Methods of Making DNA Molecules and Compositions and Uses Thereof
The present disclosure relates to methods and kits for making hairpin-ended DNA molecules through amplification (e.g., isothermal amplification, e.g., rolling circle amplification (RCA), multiple displacement amplification (MDA)) of a circular DNA template, compositions comprising such made hairpin-ended DNA molecules, and uses thereof. Methods disclosed herein can produce transfection/transcription-ready high fidelity and high purity DNA molecules that are suitable for various uses (e.g., gene therapies).
1 . A method for preparing hairpin-ended DNA molecules, wherein the method comprises:
a. providing a circular DNA molecule as a template;
b. incubating the template with a polymerase and a primer pair under conditions suitable for amplification to produce at least one amplification product and suitable for at least 2-fold amplification of the template, wherein the amplification product comprises a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
i. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
ii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
iii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
iv. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
c. incubating the amplification product with one or more nicking endonucleases recognizing the four restriction sites;
d. denaturing and thereby creating a DNA fragment that comprises the two single strand DNA overhangs as specified in step b upon separation of the top from the bottom strand; and
e. annealing the single strand DNA overhangs and thereby creating a hairpinned inverted repeat on each end of the DNA fragment resulting from the denaturing step to produce the hairpin-ended DNA molecule.
2 . A method for amplifying precursors of hairpin-ended DNA molecules, wherein the method comprises:
a. providing a circular DNA molecule as a template; and
b. incubating the template with a polymerase and a primer pair under conditions suitable for amplification to produce at least one amplification product and suitable for at least 2-fold amplification of the template, wherein the amplification product comprises a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
i. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
ii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
iii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
iv. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand.
3 . The method of claim 2 , further comprising
a. incubating the amplification product with one or more nicking endonucleases recognizing the four restriction sites;
b. denaturing and thereby creating a DNA fragment that comprises the two single strand DNA overhangs as specified in step b; and
c. annealing the single strand DNA overhangs and thereby creating a hairpinned inverted repeat on each end of the DNA fragment resulting from the denaturing step to produce the hairpin-ended DNA molecule.
4 . A method for preparing precursors of hairpin-ended DNA molecules, wherein the method comprises:
a. providing a circular DNA molecule as a template;
b. incubating the template with a polymerase and a primer pair under conditions suitable for amplification to produce at least one amplification product and suitable for at least 2-fold amplification of the template, wherein the amplification product comprises
i. a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
(1) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(2) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(3) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
(4) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
ii. a restriction enzyme site wherein the restriction enzyme site is located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end; and
c. incubating the amplification product with a restriction enzyme that cleaves the restriction enzyme site to produce the precursor of the hairpin-ended DNA molecule comprising the first inverted repeat, the sequence of interest, and the second inverted repeat.
5 . The method of claim 4 , wherein the template comprises no more than one type of restriction enzyme site, wherein the restriction enzyme site is present 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times in the template.
6 . A method for preparing precursors of a hairpin-ended DNA molecules, wherein the method comprises:
a. providing a circular DNA molecule as a template;
b. incubating the template with a polymerase and a primer pair under conditions suitable for amplification to produce at least one amplification product and suitable for at least 2-fold amplification of the template, wherein the amplification product comprises
i. a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
(1) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(2) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(3) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
(4) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
ii. a fifth and a sixth restriction sites for nicking endonuclease arranged on opposite strands, wherein the fifth and sixth restriction sites are located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end; and
c. incubating the amplification product with a nicking endonuclease that nicks the fifth and sixth restriction sites to produce the precursor of the hairpin-ended DNA molecule comprising the first inverted repeat, the sequence of interest, and the second inverted repeat.
7 . The method of claim 6 , wherein the template comprises no additional restriction sites for nicking endonuclease, optionally wherein each of the fifth and sixth restriction sites for nicking endonuclease is present 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times in the template.
8 . The method of claim 6 or 7 , wherein (i) nicks created by nicking at the fifth and sixth restriction sites are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides apart; and (ii) single strand DNA overhangs created by nicking at the fifth and sixth restriction sites do not anneal at detectable levels inter- or intramolecularly under conditions that favor annealing of the first and/or second inverted repeat.
9 . The method of any one of claims 4 to 8 , further comprising:
d. incubating the precursor of the hairpin-ended DNA molecule with one or more nicking endonucleases recognizing the first, second, third, and fourth restriction site;
e. denaturing and thereby creating a DNA fragment that comprises the two single strand DNA overhangs as specified in step b upon separation of the top from the bottom strand; and
f. annealing the single strand DNA overhangs of the DNA fragment and thereby creating a hairpinned inverted repeat on each end of the DNA fragment resulting from the denaturing step to produce the hairpin-ended DNA molecule.
10 . The method of claim 9 , wherein the method produces non-hairpin-ended DNA molecules comprising at least one non-hairpin end, and the method further comprises digesting the non-hairpin-ended DNA molecules with an exonuclease, wherein the hairpin-ended DNA molecule is resistant to digestion by the exonuclease.
11 . The method of any one of claims 4-10 , wherein the amplification product comprises an additional restriction enzyme site and/or additional restriction sites for nicking endonuclease located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end, and the method further comprises creating additional non-hairpin-ended DNA molecules by cleaving the additional restriction enzyme site and/or nicking the additional restriction sites for nicking endonuclease.
12 . A method for preparing a composition comprising pure hairpin-ended DNA molecules, wherein the method comprises:
a. providing a circular DNA molecule as a template;
b. incubating the template with a polymerase and a primer pair under conditions suitable for amplification to produce at least one amplification product and suitable for at least 2-fold amplification of the template, wherein the amplification product comprises
i. a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
(1) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(2) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(3) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
(4) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
ii. a restriction enzyme site wherein the restriction enzyme site is located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end;
c. incubating the amplification product with one or more nicking endonucleases recognizing the four restriction sites;
d. denaturing and thereby creating a DNA fragment that comprises the two single strand DNA overhangs as specified in step b upon separation of the top from the bottom strand;
e. annealing the single strand DNA overhangs and thereby creating a hairpinned inverted repeat on each end of the DNA fragment resulting from the denaturing step to produce a hairpin-ended DNA molecule comprising the sequence of interest and a hairpin-ended DNA molecule comprising the restriction enzyme site;
f. incubating the hairpin-ended DNA molecule comprising the restriction enzyme site with a restriction enzyme that cleaves at the restriction enzyme site to produce a non-hairpin-ended DNA molecule comprising at least one non-hairpin end; and
g. digesting the non-hairpin-ended DNA molecules with an exonuclease, wherein the hairpin-ended DNA molecule comprising the sequence of interest is resistant to digestion by the exonuclease.
13 . The method of claim 12 , wherein the template comprises no more than one of the restriction enzyme site.
14 . A method for preparing a composition comprising pure hairpin-ended DNA molecules, wherein the method comprises:
a. providing a circular DNA molecule as a template;
b. incubating the template with a polymerase and a primer pair under conditions suitable for amplification to produce at least one amplification product and suitable for at least 2-fold amplification of the template, wherein the amplification product comprises
i. a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
(1) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(2) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(3) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
(4) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
ii. a fifth and a sixth restriction sites for nicking endonuclease arranged on opposite strands, wherein the fifth and sixth restriction sites are located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end; and
c. incubating the amplification product with one or more nicking endonucleases recognizing the four restriction sites;
d. denaturing and thereby creating a DNA fragment that comprises the two single strand DNA overhangs as specified in step b upon separation of the top from the bottom strand;
e. annealing the single strand DNA overhangs and thereby creating a hairpinned inverted repeat on each end of the DNA fragment resulting from the denaturing step to produce a hairpin-ended DNA molecule comprising the sequence of interest and a hairpin-ended DNA molecule comprising the fifth and sixth restriction site;
f. incubating the hairpin-ended DNA molecule comprising the fifth and sixth restriction sites with a nicking endonuclease that nicks the fifth and sixth restriction sites to produce a non-hairpin-ended DNA molecule comprising at least one non-hairpin end; and
g. digesting the non-hairpin-ended DNA molecules with an exonuclease, wherein the hairpin-ended DNA molecule comprising the sequence of interest is resistant to digestion by the exonuclease.
15 . The method of claim 14 , wherein (i) the nicks created by nicking at the fifth and sixth restriction sites are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides apart; and (ii) the single strand DNA overhangs created by nicking at the fifth and sixth restriction sites do not anneal at detectable levels inter- or intramolecularly under conditions that favor annealing of the first and/or second inverted repeat.
16 . The method of any one of claims 12-15 , wherein the amplification product comprises an additional restriction enzyme site and/or additional restriction sites for nicking endonuclease located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end, and the method further comprises creating additional non-hairpin-ended DNA molecules by cleaving the additional restriction enzyme site and/or nicking the additional restriction sites for nicking endonuclease.
17 . A method for amplifying precursors of hairpin-ended DNA molecules, wherein the method comprises:
a. providing a circular DNA molecule as a template comprising a methylated methylation-sensitive restriction enzyme (MSRE)-recognition site; and
b. incubating the template with a polymerase and a primer pair under conditions suitable for amplification to produce at least one amplification product and suitable for at least 2-fold amplification of the template, wherein the amplification product comprises a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
i. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
ii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
iii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
iv. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
c. incubating the template with an MSRE, wherein the amplification product comprises an unmethylated MSRE-recognition site located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end, and the MSRE cleaves the amplification product at the unmethylated MSRE-recognition site.
18 . The method of claim 17 , wherein the circular DNA molecule is incubated with the polymerase and the MSRE concurrently, or the circular DNA molecule is incubated with the polymerase prior to the MSRE.
19 . The method of claim 17 or 18 , further comprising:
d. incubating the MSRE-cleaved amplification products with one or more nicking endonucleases recognizing the four restriction sites, thereby creating the two single strand DNA overhangs as specified in step b upon separation of the top from the bottom strand;
e. denaturing and thereby creating a DNA fragment that comprises the two single strand DNA overhangs; and
f. annealing the single strand DNA overhangs and thereby creating a hairpinned inverted repeat on each end of the DNA fragment resulting from the denaturing step to produce a hairpin-ended DNA molecule.
20 . A method for preparing hairpin-ended DNA molecules, wherein the method comprises:
a. providing a circular DNA molecule comprising a methylated methylation-sensitive nicking endonuclease (MSNE)-restriction site;
b. incubating the template with a polymerase and a primer pair under conditions suitable for amplification to produce at least one amplification product and suitable for at least 2-fold amplification of the template, wherein the amplification product comprises a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
i. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
ii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
iii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
iv. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
c. incubating the template with an MSNE, wherein the amplification product comprises two unmethylated MSNE-recognition sites located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end, and the MSNE cleaves the amplification product at the two unmethylated MSNE-recognition sites.
21 . The method of claim 20 , wherein the circular DNA molecule is incubated with the polymerase and the MSNE concurrently, or the circular DNA molecule is incubated with the polymerase prior to the MSNE.
22 . The method of claim 20 or 21 , further comprising:
d. incubating the MSNE-cleaved amplification products with one or more nicking endonucleases recognizing the four restriction sites, thereby creating the two single strand DNA overhangs as specified in step b upon separation of the top from the bottom strand;
e. denaturing and thereby creating a DNA fragment that comprises the two single strand DNA overhangs; and
f. annealing the single strand DNA overhangs and thereby creating a hairpinned inverted repeat on each end of the DNA fragment resulting from the denaturing step to produce a hairpin-ended DNA molecule.
23 . A method for preparing hairpin-ended DNA molecules, wherein the method comprises:
a. providing a circular DNA molecule as a template comprising a methylated methylation-sensitive restriction enzyme (MSRE)-recognition site; and
b. incubating the template with a polymerase and a primer pair under conditions suitable for amplification to produce at least one amplification product and suitable for at least 2-fold amplification of the template, wherein the amplification product comprises a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
i. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
ii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
iii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
iv. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
c. incubating the template with an MSRE, wherein the amplification product comprises an unmethylated MSRE-recognition site located outside the segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end, and the MSRE cleaves the amplification product at the unmethylated MSRE-recognition site;
d. incubating the MSRE-cleaved amplification products with one or more nicking endonucleases recognizing the four restriction sites, thereby creating the two single strand DNA overhangs as specified in step b upon separation of the top from the bottom strand;
e. denaturing and thereby creating a DNA fragment that comprises the two single strand DNA overhangs; and
f. annealing the single strand DNA overhangs and thereby creating a hairpinned inverted repeat on each end of the DNA fragment resulting from the denaturing step to produce a hairpin-ended DNA molecule.
24 . The method of claim 23 , wherein the method produces non-hairpin-ended DNA molecules comprising at least one non-hairpin end, and the method further comprises digesting the non-hairpin-ended DNA molecules with an exonuclease, wherein the hairpin-ended DNA molecule is resistant to digestion by the exonuclease.
25 . A method for preparing hairpin-ended DNA molecules, wherein the method comprises:
a. providing a circular DNA molecule comprising a methylated methylation-sensitive nicking endonuclease (MSNE)-restriction site;
b. incubating the template with a polymerase and a primer pair under conditions suitable for amplification to produce at least one amplification product and suitable for at least 2-fold amplification of the template, wherein the amplification product comprises a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
i. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
ii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
iii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
iv. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
c. incubating the template with an MSNE, wherein the amplification product comprises two unmethylated MSNE-recognition sites located outside the segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end, and the MSNE cleaves the amplification product at the two unmethylated MSNE-recognition sites;
d. incubating the MSNE-cleaved amplification products with one or more nicking endonucleases recognizing the four restriction sites, thereby creating the two single strand DNA overhangs as specified in step b upon separation of the top from the bottom strand;
e. denaturing and thereby creating a DNA fragment that comprises the two single strand DNA overhangs; and
f. annealing the single strand DNA overhangs and thereby creating a hairpinned inverted repeat on each end of the DNA fragment resulting from the denaturing step to produce a hairpin-ended DNA molecule.
26 . The method of claim 25 , wherein (i) the nicks created by nicking at the two unmethylated restriction sites are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides apart; and (ii) the single strand DNA overhangs created by nicking at the two unmethylated restriction sites do not anneal at detectable levels inter- or intramolecularly under conditions that favor annealing of the first and/or second inverted repeat.
27 . The method of claim 25 or 26 , wherein the method further produces non-hairpin-ended DNA molecules comprising at least one non-hairpin end, and the method further comprises digesting the non-hairpin-ended DNA molecules with one or more exonucleases, wherein the hairpin-ended DNA molecule is resistant to digestion by the one or more exonucleases.
28 . The method of any one of claims 1 to 16 , further comprising exchanging a buffer, concentrating the amplification product, and/or removing the circular DNA molecule, polymerase, and/or primer pair, after completion of step b and before initiation of step c.
29 . The method of any one of claims 17 to 27 , further comprising exchanging a buffer, concentrating the amplification product, and/or removing the circular DNA molecule, polymerase, and/or primer pair, after completion of step c and before initiation of step d.
30 . The method of claim any one of claims 1 to 29 , wherein the sequence of interest comprises a transcription unit encoding a therapeutic protein.
31 . The method of any one of claims 1 to 29 , wherein the sequence of interest comprises a transcription unit encoding an RNA for in vitro transcription (IVT).
32 . The method of any one of claims 1 to 29 , wherein the sequence of interest comprises a gene promoter, an AAV ITR, or a synthetic DNA template to be integrated into a genome.
33 . The method of any one of claims 1-32 , wherein the circular DNA molecule is a single-stranded circular DNA molecule or a double stranded circular DNA molecule.
34 . The method of any one of claims 1-33 , wherein the amplification is an isothermal amplification.
35 . The method of claim 34 , wherein the isothermal amplification is rolling circle amplification (RCA) and/or multiple displacement amplification (MDA).
36 . A kit for preparing hairpin-ended DNA molecules, comprising:
a. a circular DNA molecule as a template, wherein an amplification product amplified from the template comprises a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
i. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
ii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
iii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
iv. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
b. a DNA polymerase suitable for amplification;
c. a primer pair; and
d. one or more nicking endonucleases recognizing the four restriction sites in the amplification product.
37 . A kit for amplifying precursors of hairpin-ended DNA molecules, comprising:
a. a circular DNA molecule as a template, wherein an amplification product amplified from the template comprises a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
i. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
ii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
iii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
iv. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
b. a DNA polymerase suitable for amplification; and
c. a primer pair.
38 . The kit of claim 37 , further comprising one or more nicking endonucleases recognizing the four restriction sites in the amplification product.
39 . A kit for preparing precursors of hairpin-ended DNA molecules, comprising:
a. a circular DNA molecule as a template, wherein an amplification product amplified from the template comprises:
i. a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
(1) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(2) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(3) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
(4) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
ii. a restriction enzyme site wherein the restriction enzyme site is located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end;
b. a DNA polymerase suitable for amplification;
c. a primer pair; and
d. a restriction enzyme that recognizes the restriction enzyme site.
40 . A kit for preparing precursors of hairpin-ended DNA molecules, comprising:
a. a circular DNA molecule as a template, wherein an amplification product amplified from the template comprises:
i. a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
(1) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(2) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(3) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
(4) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
ii. a fifth and a sixth restriction sites for nicking endonuclease arranged on opposite strands, wherein the fifth and sixth restriction sites are located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end;
b. a DNA polymerase suitable for amplification;
c. a primer pair; and
d. a nicking endonuclease that recognizes the fifth and a sixth restriction site.
41 . The kit of claim 39 or 40 , further comprising one or more nicking endonucleases recognizing the first, second, third, and forth restriction sites in the amplification product.
42 . A kit for preparing a composition comprising pure hairpin-ended DNA molecules, comprising:
a. a circular DNA molecule as a template, wherein an amplification product amplified from the template comprises:
i. a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
(1) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(2) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(3) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
(4) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
ii. a restriction enzyme site wherein the restriction enzyme site is located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end;
b. a DNA polymerase suitable for amplification;
c. a primer pair;
d. a restriction enzyme that recognizes the restriction enzyme site;
e. one or more nicking endonucleases that recognizes the first, second, third, and forth restriction sites in the amplification product; and
f. an exonuclease.
43 . A kit for preparing a composition comprising pure hairpin-ended DNA molecules, comprising:
a. a circular DNA molecule as a template, wherein an amplification product amplified from the template comprises:
i. a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
(1) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(2) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
(3) the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
(4) the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
ii. a fifth and a sixth restriction sites for nicking endonuclease arranged on opposite strands, wherein the fifth and sixth restriction sites are located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end;
b. a DNA polymerase suitable for amplification;
c. a primer pair;
d. a nicking endonuclease that recognizes the fifth and a sixth restriction site;
e. one or more nicking endonucleases that recognizes the first, second, third, and forth restriction sites in the amplification product; and
f. an exonuclease.
44 . A kit for amplifying precursors of hairpin-ended DNA molecules, comprising:
a. a circular DNA molecule as a template comprising a methylated methylation-sensitive restriction enzyme (MSRE)-recognition site, wherein an amplification product amplified from the template comprises a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
i. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
ii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
iii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
iv. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
b. an MSRE that recognizes and cleaves the amplification product at an unmethylated MSRE-recognition site located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end;
c. a DNA polymerase suitable for amplification; and
d. a primer pair.
45 . A kit for preparing hairpin-ended DNA molecules, comprising:
a. a circular DNA molecule comprising a methylated methylation-sensitive nicking endonuclease (MSNE)-restriction site, wherein an amplification product amplified from the template comprises a top strand and a bottom strand and, in 5′ to 3′ direction of the top strand, a first inverted repeat, a sequence of interest, and a second inverted repeat, wherein a first and a second restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the first inverted repeat and a third and a fourth restriction sites for nicking endonuclease are arranged on opposite strands in proximity of the second inverted repeat, and wherein:
i. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
ii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand;
iii. the first, second, third, and fourth restriction sites are arranged such that nicking results in a top strand 5′ overhang comprising the first inverted repeat or a fragment thereof and a bottom strand 5′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; or
iv. the first, second, third, and fourth restriction sites are arranged such that nicking results in a bottom strand 3′ overhang comprising the first inverted repeat or a fragment thereof and a top strand 3′ overhang comprising the second inverted repeat or a fragment thereof upon separation of the top from the bottom strand; and
b. an MSNE that recognizes and nicks the amplification product at the two unmethylated MSNE-recognition sites located outside a segment comprising the first inverted repeat at one end, the sequence of interest, and the second inverted repeat at the other end;
c. a DNA polymerase suitable for amplification; and
d. a primer pair.
46 . The kit of claim 44 or 45 , further comprising one or more nicking endonucleases that recognize the first, second, third, and forth restriction sites in the amplification product.
47 . The kit of claim 46 , further comprising an exonuclease.
48 . The kit of any one of claims 39-47 , wherein the amplification is an isothermal amplification.
49 . The kit of claim 48 , wherein the isothermal amplification is rolling circle amplification (RCA) and/or multiple displacement amplification (MDA).
50 . A method of producing AAV vectors for use in gene therapy comprising:
a. transfecting a host cell with at least one hairpin-ended DNA molecule for production of AAV particles, wherein the hairpin-ended DNA molecule has been produced according to the method of any one of claims 1 to 36 and/or using the kit of any one of claims 36 to 49 ; and
b. harvesting the AAV particles.
51 . The method of claim 50 , wherein step a comprises co-transfecting the host cell with (i) a hairpin-ended DNA molecule encoding an AAV vector genome and (ii) one or more DNA molecules encoding Rep protein(s), AAV capsid protein(s), and/or helper plasmid(s).
52 . The method of claim 50 , wherein step a comprises co-transfecting the host cell with (i) a hairpin-ended DNA molecule encoding an AAV vector genome; (ii) a hairpin-ended DNA molecule encoding Rep proteins and AAV capsid proteins; and (iii) a hairpin-ended DNA molecule encoding helper plasmids.
53 . A method of producing lentiviral vectors for use in gene therapy comprising:
a. transfecting a host cell with at least one hairpin-ended DNA molecule for production of lentiviral particles, wherein the hairpin-ended DNA molecule has been produced according to the method of any one of claims 1 to 36 and/or using the kit of any one of claims 36 to 49 ; and
b. harvesting the lentiviral particles.
54 . The method of claim 53 , wherein step a comprises co-transfecting the host cell with (i) a hairpin-ended DNA molecule encoding a lentiviral transfer vector and (ii) one or more DNA molecules encoding packaging and/or envelope proteins selected from the group consisting of VSV-G protein(s)), Tat proteins, Rev protein(s), Gag protein(s), and Pol protein(s).
55 . The method of claim 53 , wherein step a comprises co-transfecting the host cell with (i) a hairpin-ended DNA molecule encoding a lentiviral transfer vector; (ii) a hairpin-ended DNA molecule encoding Rev protein; (iii) a hairpin-ended DNA molecule encoding Gag and Pol proteins; and (iv) a hairpin-ended DNA molecule encoding VSV-G protein.
56 . A method of producing RNA comprising:
a. transcribing a hairpin-ended DNA molecule, or a fragment thereof, for production of RNA, wherein the hairpin-ended DNA molecule comprises a transcription unit suitable for in vitro transcription (IVT) and has been produced according to the method of any one of claims 1 to 36 ;
b. harvesting the RNA product.
57 . The method of claim 56 , wherein the transcribing comprises the contacting the hairpin-ended DNA molecule, or fragment thereof, with an in vitro transcription reaction system comprising an RNA polymerase and ribonucleotides.