Method of packing polynucleotides

Patent metadata

Field	Value
Publication number	US-10793889-B2
Application number	US-201715444605-A
Country	US
Kind code	B2
Filing date	Feb 28, 2017
Priority date	Oct 13, 2016
Publication date	Oct 6, 2020
Grant date	Oct 6, 2020

Abstract

Provided is a method of preparing nanoparticle-type polynucleotides, the method comprising forming the polynucleotides comprising modified nucleotides, in which the forming includes chemically synthesizing the polynucleotides comprising modified nucleotides, synthesizing the polynucleotides comprising modified nucleotides using an enzyme, or a combination thereof.

First claim

What is claimed is:

1. A method of preparing nanoparticle polynucleotides, the method comprising forming the polynucleotides comprising modified nucleotides, wherein the forming comprises chemically synthesizing the polynucleotides comprising modified nucleotides, synthesizing the polynucleotides comprising modified nucleotides using an enzyme, or a combination thereof, and wherein the polynucleotides comprise at least partially a nucleotide sequence forming a secondary structure, a tertiary structure, a quaternary structure, or a combination thereof as a biomolecular structure, wherein the modified nucleotides have a modification of the hydrogen of the 8-carbon site of the purine ring when a base of the modified nucleotides is adenine or guanine, of the hydrogen of the 5-carbon site of the pyrimidine ring when a base of the modified nucleotides is cytosine or uracil, and of the methyl group of the 5-carbon site of the pyrimidine ring when a base of the modified nucleotides is thymine, the modified nucleotides being modified by substituting the hydrogen or the methyl group with a cationic residue.

2. The method of claim 1 , wherein the nanoparticle polynucleotides are self-assembled.

3. The method of claim 1 , wherein the chemically synthesizing the polynucleotides comprising modified nucleotides comprises synthesizing by oligo nucleotides synthesis.

4. The method of claim 1 , wherein the synthesizing the polynucleotides comprising modified nucleotides using an enzyme comprises the steps of extending a primer by incubating a reaction mixture comprising a template nucleotide, a polymerase, modified-NTPs (nucleoside triphosphates), and the primer having a sequence at least partially complementary to the template; and amplifying polynucleotides at least partially comprising the modified nucleotides, wherein the modified-NTPs are one or more selected from the group consisting of modified-ATP, modified-TTP, modified-CTP, modified-GTP, and modified-UTP.

5. The method of claim 4 , wherein the modified-NTPs are dNTPs (deoxyribonucleoside triphosphates) or rNTPs (ribonucleoside triphosphates).

6. The method of claim 4 , wherein the template nucleotide is DNA or RNA.

7. The method of claim 4 , wherein the template nucleotide is single-stranded or double-stranded.

8. The method of claim 4 , wherein the template nucleotide is circular or linear.

9. The method of claim 1 , wherein the modified nucleotides have a modification of one or more of a five-carbon sugar, a phosphate group, and a base.

10. The method of claim 1 , wherein the cationic residue is a substituted C 1 -C 30 alkyl group, a substituted C 2 -C 30 alkenyl group, a substituted C 2 -C 30 alkynyl group, a substituted C 3 -C 30 cycloalkyl group, a substituted C 5 -C 30 aryl group, a substituted C 5 -C 30 arylalkyl group, a substituted C 5 -C 30 arylalkenyl group, a substituted C 5 -C 30 arylalkynyl group, a substituted C 5 -C 30 heteroaryl group, a substituted C 3 -C 30 heterocyclyl group, a substituted C 3 -C 30 heterocycloalkyl group, a substituted C 3 -C 30 heterocyclylalkyl group, a substituted C 5 -C 30 heteroarylalkyl group, a substituted C 5 -C 30 heteroarylalkenyl group, or a substituted C 5 -C 30 heteroarylalkynyl group, which is substituted with one or more selected from the group consisting of an amine, guanidine, pyridine, and imidazole.

11. The method of claim 1 , wherein the modified nucleotides are derived from nucleotides which have any one chemical formula of the following Formulae 4 or 6:

12. The method of claim 1 , wherein the polynucleotides are selected from the group consisting of genomic DNA, antisense oligonucleotides, mRNA, siRNA, micro RNA, sgRNA, and aptamers.

13. The method of claim 1 , wherein the method is used for controlling a surface area of the polynucleotides, used for storing the polynucleotides, or used for controlling transfection of the polynucleotides in cells, wherein
controlling the surface area of the polynucleotides is carried out by interacting between nucleotide sequences caused by modified-NTPs,
storing the polynucleotides is carried out for long-term storage, and
controlling transfection of the polynucleotides is carried out by passing the polynucleotides through cell membrane into cells without transfection reagents.

Assignees

• Korea Inst Sci & Tech

Inventors

• Ahn Dae Ro
• Kim Kyoung Ran

Classifications

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12N2310/3523

  Allyl · CPC title

• C12Q2531/125

  Rolling circle · CPC title

• C12N2310/33

  of the base · CPC title

• C12N15/115

  Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers} · CPC title