Methods For Preventing Titration Of Bimolecular Templated Assembly Reactions By Structurally-Determined Differential Hybridizations

• US 20190316131A1
• Filed: 11/16/2017
• Published: 10/17/2019
• Est. Priority Date: 11/21/2016
• Status: Active Grant

• Current Assignee: TriBiotica LLC
• Original Assignee: TriBiotica LLC
• Inventors: Dunn, Ian, Lawler, Matthew
• Granted Patent: US 11,186,839 B2
• Time in Patent Office: Days
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2523/109 chemical ligation between nucleic acids
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

The present disclosure provides nucleic acid molecules, and kits comprising the same, for producing templated assembly products for a cell.

Claims

1. A nucleic acid molecule comprising:

a) a first stem portion comprising from about 10 to about 20 nucleotide bases;

b) an anti-target loop portion comprising from about 16 to about 40 nucleotide bases and having a first end to which the first stem portion is linked, wherein the anti-target loop portion is substantially complementary to a target nucleic acid molecule;

c) a second stem portion comprising from about 10 to about 20 nucleotide bases linked to a second end of the anti-target loop portion, wherein the first stem portion is substantially complementary to the second stem portion; and

d) a reactive effector moiety linked to either the first stem portion or the second stem portion;

wherein the Tm of the anti-target loop portion;

target nucleic acid molecule is greater than the Tm of the first stem portion;

second stem portion.

2. The nucleic acid molecule of claim 1 wherein:

the Tm of the first stem portion;

second stem portion subtracted from the Tm of the anti-target loop portion;

target nucleic acid molecule is from about 10°

C. to about 40°

C.; and

/or

the Tm of the first stem portion;

second stem portion is from about 40°

C. to about 50°

C.; and

/or

the Tm of the anti-target loop portion;

target nucleic acid molecule is from about 60°

C. to about 80°

C.; and

/or

the Tm of the first stem portion;

second stem portion subtracted from the Tm of the anti-target loop portion;

target nucleic acid molecule is from about 10°

C. to about 20°

C.

3. The nucleic acid molecule of claim 1 wherein:

the first stem portion comprises from about 12 to about 18 nucleotide bases; and

/or

the anti-target loop portion comprises from about 18 to about 35 nucleotide bases; and

/or

the second stem portion comprises from about 12 to about 18 nucleotide bases.

4. The nucleic acid molecule of claim 1 wherein the nucleotide bases of any one or more of the first stem portion, anti-target loop portion, and second stem portion are selected from the group consisting of DNA nucleotides, RNA nucleotides, phosphorothioate-modified nucleotides, 2-O-alkylated RNA nucleotides, halogenated nucleotides, locked nucleic acid nucleotides (LNA), peptide nucleic acids (PNA), morpholino nucleic acid analogues (morpholinos), pseudouridine nucleotides, xanthine nucleotides, hypoxanthine nucleotides, 2-deoxyinosine nucleotides, DNA analogs with L-ribose (L-DNA), Xeno nucleic acid (XNA) analogues, or other nucleic acid analogues capable of base-pair formation, or artificial nucleic acid analogues with altered backbones, or any combination thereof.

5. The nucleic acid molecule of claim 1 further comprising a linker between any one or more of the first stem portion and the anti-target loop portion, between the anti-target loop portion and the second stem portion, and between the second stem portion and the reactive effector moiety.

6. The nucleic acid molecule of claim 1 wherein the reactive effector moiety is selected from the group consisting of a peptide, a non-active portion of a peptidomimetic structure, a non-active portion of a drug, and a bioactive compound.

7. The nucleic acid molecule of claim 1 wherein the reactive effector moiety further comprises a bio-orthogonal reactive molecule.

8. The nucleic acid molecule of claim 7 wherein the bio-orthogonal reactive molecule is selected from the group consisting of an azide, an alkyne, a cyclooctyne, a nitrone, a norbornene, an oxanorbornadiene, a phosphine, a dialkyl phosphine, a trialkyl phosphine, a phosphinothiol, a phosphinophenol, a cyclooctene, a nitrile oxide, a thioester, a tetrazine, an isonitrile, a tetrazole, and a quadricyclane, or any derivative thereof.

9. The nucleic acid molecule of claim 1 wherein the anti-target loop portion further comprises an internal hinge region, wherein the hinge region comprises one or more nucleotides that are not complementary to the target nucleic acid molecule.

10. The nucleic acid molecule of claim 9 wherein the hinge region comprises from about 1 nucleotide to about 6 nucleotides.

11. The nucleic acid molecule of claim 1 which comprises the nucleotide sequence 5′

-ACTCGAGACGTCTCCTTGTCTTTGCTTTTCTT CAGGACACAGTGGCGAGACGTCTCGAGT-3′

(SEQ ID NO;

13) or 5′

-ACTCGAGACGTCT CCTTCCTGCCCCTCCTCCTGCTCCGAGACGTCTCGAGT-3′

(SEQ ID NO;

14).

12. A kit comprising:

a first nucleic acid molecule according to claim 1; and

a second nucleic acid molecule comprising from about 6 nucleotide bases to about 20 nucleotide bases, which comprises;

a nucleotide portion that is substantially complementary to the stem portion of the first nucleic acid molecule that is linked to the reactive effector moiety; and

a reactive effector moiety which can chemically interact with the reactive effector molecule of the first nucleic acid molecule;

wherein the Tm of the second nucleic acid molecule;

first or second stem portion linked to the reactive effector moiety is less than or equal to the Tm of the first stem portion;

second stem portion.

13. The kit of claim 12, wherein:

the reactive effector moiety of the second nucleic acid molecule is linked to a bio-orthogonal reactive molecule;

the reactive effector moiety of the first nucleic acid molecule is linked to a bio-orthogonal reactive molecule;

the bio-orthogonal reactive molecule of the second nucleic acid molecule can chemically interact with the bio-orthogonal reactive molecule of the first nucleic acid molecule.

14. The kit of claim 12 wherein:

the Tm of the duplex formed by the second nucleic acid molecule and the first or second stem portion linked to the reactive effector moiety subtracted from the Tm of the first stem portion;

second stem portion is from about 0°

C. to about 20°

C.; and

/or

the Tm of the duplex formed by the second nucleic acid molecule and the first or second stem portion linked to the reactive effector moiety is from about 30°

C. to about 40°

C.; and

/or

the Tm of the duplex formed by the nucleic acid molecule and the first or second stem portion linked to the reactive effector moiety subtracted from the Tm of the first stem portion;

second stem portion is from about 5°

C. to about 10°

C.

15. The kit of claim 12 wherein the second nucleic acid molecule comprises from about 8 to about 15 nucleotide bases.

16. The kit of claim 12 wherein:

the first nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

13, and the second nucleic acid molecule comprises the nucleotide sequence 5′

-AGCTCTCGAGT-3′

(SEQ ID NO;

15);

or

the first nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

14, and the second nucleic acid molecule comprises the nucleotide sequence 5′

-GACGTCTCGAGT-3′

(SEQ ID NO;

16).

17. The kit of claim 12 wherein the bio-orthogonal reactive molecule of the first nucleic acid molecule is hexynyl and the bio-orthogonal reactive molecule of the second nucleic acid molecule is azide.

18. A method of producing a templated assembly product for a cell comprising:

contacting a target nucleic acid molecule of the cell with a first nucleic acid molecule of claim 1; and

contacting the first nucleic acid molecule with a second nucleic acid molecule, wherein the second nucleic acid molecule comprises;

a nucleotide portion that is substantially complementary to the stem portion of the first nucleic acid molecule that is linked to the reactive effector moiety; and

a reactive effector moiety which can chemically interact with the reactive effector molecule of the first nucleic acid molecule;

wherein the Tm of the second nucleic acid molecule;

first or second stem portion linked to the reactive effector moiety is less than or equal to the Tm of the first stem portion;

second stem portion;

resulting in the combination of the respective reactive effector moieties thereby producing the templated assembly product.

19. The method of claim 18 wherein:

the first nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

13, and the second nucleic acid molecule comprises the nucleotide sequence 5′

-AGCTCTCGAGT-3′

(SEQ ID NO;

15);

or

the first nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO;

14, and the second nucleic acid molecule comprises the nucleotide sequence 5′

-GACGTCTCGAGT-3′

(SEQ ID NO;

16).

20. The method of claim 18 wherein the bio-orthogonal reactive molecule of the first nucleic acid molecule is hexynyl and the bio-orthogonal reactive molecule of the second nucleic acid molecule is azide.