Novel self-cleaving dnazymes and selection process

Abstract

The present invention provides a novel in vitro method of selecting DNAzymes with self-cleaving activity, novel DNAzymes and their use for the production of single stranded DNA.

Description

HK Application No.: 62026122158.9 Our Ref: HKP / 2026 / 95387 cc * (Draft) * May 20, 2026 Invention Title: Novel Self-Cutting DNA Enzyme and Selection Method Abstract This invention relates to a novel method for in vitro selection of a self-cutting DNA enzyme, a novel DNA enzyme, and its use in the production of single-stranded DNA. Abstract

Claims

CLAIMS1 . Method for selecting a DNAzyme with self-cleaving activity, the method comprising:(a) providing a plurality of single-stranded DNA strands, wherein each single-stranded DNA strand comprises a core region of random nucleotides flanked by a constant region 1 at its 5’ end and a constant region 2 at its 3’ end, and wherein each constant region comprises a primer binding part;(b) subjecting the plurality of single-stranded DNA strands to self-cleaving conditions;(c) isolating cleavage products 2, and optionally isolating cleavage products 1 , wherein the cleavage products 2 comprise the constant region 2, the core region, and wherein the cleavage products 2 comprise a DNA self-cleaving activity, and wherein the cleavage products 1 comprise the constant region 1 , or wherein the cleavage products 2 comprise the constant region 2, the core region, and a part of constant region 1 , and wherein the cleavage products 2 comprise a DNA self-cleaving activity, and wherein the cleavage products 1 comprises the remaining part of the constant region 1 ;(d) producing complements of the cleavage products 2;(e) optionally separating the complements of the cleavage products 2;(f) ligating the 3’ end of the complements of the cleavage products 2 to complements of the cleavage products 1 , thereby obtaining complements of single-stranded DNA strands of step (a) with the DNA self-cleaving activity;(g) optionally separating the complements of the single-stranded DNA strands;(h) amplifying the complements of the single-stranded DNA strands thereby obtaining DNA strands comprising the DNA self-cleaving activity;(i) optionally separating the DNA strands comprising the self-cleaving activity;(j) optionally subjecting the DNA strands comprising the self-cleaving activity to an error prone (EP-PCR) step; and(k) optionally subjecting the DNA strands comprising the self-cleaving activity of step (h), optionally of steps (i) or (j), to one or more rounds of steps (b)-(h), and optionally to one or more rounds of steps (i) and / or (j).

2. The method of claim 1 , wherein the constant regions 1 of the single-stranded DNA strands in step (a) comprise a part which is complementary to a part of the constant regions 2, thereby forming a hybridization stem flanking the core region.

3. The method of claim 1 or 2, wherein the core region of the single-stranded DNA strands in step (a) comprises about 14-100 nucleotides, or about 20-100 nucleotides, or about 25-50 nucleotides, or about 30-40 nucleotides, or about 32-35 nucleotides.

4. The method of any one of the preceding claims, wherein the self-cleaving conditions in step (b) comprise one or more ions, preferably monovalent, divalent and / or trivalent and / or polyvalent ions, such as Na+, Zn2+, Mg2+, Cu2+, Mn2+, Ca2+, and / or PLL-g-Dex.

5. The method of any one of the preceding claims, wherein the self-cleaving conditions in step (b) comprise an incubation time of about 15 to 20 hours in the first round, preferably about 18 hours.

6. The method of any one of the preceding claims, wherein isolating in step (c) comprises polyacrylamide gel electrophoresis (PAGE).

7. The method of any one of the preceding claims, wherein the cleavage products 2 in step (c) comprise the core region, the constant region 2 and a self-cleaving activity, and wherein the cleavage products 1 comprise the constant region 1 , and wherein cleavage of the single-stranded DNA strands occurred between the catalytic core and the constant region 1.

8. The method of any of the preceding claims, wherein producing in step (d) comprises a linear polymerase chain (PCR) amplification, preferably with a primer 2 binding to the primer binding part of the constant regions 2, preferably wherein the primer 2 comprises a tag which allows discrimination from cleavage products 2 and / or wherein the primer 2 comprises a label.

9. The method of any of the preceding claims, wherein amplifying in step (h) comprises a PCR, preferably a 2-stage PCR.

10. A DNAzyme obtainable by the method according to any one of claims 1 to 9, preferably wherein the DNAzyme comprises a self-cleaving activity between the catalytic core and the constant region 1 or within constant region 1.

11. A DNAzyme comprising a sequence:5’- N&G Y$Y#GT N$Y#ACGC Y#Y$YGTCTTATCGGTT Y$Y$N#N -3’ (SEQ ID NO: 1 ), optionally wherein an additional nucleotide N#is included between nucleotide position 29 and 30 of SEQ ID NO: 1 , wherein the numbering of nucleotides is from 5’ to 3’ direction of SEQ ID NO: 1 , and whereinA is a nucleotide with the base adenine,C is a nucleotide with the base cytosine, G is a nucleotide with the base guanine, T is a nucleotide with the base thymidine, N is independently A, C, G, or T,Y is independently C or T,$is independently preferably T,#is independently preferably C,&is independently preferably G, and wherein the DNAzyme comprises a self-cleaving activity.

12. The DNAzyme of claim 10 or 11 , wherein the self-cleaving activity results in a final yield Ymax of about 92%, about 95%, about 96%, or about 98%, wherein preferably the final yield Ymaxis calculated by Y=Ymax(1-e'kobs*t) ) wherein Y is the reaction yield, kobs is the observed rate constant and t is a timepoint within 24 hours after start of the reaction, preferably after about 24 hours of the start of the reaction.

13. The DNAzyme of any of claims 10-12, wherein the self-cleaving activity is a hydrolytic cleavage.

14. Use of the DNAzyme of any of claims 10-13 in the production of single stranded DNA molecules.

15. The use of claim 14, wherein the single stranded DNAzyme is used in DNA nanotechnology, diagnosis, DNA synthesis or in gene therapy, preferably genome editing, more preferably in homology directed repair (HDR).