Mirror nucleic acid replication system

Patent metadata

Field	Value
Publication number	US-11371027-B2
Application number	US-201716092757-A
Country	US
Kind code	B2
Filing date	Feb 15, 2017
Priority date	Aug 18, 2015
Publication date	Jun 28, 2022
Grant date	Jun 28, 2022

Abstract

Provided is a method for replicating a mirror nucleic acid, comprising: reacting a mirror nucleic acid template, a mirror nucleic acid primer and mirror dNTPs/rNTPs in the presence of a mirror nucleic acid polymerase, so as to obtain the mirror nucleic acid.

First claim

The invention claimed is:

1. A method for replicating a minor-image nucleic acid, comprising: carrying out a reaction in the presence of a mirror-image nucleic acid polymerase, a mirror- image nucleic acid template, a mirror-image nucleic acid primer, and mirror-image dNTPs/rNTPs to obtain the mirror-image nucleic acid, wherein said mirror-image nucleic acid polymerase is in a D-form and selected from the group consisting of ASFV pol X of SEQ ID NO: 17 and a Dpo4 protein of Sulfolobus solfataricus which comprises mutations C31S, S86A, N123A, S207A and S313A and methionines Met1, Met76, Met89, Met157, Met216 and Met251 replaced with norleucine.

2. The method according to claim 1 , wherein the mirror-image nucleic acid, the mirror-image nucleic acid template, the mirror-image nucleic acid primer, and the mirror-image dNTPs/rNTPs are in L-form.

3. The method according claim 1 , wherein the mirror-image nucleic acid is L-DNA.

4. The method according to claim 1 , wherein the reaction is a polymerase chain reaction.

5. The method according to claim 1 , wherein the reaction is carried out in a buffer of 50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 50 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 10% glycerol, 3% DMSO and 0.1 mg/ml BSA.

6. A method for screening a mirror-image nucleic acid molecule, comprising:
contacting a library of random mirror-image nucleic acid sequences with a target molecule under a condition that allows binding of the two;
obtaining a mirror-image nucleic acid molecule that binds to the target molecule;
and amplifying the mirror-image nucleic acid molecule that binds to the target molecule
by mirror-image PCR using a mirror-image nucleic acid polymerase in D-form selected from the group consisting of ASFV pol X as set forth in SEQ ID NO: 17 and a Dpo4 protein of Sulfolobus solfataricus which comprises mutations C31S, S86A, N123A, S207A and S313A and all methionines Met1, Met76, Met89, Met157, Met216 and Met251 replaced with norleucine.

7. The method according to claim 6 , wherein after the library of random mirror-image nucleic acid sequences is contacted with the target molecule, a mirror-image nucleic acid molecule that does not bind to the target molecule is removed by washing to obtain the mirror-image nucleic acid molecule that binds to the target molecule.

8. The method according to claim 6 , wherein the mirror-image nucleic acid molecule is L-DNA.

9. The method according to claim 6 , wherein the mirror-image PCR is performed in a buffer of 50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 50 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 10% glycerol, 3% DMSO and 0.1 mg/ml BSA.

10. The method according to claim 1 , wherein the Dpo4 protein of Sulfolobus solfataricus comprises a His6 tag.

Assignees

• Univ Tsinghua

Inventors

• Zhu Ting
• Liu Lei
• Xu Weiliang
• Wang Zimou
• Jiang Wenjun
• Wang Jiaxing
• Yu Linping

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/1068

  Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis · CPC title

• C12Y207/07048

  RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase · CPC title

• C12Y207/07007 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title