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US 11,634,742 B2Granted
Compositions of DNA molecules, methods of making therefor, and methods of use thereof
AssigneeAnjarium Biosciences AG
InventorsJoel de Beer, Monique Maurer, Nicolas Meier, Lavaniya Kunalingam, Marcello Clerici
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Abstract
Provided herein are double strand DNA molecules comprising inverted repeats, expression cassette and one or more restriction sites for nicking endonucleases, the methods of use thereof, and the methods of making therefor.
Claims24
A method for preparing a hairpin-ended DNA molecule in vitro, wherein the method comprises:
- a. providing a double-stranded DNA molecule, wherein the double-stranded DNA molecule comprises in 5′ to 3′ direction of the top strand:
- i. a first inverted repeat, wherein a first and a second restriction site for nicking endonuclease are arranged on opposite strands wherein nicking results in:
- (1) a top strand 5′ single strand DNA overhang comprising the first inverted repeat or a fragment thereof upon separation of the top from the bottom strand of the first inverted repeat;
- or
- (2) a bottom strand 3′ single strand DNA overhang comprising the first inverted repeat upon separation of the top from the bottom strand of the first inverted repeat;
- and
- ii. an expression cassette;
- and
- iii. a second inverted repeat, wherein a third and a fourth restriction site for nicking endonuclease are arranged on opposite strands wherein nicking results in:
- (1) a top strand 3′ single strand DNA overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand of the second inverted repeat;
- or
- (2) a bottom strand 5′ single strand DNA overhang comprising the second inverted repeat upon separation of the top from the bottom strand of the second inverted repeat;
- b. incubating the double-stranded DNA molecule with one or more nicking endonucleases recognizing the four restriction sites, thereby creating the two single strand DNA overhangs as specified in step a upon separation of the top from the bottom strand;
- wherein the length of each single strand DNA overhang is independently at least 20 nucleotides in length;
- c. denaturing and thereby creating a DNA fragment that comprises the expression cassette and is flanked by the two single strand DNA overhangs;
- and
- d. annealing the single strand DNA overhangs and thereby creating a hairpinned inverted repeat on both ends of the DNA fragment resulting from step c.
14 more elements
Dependencies are inferred from the claim text.