# US 2019/0169602 A1 · Compositions and methods for sequencing nucleic acids

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- Number: US 2019/0169602 A1
- Document ID: US20190169602A1
- Status: Published application
- Kind: A1
- Application number: 16/069,603
- Filed: 2017-01-12
- Published: 2019-06-06
- Earliest claimed: 2016-01-12
- Assignees: SeqWell, Inc.
- Applicants: seqWell, Inc.
- Inventors: Joseph C. MELLOR; Jack T. LEONARD
- Web page: https://patentgrep.com/patent/US20190169602A1

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12Q 1/68: involving nucleic acids
- C12Q 1/6869: Methods for sequencing
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

The invention provides compositions, including tethered synaptic complexes (TSCs), artificial nucleic acids, molecular constructs that include artificial nucleic acids bound to transposases, and kits; as well as methods of using the same, for example, for preparation of nucleic acid libraries and sequencing.

## Claims

### Claim 1

A tethered synaptic complex (TSC) comprising:
a first artificial nucleic acid comprising a first end comprising a first transposase binding site (TBS), a second end comprising a second TBS, and a linking segment disposed between the first TBS and the second TBS;
a second artificial nucleic acid comprising a first end comprising a first TBS;
a third artificial nucleic acid comprising a first end comprising a first TBS;
a first synaptic complex comprising a first pair of oligomerized transposases, the first pair comprising a first transposase and a second transposase, wherein the first transposase is bound to the first TBS of the first artificial nucleic acid, and the second transposase is bound to the first TBS of the second artificial nucleic acid; and
a second synaptic complex comprising a second pair of oligomerized transposases, the second pair comprising a third transposase and a fourth transposase, wherein the third transposase is bound to the second TBS of the first artificial nucleic acid, and the fourth transposase is bound to the first TBS of the third artificial nucleic acid.

### Claim 2 (depends on 1)

The TSC of claim 1, wherein the linking segment comprises a nucleic acid.

### Claim 3 (depends on 2)

The TSC of claim 2, wherein the nucleic acid is at least partially single-stranded.

### Claim 4 (depends on 2)

The TSC of claim 2, wherein the nucleic acid is double-stranded.

### Claim 5 (depends on 1, 2, 3, 4)

The TSC of any one of claims 1-4, wherein the linking segment comprises terminal nucleotides that form phosphodiester bonds with the first TBS and the second TBS.

### Claim 6 (depends on 1, 2, 3, 4)

The TSC of any one of claims 1-4, wherein the linking segment comprises an affinity binding pair or a covalent bond resulting from a conjugation reaction that does not form a phosphodiester bond.

### Claim 7 (depends on 6)

The TSC of claim 6, wherein the affinity binding pair comprises biotin-streptavidin, biotin-avidin, ligand-receptor, antigen-antibody or antigen binding fragment, hapten-anti-hapten, or immunoglobulin (Ig) binding protein-Ig.

### Claim 8 (depends on 7)

The TSC of claim 7, wherein the affinity binding pair comprises biotin-streptavidin or biotin-avidin.

### Claim 9 (depends on 8)

The TSC of claim 8, wherein the streptavidin or avidin binds only one or two biotin molecules.

### Claim 10 (depends on 6, 7, 8, 9)

The TSC of any one of claims 6-9, wherein the affinity binding pair comprises a first affinity component that binds to two second affinity components, where one second affinity component is linked to the first end of the first artificial nucleic acid, and the other second affinity component is linked to the second end of the first artificial nucleic acid, and wherein the two second affinity reagents do not interfere with binding of transposases to the first and second TBSs of the first artificial nucleic acid.

### Claim 11 (depends on 6, 7, 8, 9)

The TSC of any one of claims 6-9, wherein the affinity binding pair comprises a first affinity component that binds a second affinity component, where the first affinity component is linked to the first end of the first artificial nucleic acid, and the second affinity component is linked to the second end of the first artificial nucleic acid.

### Claim 12 (depends on 6)

The TSC of claim 6, wherein the conjugation reaction is selected from the group consisting of a cycloaddition, amide or thioamide bond formation, a pericyclic reaction, a Diels-Alder reaction, sulfonamide bond formation, alcohol or phenol alkylation, a condensation reaction, disulfide bond formation, and a nucleophilic substitution.

### Claim 13 (depends on 12)

The TSC of claim 12, wherein the cycloaddition is an azide-alkyne Huisgen cycloaddition.

### Claim 14 (depends on 13)

The TSC of claim 13, wherein the azide-alkyne Huisgen cycloaddition is a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or a strain-promoted azide-alkyne cycloaddition (SPAAC).

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The TSC of any one of claims 1-14, wherein the linking segment further comprises one or more additional elements selected from the group consisting of an identifiable sequence tag (IST), a primer binding site, a cleavage site, and a chemical modification.

### Claim 16 (depends on 15)

The TSC of claim 15, wherein the one or more additional elements is an IST.

### Claim 17 (depends on 15, 16)

The TSC of claim 15 or 16, wherein the IST is a random IST, a semi-random IST, or a non-random IST.

### Claim 18 (depends on 15)

The TSC of claim 15, wherein the cleavage site is a restriction endonuclease recognition site or a nickase site.

### Claim 19 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The TSC of any one of claims 2-18, wherein the first artificial nucleic acid is about 50 to about 500 base pairs (bp) long.

### Claim 20 (depends on 19)

The TSC of claim 19, wherein the first artificial nucleic acid is about 100 to about 250 bp long.

### Claim 21 (depends on 20)

The TSC of claim 20, wherein the first artificial nucleic acid is about 150 to about 200 bp long.

### Claim 22 (depends on 21)

The TSC of claim 21, wherein the first artificial nucleic acid is about 175 bp long.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The TSC of any one of claims 1-22, wherein the transposases of the first pair are a different type than the transposases of the second pair.

### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The TSC of any one of claims 1-23, wherein the transposases of the first pair and/or the second pair are Tn3, Tn5, Tn9, Tn10, gamma-delta, Mu, piggyBac, Minos, Tc1, or Sleeping Beauty transposases or biologically active variants thereof.

### Claim 25 (depends on 24)

The TSC of claim 24, wherein the transposases of the first pair and/or the second pair are Tn3, Tn5, Tn9, Tn10, gamma-delta, or Mu transposases or biologically active variants thereof.

### Claim 26 (depends on 24, 25)

The TSC of claim 24 or 25, wherein the transposases of the first pair and/or the second pair are Tn5 or Mu transposases or biologically active variants thereof.

### Claim 27 (depends on 24, 25, 26)

The TSC of any one of claims 24-26, wherein the transposases of the first pair are Tn5 transposases, and the transposases of the second pair are Mu transposases.

### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

The TSC of any one of claims 1-27, wherein at least one transposase of the first pair and/or the second pair is operably linked to a targeting moiety.

### Claim 29 (depends on 28)

The TSC of claim 28, wherein the targeting moiety is a polypeptide comprising a DNA-binding domain (DBD) or an RNA-guided endonuclease.

### Claim 30 (depends on 29)

The TSC of claim 29, wherein the DBD is a zinc finger motif or a transcription activator-like (TAL) effector.

### Claim 31 (depends on 29)

The TSC of claim 29, wherein the RNA-guided endonuclease is Cas9, Cpf1, C2c2, or a biologically active variant thereof.

### Claim 32 (depends on 31)

The TSC of claim 31, wherein the biologically active variant is a nuclease-deficient variant.

### Claim 33 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)

The TSC of any one of claims 1-32, wherein the second artificial nucleic acid or the third artificial nucleic acid further comprises a second end, wherein the second end is a ligatable end.

### Claim 34 (depends on 33)

The TSC of claim 33, wherein the ligatable end is a sticky end.

### Claim 35 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)

The TSC of any one of claims 1-32, wherein the second artificial nucleic acid or the third artificial nucleic acid further comprises a component of a second affinity binding pair or a conjugating moiety.

### Claim 36 (depends on 35)

The TSC of claim 35, wherein the conjugating moiety is selected from the group consisting of a 1,3-diene, an alkene, an alkylamino, an alkyl halide, an alkyl pseudohalide, an alkyne, an amino, an anilido, an aryl, an azide, an aziridine, a carboxyl, a carbonyl, an episulfide, an epoxide, a heterocycle, an organic alcohol, an isocyanate group, a maleimide, a succinimidyl ester, a sulfosuccinimidyl ester, a sulfhydryl, a thiol, and a thioisocyanate group.

### Claim 37 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)

The TSC of any one of claims 1-32, wherein the second artificial nucleic acid further comprises a second end comprising a second TBS, and a linking segment disposed between the first TBS and the second TBS of the second artificial nucleic acid.

### Claim 38 (depends on 37)

The TSC of claim 37, wherein the linking segment of the second artificial nucleic acid comprises a second affinity binding pair or a covalent bond resulting from a conjugation reaction.

### Claim 39 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 37)

The TSC of any one of claims 1-32, 37, or 38, wherein the third artificial nucleic acid further comprises a second end comprising a second TBS, and a linking segment disposed between the first TBS and the second TBS of the third artificial nucleic acid.

### Claim 40 (depends on 39)

The TSC of claim 39, wherein the linking segment of the third artificial nucleic acid comprises a third affinity binding pair or a covalent bond resulting from a conjugation reaction.

### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)

The TSC of any one of claims 1-32, or 37-40, further comprising:
one or more additional synaptic complexes, each additional synaptic complex comprising a pair of oligomerized transposases, and/or
one or more additional artificial nucleic acids, each additional artificial nucleic acid comprising a TBS at each end and an intervening linking segment,
wherein each of the first synaptic complex, the second synaptic complex, and the one or more additional synaptic complexes is tethered to at least one other synaptic complex of the TSC by binding to TBSs at either end of the same artificial nucleic acid.

### Claim 42 (depends on 41)

The TSC of claim 41, wherein the TSC comprises between one and ten thousand additional synaptic complexes.

### Claim 43 (depends on 41, 42)

The TSC of claim 41 or 42, wherein each artificial nucleic acid of the TSC comprises an IST.

### Claim 44 (depends on 43)

The TSC of claim 43, wherein each IST is identical.

### Claim 45 (depends on 43)

The TSC of claim 43, wherein each IST is not identical.

### Claim 46 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)

The TSC of any one of claims 1-45, wherein the linking segment of the first artificial nucleic acid is soluble in an aqueous solution.

### Claim 47 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)

The TSC of any one of claims 1-46, wherein the linking segment of the first artificial nucleic acid has a mass of less than 1 femtogram.

### Claim 48

An artificial nucleic acid comprising a first end comprising a first transposase binding site (TBS), a second end comprising a second TBS, and a linking segment disposed between the first TBS and the second TBS, wherein upon binding of a first transposase to the first TBS and a second transposase to the second TBS, the first transposase does not oligomerize with the second transposase.

### Claim 49 (depends on 48)

The nucleic acid of claim 48, wherein the linking segment comprises a nucleic acid.

### Claim 50 (depends on 49)

The nucleic acid of claim 49, wherein the nucleic acid is at least partially single-stranded.

### Claim 51 (depends on 49)

The nucleic acid of claim 49, wherein the nucleic acid is double-stranded.

### Claim 52 (depends on 48, 49, 50, 51)

The nucleic acid of any one of claims 48-51, wherein the linking segment comprises terminal nucleotides that form phosphodiester bonds with the first TBS and the second TBS.

### Claim 53 (depends on 48, 49, 50, 51)

The nucleic acid of any one of claims 48-51, wherein the linking segment comprises an affinity binding pair or a covalent bond resulting from a conjugation reaction.

### Claim 54 (depends on 53)

The nucleic acid of claim 53, wherein the affinity binding pair comprises biotin-streptavidin, biotin-avidin, ligand-receptor, antigen-antibody or antigen binding fragment, hapten-anti-hapten, or Ig binding protein-lg.

### Claim 55 (depends on 54)

The nucleic acid of claim 54, wherein the affinity binding pair comprises biotin-streptavidin or biotin-avidin.

### Claim 56 (depends on 55)

The nucleic acid of claim 55, wherein the streptavidin or avidin binds only one or two biotin molecules.

### Claim 57 (depends on 53, 54, 55, 56)

The nucleic acid of any one of claims 53-56, wherein the affinity binding pair comprises a first affinity component that binds to two second affinity components, where one second affinity component is linked to the first end of the first artificial nucleic acid, and the other second affinity component is linked to the second end of the first artificial nucleic acid.

### Claim 58 (depends on 53, 54, 55, 56)

The nucleic acid of any one of claims 53-56, wherein the affinity binding pair comprises a first affinity component that binds a second affinity component, where the first affinity component is linked to the first end of the first artificial nucleic acid, and the second affinity component is linked to the second end of the first artificial nucleic acid.

### Claim 59 (depends on 53)

The nucleic acid of claim 53, wherein the conjugation reaction is selected from the group consisting of a cycloaddition, amide or thioamide bond formation, a pericyclic reaction, a Diels-Alder reaction, sulfonamide bond formation, alcohol or phenol alkylation, a condensation reaction, disulfide bond formation, and a nucleophilic substitution.

### Claim 60 (depends on 59)

The nucleic acid of claim 59, wherein the cycloaddition is an azide-alkyne Huisgen cycloaddition.

### Claim 61 (depends on 60)

The nucleic acid of claim 60, wherein the azide-alkyne Huisgen cycloaddition is a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or a strain-promoted azide-alkyne cycloaddition (SPAAC).

### Claim 62 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61)

The nucleic acid of any one of claims 48-61, wherein the linking segment further comprises one or more additional elements selected from the group consisting of an IST, a primer binding site, a cleavage site, and a chemical modification.

### Claim 63 (depends on 62)

The nucleic acid of claim 62, wherein the one or more additional elements comprises an IST.

### Claim 64 (depends on 62, 63)

The nucleic acid of claim 62 or 63, wherein the IST is a random IST, a semi-random IST, or a non-random IST.

### Claim 65 (depends on 62)

The nucleic acid of claim 62, wherein the cleavage site is a restriction endonuclease recognition site or a nickase site.

### Claim 66 (depends on 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65)

The nucleic acid of any one of claims 49-65, wherein the nucleic acid is about 50 bp to about 500 bp in length.

### Claim 67 (depends on 66)

The nucleic acid of claim 66, wherein the artificial nucleic acid is about 100 to about 250 bp long.

### Claim 68 (depends on 67)

The nucleic acid of claim 67, wherein the artificial nucleic acid is about 150 to about 200 bp long.

### Claim 69 (depends on 68)

The nucleic acid of claim 68, wherein the artificial nucleic acid is about 175 bp long.

### Claim 70 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69)

The nucleic acid of any one of claims 48-69, wherein the linking segment prevents the first transposase and the second transposase from oligomerizing when bound to the first TBS and the second TBS.

### Claim 71 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70)

The nucleic acid of any one of claims 48-70, wherein the first transposase and the second transposase do not oligomerize with each other.

### Claim 72 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71)

The nucleic acid of any one of claims 48-71, wherein the first TBS or the second TBS is double-stranded.

### Claim 73 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72)

The nucleic acid of any one of claims 48-72, wherein more than one transposase binds to the first TBS or the second TBS.

### Claim 74 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73)

A molecular construct comprising a first transposase, a second transposase, and the artificial nucleic acid of any one of claims 48-73, wherein the first transposase is bound to the first TBS and the second transposase is bound to the second TBS.

### Claim 75 (depends on 74)

The molecular construct of claim 74, wherein the linking segment prevents the first transposase and the second transposase from oligomerizing with each other.

### Claim 76 (depends on 75)

The molecular construct of claim 75, wherein the first transposase and the second transposase do not oligomerize with each other.

### Claim 77 (depends on 74, 75, 76)

The molecular construct of any one of claims 74-76, wherein the first transposase or the second transposase is a Tn3, Tn5, Tn9, Tn10, gamma-delta, Mu, piggyBac, Minos, Tc1, or Sleeping Beauty transposase or a biologically active variant thereof.

### Claim 78 (depends on 77)

The molecular construct of claim 77, wherein the first transposase or the second transposase is a Tn3, Tn5, Tn9, Tn10, gamma-delta, or Mu transposase or a biologically active variant thereof.

### Claim 79 (depends on 77, 78)

The molecular construct of claim 77 or 78, wherein the first transposase or the second transposase is a Tn5 transposase, a Mu transposase, or a biologically active variant thereof.

### Claim 80 (depends on 74, 75, 76, 77, 78, 79)

The molecular construct of any one of claims 74-79, wherein the first transposase is a Tn5 transposase and the second transposase is a Mu transposase.

### Claim 81 (depends on 74, 75, 76, 77, 78, 79, 80)

The molecular construct of any one of claims 74-80, wherein more than one transposase binds to the first TBS or the second TBS.

### Claim 82 (depends on 74, 75, 76, 77, 78, 79, 80, 81)

The molecular construct of any one of claims 74-81, wherein the first transposase or the second transposase is operably linked to a targeting moiety.

### Claim 83 (depends on 82)

The molecular construct of claim 82, wherein the targeting moiety is a polypeptide comprising a DNA-binding domain (DBD) or an RNA-guided endonuclease.

### Claim 84 (depends on 83)

The molecular construct of claim 83, wherein the DBD is a zinc finger motif or a transcription activator-like (TAL) effector.

### Claim 85 (depends on 83)

The molecular construct of claim 83, wherein the RNA-guided endonuclease is Cas9, Cpf1, C2c2, or a biologically active variant thereof.

### Claim 86 (depends on 85)

The molecular construct of claim 85, wherein the biologically active variant is a nuclease-deficient variant.

### Claim 87 (depends on 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86)

A TSC comprising at least three of the molecular constructs of any one of claims 74-86, wherein the constructs are concatenated by oligomerization of a transposase in each construct with a transposase in another construct, and wherein at least two synaptic complexes are present in the TSC.

### Claim 88 (depends on 87)

The TSC of claim 87, wherein the TSC comprises between two and ten thousand synaptic complexes.

### Claim 89 (depends on 87, 88)

The TSC of claim 87 or 88, wherein each artificial nucleic acid in the TSC comprises an IST.

### Claim 90 (depends on 89)

The TSC of claim 89, wherein each IST is identical.

### Claim 91 (depends on 89)

The TSC of claim 89, wherein each IST is not identical.

### Claim 92

A method of preparing a TSC, the method comprising:
(a) providing at least a first synaptic complex and a second synaptic complex, wherein
(i) the first synaptic complex comprises a first pair of oligomerized transposases, wherein one member of the pair is bound to a first nucleic acid comprising a TBS and a sticky end; and
(ii) the second synaptic complex comprises a second pair of oligomerized transposases, wherein one member of the pair is bound to a second nucleic acid comprising a TBS and a sticky end; and
(b) ligating the first synaptic complex to the second synaptic complex by ligating the sticky ends of the first nucleic acid and the second nucleic acid in a ligation reaction,
thereby preparing a TSC.

### Claim 93 (depends on 92)

The method of claim 92, wherein the first nucleic acid and the second nucleic acid have the same nucleic acid sequence.

### Claim 94 (depends on 92)

The method of claim 92, wherein the first nucleic acid and the second nucleic acid have different nucleic acid sequences.

### Claim 95 (depends on 92, 93, 94)

The method of any one of claims 92-94, wherein (a) further comprises providing a linking segment nucleic acid comprising a first sticky end and a second sticky end, wherein the first sticky end is compatible with the sticky end of the first nucleic acid and the second sticky end is compatible with the sticky end of the second nucleic acid.

### Claim 96 (depends on 95)

The method of claim 95, wherein (b) further comprises ligating the linking segment nucleic acid to the first nucleic acid and the second nucleic acid.

### Claim 97 (depends on 95, 96)

The method of claim 95 or 96, wherein the linking segment comprises one or more additional elements selected from the group consisting of IST, a primer binding site, a cleavage site, or a chemical modification.

### Claim 98 (depends on 97)

The method of claim 97, wherein the chemical modification is a biotinylation.

### Claim 99 (depends on 92, 93, 94, 95, 96, 97, 98)

A TSC prepared by the method of any one of claims 92-98.

### Claim 100

A synaptic complex comprising:
(a) a first transposase;
(b) a second transposase;
(c) a first artificial nucleic acid comprising a first end comprising a TBS and being linked to a first conjugating moiety other than a 3′ hydroxyl; and
(d) a second artificial nucleic acid comprising a first end comprising a TBS and being linked to a second conjugating moiety other than a 3′ hydroxyl,
wherein the first transposase is bound to the TBS of the first artificial nucleic acid, the second transposase is bound to the TBS of the second artificial nucleic acid, and the first transposase and the second transposase are oligomerized.

### Claim 101 (depends on 100)

The synaptic complex of claim 100, wherein the first and second conjugating moieties are the same.

### Claim 102 (depends on 100)

The synaptic complex of claim 100, wherein the first and second conjugating moieties are the different.

### Claim 103 (depends on 100)

The synaptic complex of claim 100, wherein the first and second conjugating moieties are independently selected from the group consisting of a 1,3-diene, an alkene, an alkylamino, an alkyl halide, an alkyl pseudohalide, an alkyne, an amino, an anilido, an aryl, an azide, an aziridine, a carboxyl, a carbonyl, an episulfide, an epoxide, a heterocycle, an organic alcohol, an isocyanate group, a maleimide, a succinimidyl ester, a sulfosuccinimidyl ester, a sulfhydryl, a thiol, and a thioisocyanate group.

### Claim 104 (depends on 100, 101, 102, 103)

The synaptic complex of any one of claims 100-103, wherein the first or second artificial nucleic acid further includes an IST.

### Claim 105

A method of preparing a TSC, the method comprising:
(a) providing at least a first synaptic complex and a second synaptic complex, wherein
(i) the first synaptic complex comprises a first pair of oligomerized transposases, wherein one member of the pair is bound to a first nucleic acid comprising a TBS and being linked to a first component of an affinity binding pair or a first conjugating moiety; and
(ii) the second synaptic complex comprises a second pair of oligomerized transposases, wherein one member of the pair is bound to a second nucleic acid comprising a TBS and being linked to a second component of the affinity binding pair or a second conjugating moiety; and
(b) linking the first synaptic complex to the second synaptic complex, wherein linking of the first conjugating moiety to the second conjugating moiety does not result in a phosphodiester bond,
thereby preparing a TSC.

### Claim 106 (depends on 105)

The method of claim 105, wherein the linking of (b) comprises combining the first synaptic complex and the second synaptic complex under conditions suitable for binding of the first component and the second component of the affinity binding pair.

### Claim 107 (depends on 105)

The method of claim 105, wherein the linking of (b) comprises conjugating the first conjugating moiety to the second conjugating moiety in a conjugation reaction that does not produce a phosphodiester bond.

### Claim 108 (depends on 105, 106, 107)

A TSC prepared by the method of any one of claims 105-107.

### Claim 109 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 87, 88, 89, 90, 91, 99)

A method of preparing a target nucleic acid for sequencing, the method comprising:
(a) combining the TSC of any one of claims 1-47, 87-91, 99, or 108 with a target nucleic acid under conditions and for a time sufficient for the TSC to carry out a transposition event.

### Claim 110 (depends on 109)

The method of claim 109, further comprising:
(b) fragmenting the target nucleic acid and adding a polynucleotide to the resulting ends of the nucleic acid fragments.

### Claim 111 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 87, 88, 89, 90, 91, 99)

A method of sequencing a target nucleic acid, the method comprising:
(a) combining the TSC of any one of claims 1-47, 87-91, 99, or 108 with a target nucleic acid under conditions and for a time sufficient for the TSC to carry out a transposition event;
(b) fragmenting the target nucleic acid and adding a polynucleotide to the resulting ends of the nucleic acid fragments;
(c) selecting DNA fragments comprising a nucleic acid sequence resulting from the transposition event;
(d) amplifying the selected fragments; and
(e) sequencing the amplified fragments.

### Claim 112 (depends on 110, 111)

The method of claim 110 or 111, wherein (b) comprises random shearing and adapter ligation or tagmentation.

### Claim 113 (depends on 111, 112)

The method of claim 111 or 112, wherein the selecting of (c) comprises selecting nucleic acid fragments comprising an IST.

### Claim 114 (depends on 111, 112, 113)

The method of any one of claims 111-113, wherein the amplifying of (d) comprises polymerase chain reaction (PCR), multiple displacement amplification (MDA), ligase chain reaction (LCR), loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), or strand displacement amplification (SDA).

### Claim 115 (depends on 111, 112, 113, 114)

The method of any one of claims 111-114, wherein the sequencing of (e) comprises sequencing by synthesis, sequencing by ligation, or nanopore sequencing.

### Claim 116 (depends on 115)

The method of claim 115, wherein the sequencing by synthesis comprises Illumina™ dye sequencing, single-molecule real-time (SMRT™) sequencing, or pyrosequencing.

### Claim 117 (depends on 115)

The method of claim 115, wherein the sequencing by ligation comprises polony-based sequencing or SOLiD™ sequencing.

### Claim 118 (depends on 111, 112, 113, 114, 115, 116, 117)

The method of any one of claims 111-117, further comprising:
(f) analyzing the sequenced fragments to identify fragments of the target nucleic acid that can be linked due to the presence of a nucleic acid sequence resulting from the transposition event.

### Claim 119 (depends on 109, 110, 111, 112, 113, 114, 115, 116, 117, 118)

The method of any one of claims 109-118, wherein the target nucleic acid comprises genomic DNA or cDNAs from a single cell.

### Claim 120 (depends on 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119)

The method of any one of claims 109-119, wherein the target nucleic acid comprises nucleic acids from a plurality of haplotypes.

### Claim 121 (depends on 111, 112, 113, 114, 115, 116, 117, 118, 119, 120)

The method of any one of claims 111-120, wherein the sequence of the amplified fragments is used to perform de novo sequence assembly.

### Claim 122 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 87, 88, 89, 90, 91, 99)

A kit comprising the TSC of any one of claims 1-47, 87-91, 99, or 108.

### Claim 123 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73)

A kit comprising the nucleic acid of any one of claims 48-73 and a purified transposase that binds to the first TBS or the second TBS.

### Claim 124 (depends on 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86)

A kit comprising the molecular construct of any one of claims 74-86.

### Claim 125 (depends on 122, 123, 124)

The kit of any one of claims 122-124, further comprising one or more additional reagents selected from the group consisting of a cofactor, a buffered solution, and a reference nucleic acid.

### Claim 126 (depends on 125)

The kit of claim 125, wherein the cofactor is a divalent metal cation.

### Claim 127 (depends on 126)

The kit of claim 126, wherein the divalent metal cation is a magnesium cation.

### Claim 128 (depends on 122, 123, 124, 125, 126, 127)

The kit of any one of claims 122-127, further comprising a reagent for nucleic acid sequencing.

### Claim 129 (depends on 128)

The kit of claim 128, wherein the reagent is selected from the group consisting of an oligonucleotide primer, a substrate, an enzyme, and a mixture of nucleotides.