution assembly of low homology libraries using three types of test splint molecules as depicted in the oligonucleotide structures depicted in FIG. 1, FIG. 2, and FIG. 3 (further including assembly component 1 and assembly component 2, respectively). For each type of test splint molecule, three ratios of splint to construct were tested (1:1, 2:1, and 4:1). The test splint molecules in FIG. 2 and FIG. 3 were prepared with (+) and without (-) 3 nucleotide (nt) struts. The assembled constructs were characterized by next generation sequencing (NGS) of PCR amplified assembled constructs. The Y-axis in the graph is the average number of reads for correctly assembled constructs in each of the 15 sets. See Example 1 for further information. [Figure 15]FIG. 15 depicts a schematic of the test splint molecule of FIG. 5B (top panel) and data from a proof-of-concept experiment (bottom panel). The arrow indicates the 3' end of the oligonucleotide; the flat end is the 5' end of the oligonucleotide. Primer F - position of the forward primer for PCR. Primer R - position of the reverse primer for PCR. Tm is the melting temperature (i.e., melting temperature of the 5' and 3' elements) of each section of the splint hybridized to the left and right construct oligonucleotides (assembly component 1 and assembly component 2, respectively). BC1 and BC2 indicate the position of the paired barcode sequence. Index 1 and Index 2 indicate the index element in the oligonucleotide structure. The dotted line between the index sequence and the splint sequence of the indexed splint molecule indicates the spacer sequence, which in this test is a 20-base poly-T nucleotide sequence. See Example 2 for further information. [Figure 16] Figure 16 illustrates a schematic of three types of representative oligo libraries. The filled boxes represent the unique barcodes that flank the assembled constructs. [Figure 17] Figure 17 depicts representative data for testing assemblies utilizing three types of test splint molecules (see oligonucleotide structures in Figures 1, 2, and 5B) in three types of representative oligo libraries (see Figure 16). See Example 3 for further information. [Figure 18]Figure 18 depicts a schematic diagram of the test splint molecule (oligonucleotide structure in Figure 5B) and representative data on assembly using the test splint molecule at different splint lengths and ligation temperatures. In the experiment, a range of melting temperatures (Tm) of each of the hybridized elements (5' and 3' elements) of the splint pair constructs was designed and ligation at eight different temperatures was tested. The data on the median number of reads for 65.9°C ligation data is depicted in the graph. The percentage of correct and incorrect reads for each splint temperature is expressed as the median number of reads. For more information, see Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The use of "half" in any of the drawings or this specification, e.g., "half construct left," is not intended to be limiting. For example, "half construct left" and "half construct right" are not intended to indicate that assembly component 1 and assembly component 2 are the same length. Assembly components can vary in size. In this context, "half" is comparable to "portion," "part," or "oligonucleotide molecule."

[0009] Detailed Description The present disclosure relates, inter alia, to a method for generating long DNA libraries in solution by enzymatic ligation of oligonucleotides hybridized to indexed splint oligonucleotide molecules. In an embodiment, the oligonucleotides are chemically synthesized. The method is generally useful for producing long nucleic acids, including DNA or RNA, by ligating shorter components.

[0010] A.Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0011] As used herein, the term "nucleic acid assembly" refers to a method of physically linking at least two nucleic acid fragments or oligonucleotides together in a specific order to create a desired nucleic acid sequence (multicomponent construct), or the resulting nucleic acid from this process. Each of the fragments or oligonucleotides is an "assembly component" (sometimes colloquially referred to herein as a "half construct"). When two assembly components are linked, the upstream component is the "upstream assembly component" (or assembly component 1 or left half construct or half construct left) and the downstream component is the "downstream assembly component" (or assembly component 2 or right half construct or half construct right). The 3' end of the upstream assembly component (assembly component 1) is linked to the 5' end of the downstream assembly component (assembly component 2). An assembly component includes an assembly sequence and at least one non-assembly sequence. An "assembly sequence" is a sequence of interest that is included in the final multicomponent construct. Non-assembly sequences refer to sequences that are involved in the method of assembly, such as index sequences, primer sequences, restriction enzyme sequences, barcode sequences, and the like.

[0012] As used herein, the term "5' terminal sequence" refers to an oligonucleotide sequence that includes the 5' end of a nucleic acid. As used herein, the term "3' terminal sequence" refers to an oligonucleotide sequence that includes the 3' end of a nucleic acid. A terminal sequence can contain 2 or more consecutive nucleotides, 3 or more, 5 or more, or 10 or more consecutive nucleotides, 30 or more consecutive nucleotides, 50 or more consecutive nucleotides, or 100 or more consecutive nucleotides. A 3' or 5' "substantially terminal sequence" refers to a sequence that is proximal to but does not include the 3' or 5' end. For example, the 3' or 5' end of a 3' or 5' substantially terminal sequence can be within about 100 nucleotides, within about 50 nucleotides, within about 30 nucleotides, within about 25 nucleotides, within about 20 nucleotides, within about 15 nucleotides, within about 10 nucleotides, within about 5 nucleotides, within about 4 nucleotides, within about 3 nucleotides, within about 2 nucleotides, or within 1 nucleotide of the 3' or 5' terminal nucleotide, respectively.

[0013] The term "nucleic acid", "polynucleotide" or "oligonucleotide" refers to a DNA molecule, an RNA molecule, or their analogs. As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" include, but are not limited to, DNA molecules, such as cDNA, genomic DNA or synthetic DNA, and RNA molecules, such as guide RNA, messenger RNA or synthetic RNA. Furthermore, as used herein, the term includes single-stranded and double-stranded forms.

[0014] The term "hybridization" or "hybridizing" refers to the process in which fully or partially complementary polynucleotide strands come together under suitable hybridization conditions to form a double-stranded structure or region in which the two constituent strands are joined by hydrogen bonds. As used herein, the term "partial hybridization" includes cases in which the double-stranded structure or region contains one or more bulges or mismatches. Hydrogen bonds are typically formed between adenine and thymine or adenine and uracil (A and T or A and U, respectively) or cytosine and guanine (C and G), although other non-canonical base pairs may be formed (see, e.g., Adams et al., "The Biochemistry of the Nucleic Acids," 11th ed., 1992). It is contemplated that modified nucleotides can form hydrogen bonds that allow or promote hybridization in a non-canonical manner.

[0015] As used herein, the terms "portion," "segment," "element," or "fragment" of a sequence refer to any portion of a sequence (e.g., a nucleotide subsequence or an amino acid subsequence) that is smaller than the complete sequence. A portion, segment, element, or fragment of a polynucleotide can be of any length, such as two or more, e.g., at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, or 500 or more nucleotides in length.

[0016] As used herein, the term "indexed splint" refers to an oligonucleotide that includes (1) a splint sequence that can hybridize to at least one target sequence, and (2) an index sequence that can hybridize to an index sequence in a target molecule. See, for example, FIG. 5A. A "target" is an assembly component. Typically, an indexed splint is designed to hybridize to two targets to facilitate ligation of the two targets to a multi-component construct. See, for example, FIG. 5B. For that purpose, the splint sequence typically hybridizes to both targets, bringing the 3' end of one target and the 5' end of the other target into close proximity. The index sequence in the target is an extra sequence added to the target for the purpose of hybridizing to the index sequence in the indexed splint.

[0017] As used herein, the term "indexed splint-specific order ligation" refers to a protocol in which substantially single-stranded fragments are ligated together using a ligase to produce a multicomponent nucleic acid ligation product, where the order of the fragments in the ligation product is directed by an indexed splint molecule that contains splints that hybridize to the 3' and 5' terminal sequences of the two fragments to be ligated. A "substantially single-stranded fragment" refers to a fragment that contains a partial double-stranded portion, such as a 3' or 5' terminal sequence hybridized to a splint sequence in the indexed splint molecule, and / or a 3' or 5' terminal index sequence (or a 3' or 5' substantially terminal index sequence) hybridized to an index sequence in the indexed splint molecule.

[0018] "Index element" refers to a double-stranded DNA segment formed by (composed of) two strands with sequences that are reverse complements of each other. "Index sequence" refers to a sequence that is one of the two strands of a sequence that are reverse complements of each other and form an index element by hybridization to each other. In some embodiments, the index sequence is not palindromic (e.g., has no stem-loop or other secondary structure). The two index sequences in a single indexed splint do not hybridize to each other.

[0019] The methods and products of the present disclosure are based, at least in part, on the unexpected discovery that the use of indexed splint molecules as depicted diagrammatically in FIG. 5, panel A, results in improved assembly of multi-component constructs in oligonucleotide libraries. The use of indexed splint-specific order ligation, in which each pair of assembly components to be ligated has a corresponding indexed splint molecule with a splint sequence and index sequence specific to the pair of assembly components, allows for the assembly of multiple correct constructs in oligonucleotide libraries, even in the case of homologous oligo libraries (oligo libraries in which the oligos share a significant degree of sequence homology). The index sequence of the indexed splint molecule is not part of the construct sequence and is therefore not affected by the homology between library members. This is an unexpected benefit and improvement over previous high-throughput methods of multiplex assembly.

[0020] As used herein, a "mixture of single-stranded oligonucleotides" refers to an aqueous solution containing a plurality of different single-stranded oligonucleotides dissolved therein. The mixture can include at least 50, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, or at least 50,000 or more oligonucleotides, including any value between 50 and, for example, 50,000. The mixture of oligonucleotides can be made by synthesizing oligonucleotides in situ, i.e., synthesizing the oligonucleotides in place in an array, and then cleaving the oligonucleotides from the surface of the array after they have been synthesized. See, for example, Geary et al. (Nature Methods 2004 1: 241-248) and LeProust et al. (Nucleic Acids Research 2010 38: 2522-2540).

[0021] The term "oligonucleotide" as used herein refers to a polymer of nucleotides. For example, an oligonucleotide can have a length of about 2 to about 200 nucleotides, up to about 50 nucleotides, up to about 100 nucleotides, up to about 500 nucleotides, or any integer value between 2 and 500 nucleotides. In some embodiments, an oligonucleotide can be in the range of 30 to 300 nucleotides in length or 30 to 400 nucleotides in length. An oligonucleotide can contain ribonucleotide monomers (i.e., can be an oligoribonucleotide) and / or deoxyribonucleotide monomers. Oligonucleotides can be, for example, 10-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 80-100, 100-150, 150-200, 200-250, 250-300, 300-350 or 350-400 nucleotides in length, and any integer value between these ranges.

[0022] As used herein, the term "a set of single-stranded oligonucleotides that can be assembled to produce a multi-component ligation product" and its grammatical equivalents refer to a set of oligonucleotides that can be enzymatically assembled into a longer sequence, referred to herein as a "multi-component construct" or "multi-component ligation product," that contains sequences from each of the oligonucleotides in a defined order. As will be understood from the present disclosure, the single-stranded oligonucleotides of the set can contain an assembly sequence that includes (i) a 3' or 5' terminal (or substantially terminal) sequence that is an index sequence that hybridizes to a complementary sequence in the indexed splint molecule to form a double-stranded index element, and (ii) a 5' or 3' terminal sequence that hybridizes to the splint sequence in the indexed splint molecule. An "assembly sequence" is a sequence of interest that is included in the final construct, and does not include sequences related to the method of assembly, such as index sequences, primer sequences, restriction enzyme sequences, barcode sequences, etc.

[0023] As disclosed herein, a number of ranges of values ​​are provided. It is understood that each intervening value between the upper and lower limits of the range is also specifically considered. Each smaller range or intervening value encompassed by the described range is also specifically considered. The term "about" generally refers to plus or minus 10% of the indicated number. For example, "about 10%" can refer to a range of 9% to 11%, and "about 20" can mean 18 to 22. Other meanings of "about" can be clear from the context, such as rounding, so for example, "about 1" can also mean 0.5 to 1.4.

[0024] Other definitions of terms may appear throughout the specification.

[0025] B. Description of Methods and Products One method for assembling specific oligonucleotides for enzymatic ligation to form a desired construct is depicted in Figure 1. Figure 1 depicts two oligonucleotides to be ligated, one an upstream oligonucleotide ("Assembly Component 1") and one a downstream oligonucleotide ("Assembly Component 2"). The upstream construct oligonucleotide is upstream (5' to 3' base) of the ligation point, and the downstream construct oligonucleotide is downstream of the ligation point. The upstream and downstream construct oligonucleotides are hybridized to a single splint oligonucleotide. The splint oligonucleotide has two regions of complementary sequence, one of which hybridizes to the upstream construct oligonucleotide and the other of which hybridizes to the downstream construct oligonucleotide, such that the 3' end of the left construct is immediately adjacent to the 5' end of the right construct to form the intended ligation site. Under hybridization conditions, the two assembly components hybridize to a single splint oligonucleotide and a ligase joins the two pieces to form a longer multi-component construct.

[0026] In some embodiments, the disclosed methods use an indexed splint molecule (see, e.g., FIG. 5A) that includes a splint region and two index sequences, A and B, to assemble assembly components for ligation. The two index sequences of the indexed splint molecule flank the splint sequence with an intervening spacer sequence (dotted lines). The splint region has a 5' element and a 3' element, as depicted in the enlargement of the splint. The 5' element of the splint sequence hybridizes to the 5' end of the assembly component 2 oligonucleotide (see, e.g., FIG. 5B). The 3' element of the splint sequence hybridizes to the 3' end of the assembly component 1 oligonucleotide. In some aspects, the index sequences A and B are not palindromic (e.g., no stem-loop or other secondary structure) and do not hybridize to each other. One index sequence of the indexed splint molecule hybridizes to an index sequence at the end of one half construct. The other index sequence of the indexed splint molecule hybridizes to the index sequence at the end of the other half-construct, thus allowing the indexed splint to bring the two half-constructs closer together and align them in position for ligation, with the splint sequence and index sequence both hybridizing to the two half-constructs.

[0027] Index sequences are not part of the sequence of interest. They are extra sequences added to the ends of both assembly components. As such, they are designed to be as different as possible from other sequences in the reaction mixture, especially when multiple different assembly components are ligated in the same reaction (i.e., multiplexed), thus reducing the chance of unintended pairing of assembly components.

[0028] The 3' and 5' elements of the splint are each about 5-20 nucleotides in length, and optionally have a melting temperature below the melting temperature of the index element. In some embodiments, to substantially reduce or eliminate unintended pairing of two assembly components, the splint element alone (i.e., in the absence of the index element) should not be sufficient to drive hybridization under the reaction conditions, but will only transiently hybridize when the assembly components are held in place by hybridization of the index sequence of the splint indexed to the two assembly components. This is particularly important when the assembly components in the reaction share a significant degree of homology (e.g., a homologous oligo library).

[0029] As illustrated in Figures 5B and 5C, there are two exemplary orientations for the indexed splint molecule that binds to the two assembly components in the oligonucleotide structure.

[0030] Figure 5B depicts the orientation in which index sequence A (the index sequence at the 5' end of the indexed splint molecule) hybridizes to the index sequence at the 3' end of assembly component 2 to form a double-stranded index element (index 2). Index sequence B (the index sequence at the 3' end of the indexed splint molecule) hybridizes to the index sequence at the 5' end of assembly component 1 to form a double-stranded index element (index 1).

[0031] 5C depicts a second orientation in which index sequence A (index sequence at the 5' end of the indexed splint molecule) hybridizes to an index sequence at the 5' end of assembly component 1 to form a double-stranded index element (index 2). Index sequence B (index sequence at the 3' end of the indexed splint molecule) hybridizes to an index sequence at the 3' end of assembly component 2 to form a double-stranded index element (index 1). Note that the indexed splint molecule is illustrated with a 180° twist to accommodate the binding of the 5' element of the splint hybridizing to the 5' end of the assembly component 2 oligonucleotide and the 3' element of the splint sequence hybridizing to the 3' end of the assembly component 1 oligonucleotide. The twist is for illustrative purposes only and is not intended to be limiting.

[0032] Exemplary advantages and distinguishing features of the present invention in assembling polynucleotides into multi-component constructs include the following: The present invention does not require an array or solid support for the assembly method. The method can be used to assemble single-stranded oligonucleotides. In one embodiment, the method is carried out using synthetic oligonucleotides. In one embodiment, the method is carried out using an oligonucleotide library that includes a mixture of single-stranded oligonucleotides. The mixture can include at least 50, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, or at least 50,000 or more oligonucleotides. Moreover, in embodiments, the method can be carried out as an iterative process in which non-assembled sequences can be cleanly removed before the next cycle, such as by type II restriction (i.e., no "scar" at the site of fragment ligation). The method can be utilized to assemble composite libraries, low homology libraries, and high homology libraries, e.g., libraries with sequences that are substantially identical except for minor variations.

[0033] Indexed splints can be used in other formats. In one variation shown in FIG. 2, two oligonucleotides representing the left and right halves of the intended construct are hybridized to a pair of splint oligonucleotide members. Each member of the pair of splint oligonucleotide members further contains an index sequence that is the reverse complement of the index sequence in the other member of the pair and thus can hybridize to each other to form a double-stranded DNA portion referred to herein as an index element (labeled "index" in FIG. 2). The two index sequences indirectly serve to bring the two assembly components together. The index sequence is not part of the assembly sequence itself. In FIG. 2, the right-indexed splint contains a splint sequence (R-sprint) that is homologous to the 3' end of the left construct oligonucleotide, and a section that is homologous to the 5' end of the right construct oligonucleotide. In FIG. 2, the R-sprint contains about 10 nucleotides (nt), but this can range from about 5 nt to about 20 nt, for example.

[0034] Not all splint sequences need to hybridize to the target terminal sequence. Another variation of indexed splints is illustrated in FIG. 3. This oligonucleotide structure is a variant of the oligonucleotide structure in FIG. 2. The inventors considered that this structure can reduce or eliminate interference caused by truncated synthesis products that lack the 5' end of the designed assembly component. In this structure, the splint oligonucleotide member has a splint sequence region (L-sprint) that is complementary to a sequence in the assembly component 1 oligonucleotide that is not proximal to the 3' end of the assembly component 1 oligonucleotide, resulting in a looped portion of assembly component 1. The other splint oligonucleotide member has a splint sequence region (R-sprint) that is complementary to the 3' end portion of assembly component 1 and the 5' end portion of assembly component 2 oligonucleotide.

[0035] In Figure 4, the assembly component 1 oligonucleotide has a 5' index sequence at its 5' end assembly component 1. The index sequence hybridizes to the index sequence of the indexed splint molecule to form a double-stranded index element. The splint molecule has a sequence complementary to the 3' end portion of the assembly component 1 and the 5' end portion of the assembly component 2 oligonucleotide, resulting in a loop portion of assembly component 1 (as in Figure 3, but with only one indexed splint used).

[0036] As shown herein, embodiments such as those illustrated in Figures 5A-5C provided significant advantages. Assembly of low homology or composite libraries using splint molecules indexed in Figure 5 produced high accuracy (i.e., total number of correct reads compared to total number of reads). See Figure 17. "Composite library" refers to library sequences with little sequence similarity, except for peripheral primer binding sites. In addition, there were no dropout constructs. Thus, the data indicates that assembling assembly components using splint molecules indexed in Figure 5 allows for much higher discrimination. Thus, the present invention allows for correct assembly of constructs that would otherwise be impossible due to random hybridization of splint molecules, such as those in Figure 1.

[0037] Thus, the present disclosure provides a method for assembling a multi-component construct using specific oligonucleotides with indexed splints for enzymatic ligation. The present disclosure provides, inter alia, a method for assembling two or more specific components by indexed splint-specific order ligation to form a multi-component ligation product with a predefined order. The method can be performed in solution and in the absence of a solid support. The method can be used to produce a synthetic multi-component construct by ligating sequences from two, three or four or more single-stranded oligonucleotides in a defined order. The method can be multiplexed in that multiple different synthetic multi-component constructs (e.g., at least 2, at least 10, at least 100, at least 1,000 or at least 10,000 or at least 100,000 types of multi-component constructs) can be produced in the same solution. This process can also be repeated several times to produce longer nucleic acids.

[0038] In certain embodiments, the oligonucleotide molecules are produced by chemical synthesis using methods well known in the art of synthetic organic chemistry. Some oligonucleotide molecules described herein can alternatively be produced using methods known in the art, including in vitro transcription and cell-based expression.

[0039] The methods of this disclosure, along with the various oligonucleotide molecules used in the methods, are described in more detail in the following disclosures.

[0040] Indexed Sprints In some embodiments, the disclosed method uses an indexed splint as illustrated in FIG. 5A. The indexed splint includes a splint region, a first index sequence, and a second index sequence. In some embodiments, the first index sequence and the second index sequence flank the splint region. The first index sequence and the second index sequence can be located at the 5' and 3' ends of the indexed splint. In one aspect, one or both of the first index sequence and the second index sequence include terminal sequences (e.g., 5' terminal sequences and / or 3' terminal sequences) of the indexed splint. In some embodiments, one or both of the first index sequence and the second index sequence include substantially terminal sequences (e.g., 5' substantially terminal sequences and / or 3' substantially terminal sequences) of the indexed splint, but do not include terminal nucleotides. The splint region has 5' and 3' sequence elements that hybridize to each of the two assembly components, respectively, and bring the ends of the assembly components closer together. As illustrated in Figures 5A-5C, the 5' element of the splint sequence hybridizes to the 5' end of the downstream assembly component (assembly component 2), while the 3' element of the splint sequence hybridizes to the 3' end of the upstream assembly component (assembly component 1). As a result, the 5' end of the downstream assembly component and the 3' end of the upstream assembly component are brought together and can be ligated by a ligase.

[0041] There are two exemplary orientations for the indexed splint molecule that binds two assembly components in an oligonucleotide structure for ligation, shown in Figures 5B and 5C, respectively.

[0042] Index sequences are not part of the sequence of interest and will not appear in the final assembly. They are extra sequences added to the ends of both assembly components. As such, they are designed to be as different as possible from other sequences in the reaction mixture, especially when multiple different assembly components are ligated in the same reaction (i.e., multiplexed), thus reducing the chance of unintended pairing of assembly components.

[0043] Features of index elements include: The index sequence is designed to hybridize only to its complement. Thus, for any given index element, neither sequence (complement or reverse complement) is present in the final construct molecule. Similarly, for indexed splint molecules that contain one or more spacer sequences, neither index sequence (complement or reverse complement) is present in the spacer. The index sequence is preferably designed not to have a stable secondary structure of its own, such as a stem-loop structure, and thus is not palindromic. Furthermore, the nucleotide composition of the index element is preferably designed to have a melting temperature (Tm) suitable for use in the assembly method. In some embodiments, the index element is designed to remain hybridized in a temperature range where the splint element can only transiently hybridize to the assembly component, except for splint elements that have 100% identity to the assembly component. Mismatches in the splint element sequence to the construct element destabilize the hybridization. Without being bound by theory, it is believed that this dynamic hybridization allows for sampling that favors the formation of 100% identity splint element to assembly component hybridization. This is believed to contribute to the high accuracy of the multi-component constructs formed in the methods of the present disclosure. In some embodiments, the assembly accuracy (i.e., the total number of correct reads compared to the total number of reads) is at least 80%, 85% or at least 90%, for example, at least 91, 92%, 93%, 94%, 95% or at least 96%. Without being bound by theory, it is also believed that this contributes to little or no dropout of the intended construct, and thus the intended construct is represented at a high percentage.In some embodiments, the percentage of intended constructs is at least 80%, 85% or at least 90% of the total number of intended constructs, e.g., at least 91, 92%, 93%, 94%, 95%, 96%, 97% or at least 98%.

[0044] Optionally, the index sequence is a random high complexity sequence that is base balanced (A=T=G=C) to aid in predicting melting temperature. Homopolymer repeats may be included. In other embodiments, the index sequence does not include repetitive elements, e.g., homopolymer and / or dinucleotide repeats, e.g., GTGTGTGT. High melting temperatures are useful in the practice of the disclosed methods. The Tm of each index sequence can be about 50°C to about 80°C, about 65°C to about 72°C, about 55°C to about 75°C, about 50°C to about 70°C, or 60°C to about 80°C. In one aspect, the Tm of each index sequence is about 65°C to about 69°C. In one aspect, the index sequence can be 20 nt to 60 nt. The index sequence can be 20 nucleotides (nt) to 50 nt, 25 nt to 45 nt, 25 to 35 nt, or 28 nt to 32 nt. In one aspect, the index sequence is about 30 nucleotides. The reason for keeping index sequences shorter is to maximize the length of construct sequences in assembly components. The main obstacle to making very short index sequences is that specificity and duplex stability are compromised. Index sequences can be prepared as a library of sequences. Potential index sequences are screened against the appropriate genome to eliminate sequences that may hybridize. In one embodiment, potential index sequences are screened against the human genome. Index sequences in a library of sequences can be designed to share some features, such as length, base balance, or melting temperature, while being designed not to support hybridization between different index sequences. In one embodiment, a library of sequences can contain multiple sets of index sequences that are different from other sets but share features within the set. For example, a library of sequences can include one set of index sequences designed with one predicted melting temperature, while a second set of index sequences in the library can be designed with a different melting temperature.

[0045] Sprint The indexed splint comprises a splint sequence. The splint sequence is intended to guide the specificity of pairing of two assembly components. As illustrated in FIG. 5A, the splint sequence (sprint) has two sequence elements: a 5' element (5'-element) and a 3' element (3' element). The 5' element of the splint sequence hybridizes to the 5' end of the downstream assembly component. More specifically, the 5' element hybridizes to the 5' end or 5' substantially end sequence of the downstream assembly component (assembly component 2) (see, e.g., FIG. 5B). The 3' element of the splint sequence hybridizes to the 3' end of the upstream assembly component (assembly component 1). More specifically, the 3' element hybridizes to the 3' end or 3' substantially end sequence of segment D of the upstream assembly component (assembly component 1) (see, e.g., FIG. 5B).

[0046] A splint is typically a short sequence or sequence with a melting temperature lower than the melting temperature of the index region. In some embodiments, each splint element is designed such that it is insufficient by itself to drive hybridization under the reaction conditions. Instead, the splint sequence is designed to only transiently hybridize to the assembly component oligonucleotide when the assembly component is held in place by hybridization of the indexed splint oligonucleotide to the index sequence. In some aspects, the 5' and 3' elements of the splint sequence are contiguous with no intervening nucleotides. In some aspects, the 5' and 3' elements of the splint sequence are substantially contiguous, e.g., there are several (1, 2 or 3) intervening nucleotides between the 5' and 3' elements, and the several intervening nucleotides are not intended to bind to the assembly sequence of any of the assembly components. The 5' and 3' elements of the splint sequence can independently be from about 5 nucleotides to about 25 nucleotides in length, or from about 5 nucleotides to about 20 nucleotides in length, or from about 8 nucleotides to about 15 nucleotides or from about 10 nucleotides to about 13 nucleotides in length. The 5' and 3' elements of the splint generally have similar melting temperatures. The melting temperature of the splint element is less than the melting temperature of the index element. The choice of melting temperature of the splint element is influenced by the ligase used in the assembly step. In some aspects, the melting temperature (Tm) of the splint element is determined by the following formula: Tm=(2*(A 数値 +T 数値 ))+(4*(C 数値 +G 数値))-7), can be in the range of 10° C. to 80° C., 14° C. to 44° C., 20° C. to 42° C., or 22° C. to 40° C. One of skill in the art will recognize that different splint melting temperatures can be selected for different ligases, e.g., a ligase that can be used at higher temperatures, e.g., a thermostable ligase such as 9°N™ DNA ligase (New England Biolabs, Ipswich, Mass.), can be used with a splint having a higher melting temperature.

[0047] In some aspects, the indexed splint comprises a spacer sequence that is a linker between the specific structural features of the indexed splint (e.g., the index sequence and the splint sequence). The spacer can be 1-30 nucleotides in length in some embodiments. The spacer is any sequence or non-nucleic acid component designed not to hybridize to the assembly sequence or index sequence. In some embodiments, the spacer can be a simple sequence, e.g., a homopolymer sequence or a short repeating sequence, e.g., a di-nucleotide or tri-nucleotide repeat. In some aspects, the indexed splint has two spacer sequences, as illustrated in FIG. 5A. In some aspects, the indexed splint has more than two spacer sequences to link one or more additional structural features. For example, as described elsewhere herein, an indexed splint for iterative assembly applications can contain a second splint element and a third index sequence, and the spacer sequence can optionally separate the specific structural features.

[0048] Assembly Components In some embodiments, the assembly components are oligonucleotide molecules that contain the sequences desired to be present in the final construct assembly, and an index sequence. The location of the index sequence in the assembly components depends on whether the component is the left (upstream) or right (downstream) component in the intended ligation reaction.

[0049] Typically, in the upstream assembly component, the indexed splint sequence is located at or substantially at the 5' end of the assembly component oligonucleotide. The assembly sequence is located at the 3' end region of the assembly component oligonucleotide. In some embodiments, the index sequence borders the assembly sequence. In other embodiments, additional sequences (structural features) can be located between the index sequence and the assembly sequence. Such structural features can be one or more of at least one primer sequence, at least one barcode sequence, and / or at least one restriction enzyme cleavage site. In embodiments, the type IIS restriction enzyme site is directly upstream of the assembly sequence. See, for example, FIG. 8, molecule (c). In this embodiment, the type IIS restriction enzyme site is positioned such that cleavage by the enzyme at the cleavage site separates all non-assembly sequences from the assembly sequence. This embodiment is useful for iterative versions of the method, for example, where the products of a first assembly step are then used as components in a subsequent assembly step.

[0050] Typically, in downstream assembly components, the indexed splint sequence is located at or substantially at the 3' end of the assembly component oligonucleotide. The assembly sequence is located at the 5' end sequence of the assembly component oligonucleotide. In some embodiments, the index sequence borders the assembly sequence (segment E). In other embodiments, additional sequences can be located between the assembly sequence (segment E) and the index sequence (segment F). Such sequences can be one or more of at least one primer sequence, at least one barcode sequence, and at least one restriction enzyme cleavage site. In embodiments, the type IIS restriction enzyme site is directly downstream of the assembly sequence. See, for example, FIG. 8, molecule (a). The type IIS restriction enzyme site is positioned such that cleavage by the enzyme at the cleavage site removes all non-assembly sequences from the assembly sequence. This embodiment is useful for iterative versions of the method. This embodiment is useful, for example, for iterative versions of the method in which the products of a first assembly step are then used as components in a subsequent assembly step.

[0051] method The present disclosure provides a method for assembling two nucleic acid assembly components to produce a ligation product (multi-component assembly). In some embodiments, the method includes hybridizing an upstream assembly component and a downstream assembly component to an indexed splint present in a reaction mixture. In some embodiments, in the hybridized structure, both the 5' and 3' ends of the upstream assembly component are hybridized to the indexed splint, and both the 5' and 3' ends of the downstream assembly component are hybridized to the indexed splint. The hybridizing step is followed by a ligating step. In the ligating step, the 3' end of the upstream assembly component is ligated to the 5' end of the downstream assembly component, thereby producing a first ligation product. In some aspects, the reaction mixture is multiplexed and includes a mixture of single-stranded oligonucleotides including a plurality of upstream assembly component species, a plurality of downstream assembly component species, and a plurality of indexed splint molecular species. Each of the multiple indexed splint molecular species is capable of hybridizing to a given upstream assembly component and a given downstream assembly component in the mixture.

[0052] The method is carried out in an aqueous solution. The aqueous solution can be the same for both the hybridizing and ligating steps. The aqueous solution for the ligation step can be any aqueous solution with a suitable pH and salt concentration suitable for the ligase used in the method. The reaction solution is typically buffered and contains one or more salts, and can optionally contain a molecular crowding agent, such as PEG (polyethylene glycol), if suitable for the ligase. In some embodiments, when the same aqueous solution is used for both the hybridization and ligation steps, the components of the buffer can be selected to be more favorable or unfavorable to the ligase enzyme, or more favorable or unfavorable to the hybridization reaction, to achieve the best results. For example, the concentration of magnesium in the solution can be selected to be slightly lower or significantly higher than ideal for the ligase reaction, if that concentration of magnesium supports a more stringent hybridization step.

[0053] The hybridization step can be carried out by incubation in a water bath, cooling the reaction from an elevated temperature to a cooler temperature for a period of time sufficient to form an oligonucleotide structure comprising an indexed splint and two assembly components. The temperature profile of the hybridizing step is guided by the Tm of the index sequence and the Tm of the splint region. For example, the hybridization step can start at a higher temperature followed by a cooling period. The starting temperature is above the Tm of the index sequence so as to melt any secondary structure. An exemplary temperature profile is from about 95°C to about 40°C, e.g., 93°C to 44°C. The duration of the hybridization cooling can range from about 15 minutes to several hours. An exemplary duration is 1.75 hours. In one aspect, the hybridization step includes a step of cooling from 93°C to 44°C for 1.75 hours. In some embodiments, the temperature of the hybridization step can be varied over time or cycled from higher to lower temperatures for several cycles.

[0054] The ligation step is carried out by contacting the hybridized oligonucleotide structure with a ligase. The temperature of the reaction mixture can be changed to a temperature appropriate for the ligase used, and in some embodiments, the temperature is below the melting temperature of the index element and above the melting temperature of the splint element. For example, after hybridization, the temperature of the reaction mixture is increased to a range of about 45° to about 70°C, about 45° to 65°C, about 50° to 65°C, or about 61° to 65°C, etc., and then the hybridized oligonucleotide structure is contacted with a thermostable ligase. Lower temperatures can be used for non-thermostable ligases. The ligation step is carried out for a suitable length of time, which can range from a few minutes to a few hours. Optionally, the ligation step is terminated by methods known in the art. For example, the ligase can be denatured by high temperature, chemical denaturants, or EDTA can be added (to bind to magnesium required for the ligation reaction).

[0055] The method can be carried out with any ligase. DNA and RNA ligases are commercially available. Alternatively, the ligase can be chemically synthesized (see, e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, 2nd Edition, WH Freeman & Co., NY, 1992) or produced recombinantly by molecular biology methods, such as expression in cells. Recombinant ligase can be prepared as a fusion protein, for example, to aid in purification. In certain aspects, the ligase is provided in a purified or isolated form. In certain embodiments, the ligase is provided at about 80%, about 90%, about 95% or about 99% purity. In certain embodiments, the ligase is provided as part of a composition. In certain embodiments, the ligase is provided in an aqueous composition suitable for use as or inclusion in a composition for assembly of a multi-component construct. Those skilled in the art will be aware of the various materials that can be included in such a ligase reaction composition.

[0056] The choice of ligase will affect the temperature of the ligation step. Different ligases can have different efficiencies, different activities at different temperatures, and different sensitivities to mismatches at the ligation site. There are many examples of mesophilic DNA ligases that function at lower temperature ranges. Thermostable ligases are also available. Thermostable ligases allow for ligase reaction conditions at higher temperatures, for example, up to 65°C. Higher temperature reaction conditions can contribute to more accurate assembled constructs. In some embodiments, thermostable ligases have a larger footprint requirement (e.g., about 25 nucleotides of hybridized sequence) before ligation occurs at the ligation site. This feature can contribute to greater specificity and result in better purity of the ligated product. In some embodiments, multiple ligases can be used. In some embodiments, different ligases can be added sequentially to the same hybridization reaction. In some embodiments, different ligases can be included in the same reaction. For example, a mesophilic ligase and a thermostable ligase may be included in the reaction, and the mesophilic ligase may be used at a lower temperature before increasing the temperature for the thermophilic ligase. Such an embodiment may be applicable by practice of a method using a library of sequences that includes one set of index sequences designed with one predicted melting temperature, where a second set of index sequences in the library can be designed with a different melting temperature.

[0057] The concentration of oligonucleotide components is a reaction parameter that affects the accuracy of ligation. High concentrations of oligonucleotide components increase the likelihood of erroneous ligation results. Thus, lower concentrations can result in improved accuracy of the ligation product.

[0058] In general, hybridization conditions, the length of the hybridized sequence, and ligation conditions can be varied to achieve the highest yield of correctly assembled constructs. For example, if a set of conditions results in incorrect assembly of a particular set of components, a higher ligation temperature and / or more stringent hybridization conditions can be used. Optimization of hybridization and ligase conditions is within the scope of the art in view of this disclosure and prior art knowledge in the art. See, for example, Sambrook et al., Molecular Cloning A Laboratory Manual, 2001, 144: 111-115, 1999. nd Ed. Cold Spring Harbor Press (1989) or Anderson, Nucleic Acid Hybridization, 1 st Ed., BIOS Scientific Publishers Limited (1999).

[0059] In certain aspects, the method can be performed using an indexed splint of FIG. 2 and further comprising at least one blocking nucleotide. FIG. 6 illustrates an oligonucleotide structure that includes at least one blocking oligonucleotide hybridized to a single-stranded region in an assembly sequence of an assembly component. This variation is contemplated to prevent unintended hybridization. The blocking oligonucleotide can be designed to block some or all of the single-stranded region. In the depicted embodiment of FIG. 6, the oligonucleotide structure includes two additional oligonucleotide strands, one of which hybridizes to the assembly component 1 sequence and the other of which hybridizes to the assembly component 2 sequence. It is contemplated that the blocking oligonucleotide can be synthesized as part of the two oligonucleotide molecules of the indexed splint molecule depicted in FIG. 6. The blocking oligonucleotide can also be used in the oligonucleotide structure of FIG. 5 or other embodiments of the invention. It is contemplated that the blocking oligonucleotide can be synthesized as part of the indexed splint molecule depicted in FIG. 5.

[0060] The method can be carried out iteratively. In one embodiment, the downstream assembly component comprises a removable (removable) index sequence at its 3' end. After the hybridization and ligation steps to provide a ligation product comprising the assembly components, the index sequence is removed (e.g., by enzymatic cleavage or cleavage of a cleavable linker) so that the 3' terminal sequence of the ligated product is the 3' sequence of the assembly sequence. This first ligated product is then utilized as an upstream assembly component in further iterations of the method to produce a second ligated product. The reaction mixture comprises the first ligated product and an indexed splint molecule that hybridizes to another downstream assembly component.

[0061] In one embodiment, the removable (or detachable) index sequence comprises a restriction enzyme site. In one embodiment, the restriction enzyme site is for a Type IIS restriction enzyme. Type IIS restriction enzymes comprise a specific group of enzymes that recognize asymmetric DNA sequences and cut at a defined distance outside their recognition sequence, usually within 1-20 nucleotides. This specific cleavage mechanism of action of Type IIS restriction enzymes allows for cleavage such that the desired assembly sequence is retained and non-assembly sequences are removed. This allows for DNA manipulations that do not alter the final assembly sequence, thus allowing for the generation of scarless seams in multi-component constructs.

[0062] In some embodiments, the removable (or detachable) index sequence can be an index sequence linked to the assembly sequence by a cleavable linker. Photocleavable linker chemistry is known in the art and is commercially available from TriLink Biotechnologies, Integrated DNA Technologies, and Glen Research.

[0063] In one embodiment, the method is a four-part assembly as depicted in Figures 7-9, where the assembly process is repeated on the ligation product of the first assembly. In this embodiment, there are two ligation steps separated by a restriction enzyme digestion step. The hybridization step forms two oligonucleotide structures depicted in Figure 7. In the top oligonucleotide structure depicted in Figure 7, the oligonucleotide structure is in the orientation depicted in Figure 5B (but can be designed in the orientation depicted in Figure 5C). The indexed splint molecule includes, in a 5' to 3' orientation, a first index sequence hybridized to downstream assembly component 2 to form index 2, a splint sequence (sprint 1), and a second index sequence hybridized to upstream assembly component 1 to form index 1. Splint 1 hybridizes to the 3' end sequence of upstream assembly component 1 and the 5' end sequence of downstream assembly component 2. In addition to the first splint structure (Sprint 1), the indexed splint of the upper oligonucleotide structure further comprises a second splint sequence (Sprint 2) in the spacer section, and another index sequence (forming Index 3) between the 5' index sequence (Index 2) and the first splint structure (Sprint 1). Downstream assembly component 2 comprises yet another structural feature, a restriction enzyme sequence 3' to the assembly sequence (depicted in Figure 8).

[0064] In the lower oligonucleotide structure depicted in Figure 7, the oligonucleotide structure is in the orientation depicted in Figure 5C (although it can be designed in the orientation of Figure 5B). The indexed splint molecule comprises, in a 5' to 3' orientation, a first index sequence hybridized to upstream assembly component 3 to form index 4, a splint sequence (sprint 3), and a second index sequence hybridized to downstream assembly component 4 to form index 5. Splint 3 hybridizes to the 3' terminal sequence of upstream assembly component 3 and the 5' terminal sequence of downstream assembly component 4. The indexed splint of the lower oligonucleotide structure further comprises a complementary sequence of index 3 (index 3 complement) between the 5' index sequence (index 4) and splint 3.

[0065] The ligation step produces a ligation product of components 1&2, assembled product (1-2), and a second ligation product of components 3&4, assembled product (3-4), as illustrated in FIG. 8, structures (a) and (c), respectively. As illustrated in FIG. 8, the indexed splint of superstructure (a) contains a restriction enzyme site (RE cut site) that is 3' of the assembly sequence of assembly component 2 and 5' to index 2. The indexed splint of structure (c) contains a restriction enzyme site that is 5' of the assembly sequence of assembly component 3 and 3' to index 3. The indexed splint contains two additional structural features, an index sequence that is the complementary sequence to form index 3, located between the index sequence of splint 2 and index 4, and a restriction enzyme site that is 5' to the index sequence of index 4. The ligation products are subjected to type IIS restriction enzyme digestion to cleave the restriction enzyme sites in assembled product 1-2 and assembled product 3-4 to remove index 2 and index 4, respectively. Consequently, in further iterations of the method, the digestion product of ligation product 1-2 can become the upstream assembly component, and the digestion product of ligation product 3-4 can become the downstream assembly component. The digestion products can be purified, for example, by SPRI purification (e.g., SPRI is an abbreviation for solid phase reversible immobilization), and then subjected to a hybridization step. The hybridization step produces the oligonucleotide structure illustrated in FIG. 9. As illustrated, a double-stranded index 3 is formed by hybridization of the index 3 sequences of the two indexed splint molecules. Splint 2 acts to hybridize to the assembly components to form ligation sites; the 5' element binds to the 5' end of the assembled product (3-4) and the 3' element binds to the 3' end of the assembled product (1-2).A second ligation step is performed, producing the multi-component ligation product 1-2-3-4 (not shown). The product of the second ligation step can optionally be SPRI purified and amplified by PCR.

[0066] As shown herein, in index-based ligation, hybridization of opposite ends of two assembly components to a common indexed splint molecule with two complementary index sequences provides excellent specificity. For example, the melting temperature (Tm) of the index element cannot be higher than the melting temperature (Tm) of each of the two hybridized splint elements. In this way, the index bond is stable under hybridization conditions, while the splint bond is metastable. Thus, a specifically designed index sequence can drive the hybridization reaction and avoid undesired binding between the splint sequence and unintended targets in the reaction mixture.

[0067] The same process can be repeated for the assembly of further larger products.

[0068] It is further contemplated that there may be conditions under which specificity may be improved by variations of the disclosed method. Next, methods for increasing the melting temperature of the index sequence and thus increasing specificity are described. This embodiment also relies on a two-step ligation process. Briefly, and as illustrated in Figures 10-13, in the first ligation step, two assembly component oligos are hybridized to a molecule containing two index sequences adjacent to each other but not including a splint region (see "index splint" in Figure 10) and ligated together at their indexing sequences to form a much longer oligo in the first ligation step (see Figure 11). After the first ligation step (ligating the ends of the indexing sequences), the temperature can be increased above the melting temperature of the indexing sequences with their complementary splint sequences, and a second molecule (a "construct splint" that hybridizes to the two assembly targets) is added at this higher temperature (see FIG. 12B), allowing for higher stringency and reducing the chance of cross-hybridization between homologous constructs, thus potentially resulting in even better specificity than previously described embodiments. A second ligation event per molecule occurs when the free end of the ligation product of the first ligation hybridizes to a splint oligo and is then ligated to form a single-stranded loop (see FIG. 13). To stabilize the formation of the loop, one of the sections of the second splint is made substantially longer than the other section, allowing it to form a stable duplex at one end, while the other end only needs to be in contact with the duplex end for a short time in order for it to be ligated to form a loop. Moreover, chimera formation can be inhibited by diluting the solution after the first ligation step by adding more buffer solution, which increases the entropy of the solution, thereby increasing the probability of intramolecular ligation and loop formation over intermolecular ligation.

[0069] As illustrated in FIG. 10, two assembly components are hybridized to a splint molecule with an index sequence at the 5' end of one assembly component and an index sequence at the 3' end of the other assembly component. After ligation, the ligation product contains two assembly components, where the two index sequences are adjacent and ligated to each other in the middle of the ligated product. See FIG. 11. After ligation, the ligation product remains hybridized with the two index sequences (index1-index2) now ligated to the index splint and has a significantly increased melting temperature. For example, in one embodiment, if each index sequence is 30 nucleotides long and each index element has a melting temperature of about 69° C., the combined melting temperature of the two index sequences in the duplex is about 17° C. higher, resulting in an average temperature of about 86° C. This means that the temperature of the reaction solution can be raised to temperatures in the low 80s without denaturing the duplex.

[0070] Optionally, between steps of the method, unbound fragments or unligated products can be removed from the reaction solution, for example, by washing away the hybridization solution to clean the reaction solution. Optionally, this cleaning can be done by binding the indexing splint oligo to a solid support. For example, the index splint oligo can have a biotin attached to either its 3' end (as shown in Figure 11) or its 5' end. The oligo duplexes can then be bound to magnetic streptavidin beads, which are commonly used for target enrichment. As is commonly done with SPRI beads, they need to be mixed in a tube, incubated for a few minutes, and then pulled to the side or bottom of the tube using a strong gradient magnetic field. A wash step can then be accomplished by removing the hybridization solution, refilling the container, and rinsing the ligated products in the tube one or more times with a wash buffer. This will reduce the concentration of unligated oligonucleotides and reduce the likelihood of incorrect ligation events in the second ligation step.

[0071] The ligated products can be released from the duplex (and thus from the beads) by adding water or a more stringent ligation buffer, or by heating above the appropriate melting temperature for the buffer. A thermostable ligase, e.g., 9°N™ Ligase (NEB, Ipswich, MA), can be added to the ligase buffer and the temperature increased to above 80° C. or the highest temperature at which another thermostable ligase remains active, at which point a splint molecule for the free end of the ligation product can be added to the tube. Alternatively, a splint can be added to the mixture before heating and a ligase added after heating. This step can be done with the oligos in solution or while still attached to the magnetic beads. This second ligation event is depicted in FIG. 12 and FIG. 13. After a period of ligation reaction, the ligation reaction can be stopped by adding EDTA or simply by quickly cooling to or at room temperature.

[0072] Figure 13 shows optional additional structural features in the oligos, including, for example, primer binding sites (PBS1 and BPS1) and optional restriction enzyme sites. Amplification can be performed by either PCR or rolling circle amplification. Rolling circle amplification is performed by adding a single primer complementary to either primer binding site in the looped oligo and a highly processive DNA polymerase, such as bacteriophage phi29 DNA polymerase, to a tube. Alternatively, PCR can be performed with a polymerase, a pair of primers and dNTPs, followed by thermal cycling in a thermal cycler.

[0073] Sometimes, during PCR amplification of the assembled products, non-ligated oligonucleotides may be extended by the polymerase, creating unwanted chimeras. The embodiment shown in Figures 12 and 13, which creates circularized DNA, can reduce this chimera production by adding an exonuclease step. In this additional step, an exonuclease can be used to digest single-stranded and double-stranded DNA from its 5' end, leaving behind a circular DNA assembly. The exonuclease step should be performed prior to PCT amplification. The digestion process can be stopped by adding EDTA or by killing the enzyme by heating the reaction mixture to 75°C, or both. Such circular constructs are then amplified by PCR or by rolling circle amplification using a high processivity polymerase, e.g., phi29.

[0074] In one embodiment, the first ligation step for ligating the two assembly components in Figure 10 is omitted. A second splint ("construct splint") hybridizes to the construct sequences at the 5' and 3' free ends of the two assembly components, which are then ligated.

[0075] In another embodiment related to Figures 10-13, the index splint and construct splint are connected using a linker molecule or sequence ("Optional Tether" in Figure 12A). The molecule has the components of the indexed splint of Figure 5A but in a different 5' to 3' order.

[0076] In some embodiments of the present disclosure, the multi-component constructs can be first assembled in solution according to the present disclosure and attached to a solid substrate after assembly by any method known in the art. Thus, the present disclosure can be used to prepare an array of multi-component constructs, whereby the multi-component constructs are attached to an array substrate after assembly. The multi-component constructs or a pool or pools of multi-component constructs can then be optionally and selectively cleaved from the array substrate and used as a library or libraries. Alternatively, the multi-component constructs can be attached to beads. The constructs can be attached to the beads by chemical means or by specific hybridization. Depending on the attachment method, different beads can be used to capture specific different constructs, or one bead can capture multiple constructs. Attaching the library to beads can provide advantages for storing, washing, concentrating, or enzymatically manipulating the library.

[0077] kit The present disclosure also provides kits useful for carrying out the disclosed methods. In one aspect, a kit is provided that contains reagents for carrying out the above-mentioned methods, including at least one type of upstream assembly component, one type of downstream assembly component, and an oligonucleotide library comprising a mixture of single-stranded oligonucleotides with indexed splints to direct ligation of the assembly components, and DNA ligase. In certain embodiments, the kit includes one or more other reaction components. In certain embodiments, an appropriate amount of one or more reaction components is provided in one or more containers or held on a substrate. Examples of additional components of the kit include, but are not limited to, reaction buffers, one or more reagents for detecting ligation products (e.g., probes or PCR primers), etc. The reaction components used can be provided in various forms. For example, the components (e.g., enzymes, oligonucleotides, probes and / or primers) can be suspended in aqueous solutions or attached to beads, or provided as freeze-dried or lyophilized powders or pellets. The kits of the present disclosure can be provided at any suitable temperature. For example, for storage of kits containing protein components or complexes thereof in liquid form, it is preferred that they be provided and maintained below 0°C, preferably at about -20°C, in a freeze-resistant solution, possibly containing glycerol or other suitable freeze-antiseptic.

[0078] The kit or system can contain any combination of components described herein in an amount sufficient for at least one assay. In some applications, one or more reaction components can be provided in pre-measured single-use amounts in individual, typically disposable, tubes or equivalent containers. The amount of components provided in the kit can be any suitable amount and can depend on the market the product is targeted to. The container(s) in which the components are provided can be any conventional container capable of retaining the form in which they are provided, such as a microcentrifuge tube, a microtiter plate, an ampoule, a bottle, or an integrated test device, such as a fluidic device, cartridge, lateral flow or other similar device.

[0079] The kits may also include packaging materials for holding the container or combination of containers. Exemplary packaging materials for such kits and systems include solid matrices (e.g., glass, plastic, paper, foil, microparticles, etc.) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in vials, microtiter plate wells, microarrays, etc.). The kits may further include instructions recorded in tangible form for the use of the components.

[0080] The disclosed method of assembly relies on stable and specific hybridization between the index sequence of the indexed splint molecule and the assembly component molecule. In contrast, prior art methods utilizing splints to direct assembly rely on stable and specific hybridization between the splint molecule and two assembly components. Without being bound by theory, it is believed that the fundamental distinction between the disclosed approach (FIGS. 5A-5C) and the prior art approaches is that in the disclosed methods, the desired assembly sequence is not used to assemble the pieces into position for ligation. The splint sequence in the disclosed method of assembly is designed to be too short for stable hybridization and thus not long enough to allow assembly-free index hybridization. The index sequence of the indexed splint molecule is not part of the construct sequence and therefore is not affected by homology between library members. This is an unexpected benefit and improvement over previous high-throughput methods of multiplex assembly. EXAMPLES

[0081] Aspects of the present teachings can be further understood in light of the following examples, which should not be construed in any way as limiting the scope of the present teachings.

[0082] [Example 1] The following experiments were designed and performed to evaluate the preparation of low homology libraries in solution using the oligonucleotide splint structures depicted in Figures 1, 2, and 3. Thus, there were three types of test splint molecules evaluated. The term "test splint molecule" refers to a single splint molecule in Figure 1. In Figures 2 and 3, "test splint molecule" refers to two oligonucleotide structures formed by hybridization of complementary index sequences that form an index element. The index elements in Figures 2 and 3 were identical and had a Tm in the range of 60°C to 75°C. The high Tm of the hybridized index effectively rendered the two nucleotide structures into a single molecule at temperatures below the Tm.

[0083] Construct sequences were obtained from a set of low homology genomic sequences, UTRs (untranslated regions). 140 UTRs were assembled. Due to the design of the splint molecules being tested, the correct construct was the UTR sequence that was ligated to the same UTR sequence. Thus, for 140 different UTRs, there were 140 different test splint molecules and all 140 possible correct constructs to assemble, as well as all 19,460 possible incorrect constructs. Typically, each of the 140 UTRs has two versions in the original oligo library: one version has a primer site at the 5' end and the other version has a primer site at the 3' end for PCR amplification and introduction of adapters required for MiSeq (Illumina).

[0084] Three ratios of test splint molecules to constructs (1:1, 2:1 and 4:1) were tested for each type of test splint molecule. For the test splint molecules of Figures 2 and 3 (both with index), a + / -3 nucleotide spacer (referred to as a support) was also tested. The 3 nucleotide support was located between the index and the splint portion of the test splint molecule. As a result, there were 15 sets assembled in this experiment. Each set had 140 correct UTR constructs that could be assembled from 420 different UTR sequences. The products from different sets were distinguishable by sequencing, whereby different sequences were printed with different molecular copy numbers for each subset in the production of DNA library material.

[0085] For each set, an oligo library was prepared from the UTR and test splint molecules. Approximately 80 ng of the library was solubilized (in a buffer suitable for both kinase and ligase reactions) and then subjected to a kinase reaction using T4 polynucleotide kinase (NEB) according to the manufacturer's recommendations to phosphorylate the 5' ends of the DNA oligos. The kinased products were hybridized by incubation in a water bath cooling from 93°C to 44°C for 1.75 hours to form the oligonucleotide structures depicted in Figures 1, 2 and 3. The hybridized products were then heated to the ligation temperature and 1 microliter (μl) of 9°N™ Ligase (New England Biolabs Inc., Ipswich, MA) was added. The ligation reaction was allowed to proceed for 1 hour. The ligated products were purified using 1.5×AMPure® XP beads (Beckman Coulter, Brea, CA) and amplified by PCR. PCR products were sequenced by NGS (next generation sequencing) to characterize the degree of correct assembly in each set.

[0086] result NGS read data is shown in FIG. 14. The data shows that all three types of test splint molecules (of the oligonucleotide structures depicted in FIG. 1, FIG. 2 or FIG. 3) worked in all 15 sets. The test splint molecules of FIG. 1 performed best. In particular, the set with a splint / construct molecule ratio of 4:1 performed best among the three ratios tested, with an average number of reads for correctly assembled constructs approaching 700. The data of FIG. 2 and FIG. 3 are similar to each other, with the FIG. 2 data showing a slightly higher average number of reads for correctly assembled constructs. The presence of the three nucleotide struts in the test splint molecules of FIG. 2 had little effect on the test splint molecules of FIG. 2. The presence of the three nucleotide struts in the test splint molecules of FIG. 3 resulted in a slightly reduced average number of reads compared to the data for the test splint molecules without struts.

[0087] [Example 2] The following experiment was designed and performed to test a different test splint molecule than that in Example 1. This experiment used a test splint molecule as illustrated in the oligonucleotide structure in Figure 5B. The test splint molecule is an indexed splint molecule, which is a single oligonucleotide sequence containing index sequences located at the 5' and 3' ends of the test splint molecule and flanking the splint region that binds to the two assembly components. The assembly components (including the assembly sequences that will be ligated to form the assembled construct) were prepared to further contain a reverse complement sequence to the index sequence of the test splint molecule, thereby allowing hybridization of the test splint molecule to the assembly components in two locations. For the left assembly component, the test splint molecule binds (1) at the splint element that binds to a specific sequence at the 3' end of the left assembly component, and (2) at the index sequence at the 5' end of the left assembly component (hybridizing to form the index element). For the right assembly component, the test splint molecule binds (1) at a splint element that binds to a specific sequence at the 5' end of the right assembly component, and (2) at an index sequence at the 5' end of the right assembly component (hybridizing to form a separate index element). Because the index element at the 3' end of the left assembly component has a different sequence than the index element at the 3' end of the right assembly component, there is no cross-hybridization between the two separate index elements. The index element at the 5' end of the left assembly component consists of 30 nucleotides and has a Tm in the range of 65-72°C, and the index element at the 3' end of the right assembly component consists of 30 nucleotides and has a Tm in the range of 65-72°C.

[0088] Construct sequences (2250 sequences) were derived from a set of low homology genomic sequences, UTRs (untranslated regions). The assembly sequences were 200 nucleotides in length. The assembly components included UTR construct sequences, index sequences, barcodes, and primer sequences. As illustrated in FIG. 15, the left assembly component (left) included, in a 5' to 3' orientation, an index sequence (to form index 1 when hybridized to the indexed splint), a primer sequence (primer F), a barcode (BC1), and an assembly sequence. The right assembly component (right) included, in a 5' to 3' orientation, an assembly sequence, a barcode (BC2), a primer sequence (primer R), and an index sequence (to form index 2 when hybridized to the indexed splint).

[0089] The test splint molecule contained, in a 5' to 3' orientation, an index sequence (which hybridizes to the right assembly component to form index 2 in the oligonucleotide structure), a spacer (dotted line), a splint sequence, another spacer (dotted line) and another index sequence (which hybridizes to form index 1 in the oligonucleotide structure). The splint sequence contains a 5' sequence element that hybridizes to the 5' terminal sequence of the assembly sequence of the right assembly component and a 3' sequence that hybridizes to the 3' terminal sequence of the assembly sequence of the right assembly component. Each of the 5' and 3' sequence elements of the splint is at least 12 nucleotides in length and each has a Tm of 28°C or greater (EQ.1: Tm=(2*(A 数値 +T 数値 ))+(4*(C 数値 +G 数値 ))-7).

[0090] Due to the design of the splint molecules utilized in the experiments, the correct construct was a UTR ligated to the same UTR, resulting in a 400 nucleotide multicomponent construct (containing no non-construct sequences). Each UTR assembly component was designed to further contain six unique barcode pairs. The correct construct is indicated by the left and right barcodes that are correctly paired in the assembled construct. Thus, there are six possible correct constructs (and 30 possible incorrect constructs) per UTR. Since 2250 different UTR sequences and six unique barcode pairs were examined, there are a total of 13,500 unique indexed splint molecules (2250 x 6) and 13,500 possible unique correct constructs.

[0091] An oligo library containing the UTR component assembly and indexed splint molecules was prepared. Approximately 80 ng of the library was solubilized and then subjected to a kinase reaction using T4 polynucleotide kinase (NEB) according to the manufacturer's recommendations to phosphorylate the 5' ends of the DNA oligos. The kinased products were hybridized by incubation in a water bath cooling from 93°C to 44°C for 1.75 hours. The hybridized products were then heated to a ligation temperature of 60.6°C and 1 microliter (μl) of 9°N™ Ligase (New England Biolabs Inc., Ipswich, MA) was added. The ligation reaction was allowed to proceed for 10 minutes. The ligated products were purified using 1.5×AMPure® XP beads (Beckman Coulter, Brea, CA) and amplified by PCR. The PCR products were sequenced by NGS (next generation sequencing) to characterize the degree of correct assembly.

[0092] result The data are shown in the table in Figure 15. Of the 13,500 possible correct constructs, 13,465 were detected by sequencing the library with fewer than 10 million reads. Based on the unique barcodes located at opposite ends of the assembly, this data indicates that 99.70% of the possible correct constructs were assembled in the reaction, but does not indicate full-length sequence accuracy of each sequence between the barcodes.

[0093] Thus, the Figure 5B oligonucleotide structure allowed for a significantly higher number of correct constructs, a number that is of great consequence for the preparation of oligonucleotide libraries.

[0094] [Example 3] The following experiments were designed and performed to examine assembly using the oligonucleotide structures of Figures 1, 2 and 5B. Experiments were designed to compare three types of oligonucleotide libraries: (a) composite libraries, (b) tiled libraries and (c) homologous libraries. The three types of libraries are depicted diagrammatically in Figure 16.

[0095] In the composite library of this experiment, the sequences have little sequence similarity except at the peripheral primer sites. The composite library (type a) had 600 distinct UTR sequences (i.e., a total of 600 possible correctly assembled constructs). The homologous library (type c) utilized 60 distinct UTR sequences, with each distinct UTR sequence having 10 uniquely barcoded constructs (a total of 600 possible correctly assembled constructs). In the tiled library of this experiment, each member has overlapping sequence identity with three other members. For the tiled library (type b), there were four tiled constructs (with 82-246 base pairs of overlapping sequence identity) for each of the 150 distinct UTR sequences (a total of 600 possible correctly assembled constructs). For each library, the assembled constructs were 400 bases long.

[0096] Each type of library was tested with each type of test splint molecule (see oligonucleotide structures in Figures 1, 2 and 5B), resulting in nine categories being tested as shown in Table 1.

[0097] [Table 1]

[0098] All nine categories were in the same library and assembled together, and the PCR products were sequenced by NGS (next-generation sequencing) to characterize the degree of correct assembly.

[0099] result The data are shown in Figure 17. Data for the total number of reads (correct and incorrect) are depicted in the top left graph. These data show that the indexed splints of Figures 1 and 2 in the homology library (type c) resulted in a majority of incorrectly assembled constructs. Percentage accuracy data (i.e., the total number of correct reads compared to the total number of reads) are depicted in the bottom left graph. All three types of indexed splint molecules produced high accuracy in the composite library (type a) and a moderate level of accuracy in the tiled library (type b). Only the splint molecule of Figure 5B had high accuracy for the homology library (type c). Median correct read data are depicted in the top right graph. Data for the number of dropout constructs are depicted in the bottom right graph. "Dropout constructs" refers to correct constructs for which no correct reads exist (there are 600 possible correct constructs per library). These data show that the splints indexed in Figure 1 and Figure 2 assemblies had dropout constructs for each type of library. For example, the Figure 1 indexed splint showed over 50 dropout constructs for the composite library (type a), over 60 dropout constructs for the tiled library (type b), and close to 70 dropout constructs for the homologous library (type c). There were no dropout constructs for any of the libraries in the Figure 5B indexed splint.

[0100] Two noteworthy aspects of the FIG. 5B data are observed in this data. First, the assembly method using the FIG. 5B indexed splint molecules was the only one that was able to assemble the c-type (composite library) library type with a useful level of accuracy (see bottom left graph). Second, the assembly method using the FIG. 5B indexed splint molecules had no dropouts (see bottom right graph). All 600 of the possible correctly assembled constructs were assembled in the FIG. 5B assembly. In contrast, the other two approaches had 50-80 dropouts, i.e., constructs that were not built in the 600 possible correct constructs.

[0101] These data indicate an effect on correct assembly using the indexed splint molecules depicted in Figure 5B and demonstrate correct assembly of constructs that would otherwise not be possible due to random hybridization of the splint molecules.

[0102] [Example 4] The following experiments were designed and performed to determine the effect of two experimental variables on the assembly of constructs utilizing indexed splint molecules as shown in FIG. 5B.

[0103] The construct sequences were obtained from a set of low homology genomic sequences, UTRs (untranslated regions). Melting temperatures (Tm = (2*(A 数値 +T 数値 ))+(4*(C 数値 +G 数値Sixteen variable lengths were designed for each splinted sequence half, organized by Tm) calculated by ΔTm−7. Temperature thresholds of 14°C to 44°C at 2°C were prepared for the splint halves. Forty UTR-specific sequences were assembled for each splint design (16 x 40 = 640 UTR sequences in total). Ten unique barcodes were used for each UTR sequence (6,400 possible correct constructs in total). A "correct" pair was called if the barcodes were correctly paired in the assembled construct (as measured by NGS). The assembly components further included sequences for a forward primer (upstream assembly component) and a reverse primer (downstream assembly component) to allow PCR amplification. Libraries were assembled at eight different ligation temperatures. Figure 18 depicts a schematic of the Figure 5B test splint molecule of this experiment.

[0104] Oligo libraries were prepared from the UTR assembly components and various test splint molecules. Approximately 80 ng of the library was solubilized (in a buffer suitable for both kinase and ligase reactions) and then subjected to a kinase reaction using T4 polynucleotide kinase (NEB) according to the manufacturer's recommendations to phosphorylate the 5' ends of the DNA oligos. The kinased products were hybridized by incubation in a water bath cooling from 93°C to 44°C for 1.75 hours to form the oligonucleotide structures depicted in Figure 5B. The hybridized products were then heated to one of the ligation temperatures being tested and 1 microliter (μl) of 9°N™ Ligase (New England Biolabs Inc., Ipswich, MA) was added. The ligation reaction was allowed to proceed for 1 hour. The ligated products were purified using 1.5x AMPure® XP beads (Beckman Coulter, Brea, CA) and amplified by PCR. PCR products were sequenced by NGS (next generation sequencing) to characterize the extent of correct assembly.

[0105] result The data generally demonstrate that the specificity of assembly increases at higher ligation temperatures. The data also generally demonstrate that the efficiency of assembly constructs with shortened splint lengths (lower Tm) decreases at higher ligation temperatures. Representative data are depicted in the graph in Figure 18. The data illustrate the high degree of correct assembly possible using the splint molecules indexed in Figure 5B across a range of Tm for the two splint segments.

[0106] Exemplary embodiments product Embodiment 1. An oligonucleotide structure for assembling two nucleic acid components to produce a multi-component ligation product, comprising a first single-stranded oligonucleotide, a second single-stranded oligonucleotide, and an indexed splint, wherein the first single-stranded oligonucleotide and the second single-stranded oligonucleotide are partially hybridized to the indexed splint. (a) an indexed splint includes a splint array and a first index array and a second index array that sandwich the splint array; (b) a splint sequence hybridizes to both the 3' end of the first single-stranded oligonucleotide and the 5' end of the second single-stranded oligonucleotide; (c) a first index sequence is hybridized to the 5' end of a first single-stranded oligonucleotide to form a first double-stranded index element, and a second index sequence is hybridized to the 3' end of a second single-stranded oligonucleotide to form a second double-stranded index element; 2. The oligonucleotide structure of embodiment 1. Embodiment 3. A splint array of indexed splints comprising: (a) a 5' element that hybridizes to (is the reverse complement of) the 5' end of a second single-stranded oligonucleotide; (b) a 3' element that hybridizes to (is the reverse complement of) the 3' end of the first single-stranded oligonucleotide; and the 5' and 3' elements of the splint sequence are contiguous, i.e., there are no intervening nucleotides. Embodiment 4. Each of the 5' hybridized splint element of (a) and the 3' hybridized splint element of (b) has a melting temperature (Tm) of about 14° C. to about 44° C.; Tm = (2*(A 数値 +T 数値 ))+(4*(C 数値 +G 数値 4. The oligonucleotide structure of embodiment 3, wherein: (1) a first index sequence is located at the 3' end of the indexed splint; (2) a second index sequence is located at the 5' end of the indexed sequence; 5. The oligonucleotide structure according to any one of embodiments 2, 3 and 4. (a) a first single-stranded oligonucleotide comprising, in a 5' to 3' direction: (1) a third index sequence comprising the 5'-terminal sequence of the first single-stranded oligonucleotide (the left assembly component); (2) a first DNA sequence component that is ligated into the multicomponent ligation product, the first DNA sequence component comprising a 3' terminal sequence of a first single-stranded oligonucleotide (left assembly component); (b) a second single-stranded oligonucleotide, in a 5' to 3' orientation: (1) a second DNA sequence component to be ligated to the multicomponent ligation product comprising a 5' terminal sequence of a second single-stranded oligonucleotide; (2) a fourth index sequence comprising the 3'-terminal sequence of the second single-stranded oligonucleotide; (c) The indexed splint is oriented 5' to 3'. (1) a 5'-terminal sequence which is a second index sequence; (2) an optional first spacer sequence; and (3) A splint arrangement, the 5' element of the splint sequence hybridizes to (is the reverse complement of) the 5' terminal sequence of a second single-stranded oligonucleotide; a splint sequence, the 3' element of which hybridizes to (is the reverse complement of) the 3' terminal sequence of the first single-stranded oligonucleotide; (4) an optional second spacer sequence; and (5) a 3'-terminal sequence which is a first index sequence; 6. The oligonucleotide structure of embodiment 5. Embodiment 7. The oligonucleotide structure of embodiment 6, wherein each spacer sequence independently consists of 1 to 30 nucleotides. (1) a first index sequence is located at the 5' end of the indexed splint; (2) a second index sequence is located at the 3' end of the indexed sequence; 5. The oligonucleotide structure according to any one of embodiments 2, 3 and 4. Embodiment 9. (a) a first single-stranded oligonucleotide comprising, in a 5' to 3' direction: (1) a third index sequence comprising the 5'-terminal sequence of the first single-stranded oligonucleotide; (2) a first DNA sequence component that is ligated into the multicomponent ligation product, the first DNA sequence component comprising a 3' terminal sequence of a first single-stranded oligonucleotide (left assembly component); (b) a second single-stranded oligonucleotide, in a 5' to 3' orientation: (1) a second DNA sequence component to be ligated to the multicomponent ligation product comprising a 5' terminal sequence of a second single-stranded oligonucleotide; (2) a fourth index sequence comprising the 3'-terminal sequence of the second single-stranded oligonucleotide; (c) The indexed splint is oriented 5' to 3'. (1) a 5'-end sequence which is a first index sequence; (2) an optional first spacer sequence; and (3) A splint arrangement, the 5' element of the splint sequence hybridizes to (is the reverse complement of) the 5' terminal sequence of a second single-stranded oligonucleotide; a splint sequence, the 3' element of which hybridizes to (is the reverse complement of) the 3' terminal sequence of a first single-stranded oligonucleotide (the left assembly component); (4) an optional second spacer sequence; and (5) a 3'-terminal sequence which is a second index sequence; 9. The oligonucleotide structure of embodiment 8. Embodiment 10. The oligonucleotide structure of embodiment 8 or 9, wherein each spacer sequence independently consists of 1 to 30 nucleotides. Embodiment 11. The oligonucleotide structure of any one of embodiments 2 to 10, wherein each double-stranded index element comprises 10 to 40 base pairs. Embodiment 12. Each double-stranded index element has a melting temperature (Tm) of about 10° C. to about 80° C. Tm = (2*(A 数値 +T 数値 ))+(4*(C 数値 +G 数値 ))-7, 12. The oligonucleotide structure according to any one of embodiments 2 to 11. Embodiment 13. The oligonucleotide structure of any one of embodiments 6, 7, 9, 10 and 11, wherein the fourth index sequence (segment F) is removably (or detachably) linked to the second DNA sequence component. Embodiment 14. The oligonucleotide structure of embodiment 13, wherein the second single-stranded oligonucleotide further comprises a type II restriction enzyme site 3' to the second DNA sequence component, the restriction enzyme site for cleavage being directly 3' to the second DNA sequence component. Embodiment 15. The oligonucleotide structure of embodiment 13, wherein the second single-stranded oligonucleotide is removably (or detachably) linked to the second DNA sequence component by a cleavable linker, and the site of cleavage is directly 3' to the second DNA sequence component. Embodiment 16. The oligonucleotide structure of any one of embodiments 2 to 15, further comprising at least one blocking oligonucleotide component that hybridizes to (is the reverse complement of) a sequence segment of Segment D or Segment E, the sequence segment excluding the sequence that hybridizes to the splint. Embodiment 17. A set or library of oligonucleotide structures according to any one of embodiments 1 to 16. Embodiment 18. A kit comprising the set or library of embodiment 17. Embodiment 19 The kit of embodiment 18, further comprising a DNA ligase.

[0107] method Embodiment 20. A method for assembling two nucleic acid components to produce a multi-component ligation product, comprising: (A) hybridizing a first single-stranded oligonucleotide comprising a first DNA sequence component and a second single-stranded oligonucleotide comprising a second DNA sequence component to an indexed splint, wherein both the 5' and 3' ends of the first single-stranded oligonucleotide are hybridized to the indexed splint, and both the 5' and 3' ends of the second single-stranded oligonucleotide are hybridized to the indexed splint; (B) ligating the 3' end of the first single-stranded oligonucleotide to the 5' end of the second single-stranded oligonucleotide, thereby producing a first multi-component ligation product. Embodiment 21. (a) An indexed splint further includes a splint array, and a first index array and a second index array that sandwich the splint array; (b) a splint sequence hybridizes to both the 3' end of the first single-stranded oligonucleotide and the 5' end of the second single-stranded oligonucleotide; 21. The method of embodiment 20, wherein (c) a first index sequence hybridizes to a 5' end of a first single-stranded oligonucleotide to form a first double-stranded index element, and a second index sequence hybridizes to a 3' end of a second single-stranded oligonucleotide to form a second double-stranded index element. 22. A splint array of indexed splints, (a) a 5' element that hybridizes to the 5' end of a second single-stranded oligonucleotide to form a double-stranded 5' splint element; (b) a 3' element that hybridizes to the 3' end of the first single-stranded oligonucleotide to form a double-stranded 3' splint element; and the 5' and 3' elements of the splint sequence are contiguous, i.e., there are no intervening nucleotides. Embodiment 23. The double-stranded 5' splint element and the double-stranded 3' splint element each have a melting temperature (Tm) of about 10°C to about 80°C, Tm = (2*(A 数値 +T 数値 ))+(4*(C 数値 +G 数値 ))-7, 23. The method of embodiment 22. (1) a first index sequence is located at the 3' end of the indexed splint; (2) a second index sequence is located at the 5' end of the indexed splint; The method of embodiment 21, 22 or 23. Embodiment 25. (a) a first single-stranded oligonucleotide comprising, in a 5' to 3' direction: (1) a third index sequence comprising the 5'-terminal sequence of the first single-stranded oligonucleotide; (2) a first DNA sequence component that is ligated into the multicomponent ligation product, the first DNA sequence component comprising a 3' terminal sequence of the first single-stranded oligonucleotide; (b) a second single-stranded oligonucleotide, in a 5' to 3' orientation: (1) a second DNA sequence component to be ligated to the multicomponent ligation product comprising a 5' terminal sequence of a second single-stranded oligonucleotide; (2) a fourth index sequence comprising the 3'-terminal sequence of the second single-stranded oligonucleotide; (c) The indexed splint is oriented 5' to 3'. (1) a 5'-terminal sequence which is a second index sequence; (2) an optional first spacer sequence; and (3) A splint arrangement, the 5' element of the splint sequence hybridizes to the 5' terminal sequence of a second single-stranded oligonucleotide; a splint sequence, the 3' element of which hybridizes to the 3' terminal sequence of the first single-stranded oligonucleotide; (4) an optional second spacer sequence; and (5) a 3'-terminal sequence which is a first index sequence; 25. The method of embodiment 24. Embodiment 26. The method of embodiment 25, wherein each spacer sequence independently consists of 1 to 30 nucleotides. (1) a first index sequence is located at the 5' end of the indexed splint; (2) a second index sequence is located at the 3' end of the indexed splint; The method of embodiment 21, 22 or 23.

[0036] Embodiment 28. (a) a first single-stranded oligonucleotide comprising, in a 5' to 3' direction: (1) a third index sequence comprising the 5'-terminal sequence of the first single-stranded oligonucleotide; (2) a first DNA sequence component that is ligated into the multicomponent ligation product, the first DNA sequence component comprising a 3' terminal sequence of the first single-stranded oligonucleotide; (b) a second single-stranded oligonucleotide, in a 5' to 3' orientation: (1) a second DNA sequence component to be ligated to the multicomponent ligation product comprising a 5' terminal sequence of a second single-stranded oligonucleotide; (2) a fourth index sequence comprising the 3'-terminal sequence of the second single-stranded oligonucleotide; (c) The indexed splint is oriented 5' to 3'. (1) a 5'-end sequence which is a first index sequence; (2) an optional first spacer sequence; and (3) A splint arrangement, the 5' element of the splint sequence hybridizes to the 5' terminal sequence of a second single-stranded oligonucleotide; a splint sequence, the 3' element of which hybridizes to the 3' terminal sequence of the first single-stranded oligonucleotide; (4) an optional second spacer sequence; and (5) a 3'-terminal sequence which is a second index sequence; 28. The method of embodiment 27. Embodiment 29. The method of embodiment 27 or 28, wherein each spacer sequence independently consists of 1 to 30 nucleotides. Embodiment 30. The method of any one of embodiments 21 to 29, wherein each double-stranded index element comprises 10 to 40 base pairs. Embodiment 31. Each double-stranded index element has a melting temperature (Tm) of about 10° C. to about 80° C. Tm = (2*(A 数値 +T 数値 ))+(4*(C 数値 +G 数値 ))-7, The method according to any one of embodiments 21 to 30. (C) denaturing an indexed splint from a multi-component ligation product; thereafter, repeating steps (A), (B) and (C); 32. The method of any one of embodiments 20 to 31, further comprising: Embodiment 33 The method of any one of embodiments 20 to 32, wherein the fourth index sequence is removably (or detachably) linked to the second DNA sequence component. Embodiment 34. The method of embodiment 33, wherein the second single-stranded oligonucleotide further comprises a type IIs restriction enzyme site 3' to the second DNA sequence component, and the site of restriction enzyme cleavage is directly 3' to the second DNA sequence component. Embodiment 35. The method of embodiment 33, wherein the fourth index sequence is linked to the second DNA sequence component by a cleavable (or removable) linker, and the site of cleavage is directly 3' to the second DNA sequence component. Embodiment 36. Removing a fourth index sequence from a second DNA component in a first ligation product; providing a further second single stranded oligonucleotide having a fourth index sequence and a further indexed splint comprising a second splint sequence; thereafter, performing steps (A), (B) and (C); wherein the first ligation product is the first single-stranded oligonucleotide of step (A); The method of any one of embodiments 33 to 35, wherein the second splint sequence of the further indexed splint contains a 5' element that hybridizes to the 5' terminal sequence in the further second single-stranded oligonucleotide and a 3' element that hybridizes to the 3' terminal sequence of the first ligation product. Embodiment 37. The method of any one of embodiments 33 to 35, wherein the indexed splint further comprises a second splint sequence adjacent to the fifth index sequence, and the 5' element of the second splint sequence is capable of hybridizing to the 3' terminal sequence of the second DNA sequence component. Embodiment 38 The method of any one of embodiments 20 to 32, wherein the third index sequence is removably (or detachably) linked to the first DNA sequence component. Embodiment 39. The method of embodiment 38, wherein the first single-stranded oligonucleotide further comprises a type IIs restriction enzyme site 5' to the first DNA sequence component, and the site of restriction enzyme cleavage is directly 5' to the first DNA sequence component. Embodiment 40. The method of embodiment 38, wherein the third index sequence is linked to the first DNA sequence component by a cleavable (or removable) linker, and the site of cleavage is directly 5' to the first DNA sequence component. Embodiment 41. A method for the preparation of a first ligation product comprising the steps of: removing a third index sequence from a first DNA sequence component in the first ligation product; providing a further first single stranded oligonucleotide having a third index sequence and a further indexed splint; thereafter, performing steps (A), (B) and (C); wherein the first ligation product is the second single-stranded oligonucleotide of step (A); The method of any one of embodiments 38 to 40, wherein the splint sequence of the further indexed splint contains a 3' element that hybridizes to the 3' terminal sequence in the further first single-stranded oligonucleotide and a 5' element that hybridizes to (is the reverse complement to) the 5' terminal sequence in the first ligation product. Embodiment 42. The hybridizing step is carried out at a range of temperatures based on the melting temperatures (Tm) of the index elements; 42. The method of any one of embodiments 20 to 41, wherein the ligation step is performed at a temperature higher than the melting temperature (Tm) of the 5' and 3' elements of the splint sequence and lower than the melting temperature (Tm) of the index element. Embodiment 43. The method of any one of embodiments 20 to 42, wherein the temperature of the ligation step is about 45° to 70° C. and the ligase is thermostable. Embodiment 44. The method of any one of embodiments 20 to 43, wherein the melting temperature of the index element is between about 60° and 90° C. Embodiment 45. The method of any one of embodiments 20 to 44, wherein the melting temperature of the 5' splint element and the 3' splint element is between about 20° and 42° C.

[0108] The above description of exemplary or preferred embodiments should be construed as illustrative rather than limiting of the present disclosure as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present disclosure as set forth in the claims. Such variations are not considered as a departure from the scope of the present disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated herein by reference in their entirety.

Claims

**Claim 1** A method for assembling two nucleic acid components to produce a multi-component ligation product, comprising: (A) hybridizing a first single-stranded oligonucleotide comprising a first DNA sequence component and a second single-stranded oligonucleotide comprising a second DNA sequence component to a spritzed index, wherein both the 5' end and the 3' end of the first single-stranded oligonucleotide are hybridized to the spritzed index, and both the 5' end and the 3' end of the second single-stranded oligonucleotide are hybridized to the spritzed index; and (B) ligating the 3' end of the first single-stranded oligonucleotide to the 5' end of the second single-stranded oligonucleotide, thereby producing a first multi-component ligation product. A method as described above. **Claim 2** (a) The spritzed index further comprises a spritzed sequence and a first index sequence and a second index sequence flanking the spritzed sequence; (b) the spritzed sequence hybridizes to both the 3' end of the first single-stranded oligonucleotide and the 5' end of the second single-stranded oligonucleotide; (c) the first index sequence hybridizes to the 5' end of the first single-stranded oligonucleotide to form a first double-stranded index element, and the second index sequence hybridizes to the 3' end of the second single-stranded oligonucleotide to form a second double-stranded index element. The method according to claim 1. **Claim 3** The spritzed sequence of the spritzed index comprises: (a) a 5' element that hybridizes to the 5' end of the second single-stranded oligonucleotide to form a double-stranded 5' spritzed element; and (b) a 3' element that hybridizes to the 3' end of the first single-stranded oligonucleotide to form a double-stranded 3' spritzed element, and the 5' element and the 3' element of the spritzed sequence are in contact, i.e., there are no intervening nucleotides. The method according to claim 2. **Claim 4** (1) The first index sequence is located at the 3' end of the spritzed index. ​ (2) The second index array is located at the 5' end of the indexed sprint. The method according to claim 2 or 3.

5. (a) The first single-stranded oligonucleotide is in the 5' to 3' direction. (1) A third index array including the 5' terminal sequence of the first single-stranded oligonucleotide. (2) The first DNA sequence component ligated to the multi-component ligation product, the first DNA sequence component including the 3' terminal sequence of the first single-stranded oligonucleotide. and includes (b) The second single-stranded oligonucleotide is in the 5' to 3' direction. (1) The second DNA sequence component ligated to the multi-component ligation product, including the 5' terminal sequence of the second single-stranded oligonucleotide. (2) A fourth index array including the 3' terminal sequence of the second single-stranded oligonucleotide. and includes (c) The indexed sprint is in the 5' to 3' direction. (1) The 5' terminal sequence which is the second index array. (2) An optional first spacer sequence. (3) The sprint sequence, where the 5' element of the sprint sequence hybridizes to the 5' terminal sequence of the second single-stranded oligonucleotide. the 3' element of the sprint sequence hybridizes to the 3' terminal sequence of the first single-stranded oligonucleotide, the sprint sequence. (4) An optional second spacer sequence. (5) The 3' terminal sequence which is the first index array. and includes The method according to claim 4.

6. The method according to claim 5, wherein each spacer sequence independently consists of 1 to 30 nucleotides.

7. (1) The first index array is located at the 5' end of the indexed sprint. (2) The second index array is located at the 3' end of the indexed sprint. The method according to claim 2 or 3.

8. (a) The first single-stranded oligonucleotide is in the 5' to 3' direction. (1) A third index array including the 5' terminal sequence of the first single-stranded oligonucleotide. (2) A first DNA sequence component that is ligated to the multi-component ligation product, the first DNA sequence component including the 3'-terminal sequence of the first single-stranded oligonucleotide, and including (b) the second single-stranded oligonucleotide, in the 5' to 3' direction, (1) A second DNA sequence component that is ligated to the multi-component ligation product including the 5'-terminal sequence of the second single-stranded oligonucleotide, and (2) A fourth index sequence including the 3'-terminal sequence of the second single-stranded oligonucleotide including (c) the indexed sprint, in the 5' to 3' direction, (1) the 5'-terminal sequence that is the first index sequence, and (2) an optional first spacer sequence, and (3) the sprint sequence, where the 5' element of the sprint sequence hybridizes to the 5'-terminal sequence of the second single-stranded oligonucleotide, the 3' element of the sprint sequence hybridizes to the 3'-terminal sequence of the first single-stranded oligonucleotide, the sprint sequence, and (4) an optional second spacer sequence, and (5) the 3'-terminal sequence that is the second index sequence including The method according to claim 7.

9. The method according to claim 7, wherein each spacer sequence independently consists of 1 to 30 nucleotides.

10. The method according to claim 2, wherein each double-stranded index element includes 10 to 40 base pairs.

11. (C) a step of denaturing the indexed sprint from the multi-component ligation product, and then, a step of repeatedly performing steps (A), (B), and (C) further including the method according to claim 1.

12. The method according to claim 5, wherein the fourth index sequence is removably linked to the second DNA sequence component.

13. The method according to claim 12, wherein the second single-stranded oligonucleotide further includes an IIS-type restriction enzyme site that is 3' to the second DNA sequence component, and the site of restriction enzyme cleavage is directly 3' to the second DNA sequence component.

14. The method according to claim 12, wherein the fourth index array is linked to the second DNA sequence component by a cleavable linker, and the cleavage site is directly 3' of the second DNA sequence component.

15. Removing the fourth index array from the second DNA component in the first ligation product; Preparing another second single-stranded oligonucleotide having the fourth index array and another sprint with an additional index including a second sprint array; Then, performing steps (A), (B), and (C); further comprising: wherein the first ligation product is the first single-stranded oligonucleotide of step (A); The method according to claim 12, wherein the second sprint array of the further indexed sprint contains a 5' element that hybridizes to the 5' terminal sequence in the further second single-stranded oligonucleotide and a 3' element that hybridizes to the 3' terminal sequence of the first ligation product.

16. The method according to claim 12, wherein the indexed sprint further includes a second sprint array adjacent to the fifth index array, and the 5' element of the second sprint array can hybridize to the 3' terminal sequence of the second DNA sequence component.

17. The method according to claim 5, wherein the third index array is removably linked to the first DNA sequence component.

18. The method according to claim 17, wherein the first single-stranded oligonucleotide further includes an IIS-type restriction enzyme site 5' to the first DNA sequence component, and the restriction enzyme cleavage site is directly 5' of the first DNA sequence component.

19. The method according to claim 17, wherein the third index array is linked to the first DNA sequence component by a cleavable linker, and the cleavage site is directly 5' of the first DNA sequence component.

20. Removing the third index array from the first DNA component in the first ligation product; The step of preparing yet another first single-stranded oligonucleotide having the third index array, and a splint with yet another index; Then, the step of performing steps (A), (B) and (C); further comprising; the first ligation product is the second single-stranded oligonucleotide of step (A); the splint sequence of the splint with the yet another index contains a 3' element that hybridizes to the 3' terminal sequence in the yet another first single-stranded oligonucleotide, and a 5' element that hybridizes to the 5' terminal sequence in the first ligation product, the method according to claim 17. **Claim 21** the hybridizing step is performed in a temperature range based on the melting temperature (Tm) of the index element; the ligating step is performed at a temperature higher than the melting temperature (Tm) of the 5' and 3' elements of the splint sequence and lower than the melting temperature (Tm) of the index element, the method according to claim 1. **Claim 22** the temperature of the ligation step is about 45° to 70°C and the ligase is thermostable, the method according to claim 1. **Claim 23** the melting temperature of the index element is about 60° to 90°C, the method according to claim 1. **Claim 24** the melting temperatures of the 5' splint element and the 3' splint element are about 20° to 42°C, the method according to claim 1. **Claim 25** An oligonucleotide structure for assembling two nucleic acid components to produce a multi-component ligation product, comprising a first single-stranded oligonucleotide, a second single-stranded oligonucleotide, and an indexed splint, wherein the first single-stranded oligonucleotide and the second single-stranded oligonucleotide are partially hybridized to the indexed splint. **Claim 26** A set or library of oligonucleotide structures, each having the oligonucleotide structure according to claim 25. **Claim 27** A kit comprising the set or library according to claim 25. **Claim 28** The kit according to claim 27, further comprising DNA ligase.

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