DUPLEX-SPECIFIC DNASES
The present disclosure relates, according to some embodiments, to enzymes, systems, compositions, methods, apparatus, and workflows for selectively cutting, cleaving, digesting and/or hydrolyzing duplex DNA strands or the DNA strand of a DNA/RNA hybrid.
1 . An engineered dsDNase having an amino acid sequence that is at least 95% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, or SEQ ID NO:13.
2 . An engineered dsDNase according to claim 1 , wherein the engineered dsDNase cleaves each strand of a DNA:DNA duplex and only the DNA strand of a DNA:RNA heteroduplex and does not cleave ssDNA.
3 . An engineered dsDNase according to claim 2 , wherein the engineered dsDNase comprises one or more bound magnesium cations.
4 . An engineered dsDNase according to claim 1 , wherein the amino acid sequence is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO:2.
5 . An engineered dsDNase according to claim 1 , wherein the dsDNase in the presence of 50 mM NaCl has at least 50% of its peak activity in the absence of NaCl.
6 . An engineered dsDNase according to claim 1 , wherein the dsDNase in the presence of 100 mM NaCl has at least 20% of its peak activity in the absence of NaCl.
7 . A composition comprising an engineered dsDNase according to claim 1 and one or more salts, wherein the composition has a total salt concentration of at least 50 mM.
8 . A composition comprising an engineered dsDNase according to claim 1 and one or more DNA hydrolysis products.
9 . A composition according to claim 8 further comprising one or more enzymes other than the dsDNase, one or more buffering agents, one or more detergents, one or more glass-forming agents, one or more salts, one or more stabilizers, one or more DNA:DNA duplexes, one or more DNA:RNA duplexes, or combinations thereof.
10 . A fusion protein comprising a single polypeptide chain, the single peptide chain comprising:
(a) a dsDNase according to claim 1 ; and
(b) at least one of an affinity tag, a secretion signal, and a linker.
11 . An engineered dsDNase comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29.
12 . An engineered dsDNase according to claim 11 , wherein the amino acid sequence is identical to SEQ ID NO:19, SEQ ID NO: 28, or SEQ ID NO: 29.
13 . A method for depleting a target sequence from a population of duplex DNA molecules, the population comprising at least one copy of a DNA strand having the target sequence, the method comprising:
(a) hybridizing to the at least one strand a polynucleotide having a sequence complementary to the target sequence to form a target duplex, and
(b) contacting the target duplex with a dsDNase according to claim 1 to form a target sequence depleted population of duplex DNA molecules and DNA hydrolysis products.
14 . A method according to claim 13 , wherein the polynucleotide having a sequence complementary to the target sequence is an oligonucleotide probe.
15 . A method according to claim 13 , wherein the polynucleotide having a sequence complementary to the target sequence comprises RNA.
16 . A method according to claim 13 , wherein the polynucleotide having a sequence complementary to the target sequence comprises DNA.
17 . A method according to claim 16 , wherein the polynucleotide having a sequence complementary to the target sequence is a hairpin probe comprising, in a 5′ to 3′ direction, a fluorophore, a first hairpin stem sequence, the sequence complementary to the target sequence, a second hairpin stem sequence complementary to the first hairpin stem sequence, and a quencher corresponding to the fluorophore.
18 . A method according to claim 17 , wherein the DNA hydrolysis products comprise the fluorophore and the quencher, wherein the fluorophore is sufficiently separated from the quencher to be fluorescently active.
19 . A method according to claim 13 , wherein the target sequence is a repetitive sequence present in the population of duplex DNA molecules.
20 . A method according to claim 19 , wherein (a) further comprises denaturing and reannealing the population of duplex DNA molecules and each target duplex comprises hybridized repetitive sequences.
21 . A method for hydrolyzing DNA comprising contacting (a) a composition comprising DNA and optionally RNA, and (b) a dsDNase according to claim 1 to form a reaction mixture comprising DNA hydrolysis products.
22 . A method according to claim 21 , wherein the reaction mixture has a total salt concentration of 0 mM to 150 mM.
23 . A method according to claim 21 , wherein the composition comprises RNA and the reaction mixture comprises at least 90% of the RNA that was in the composition.
24 . A method according to claim 21 , wherein the reaction mixture comprises less than 10% of the DNA that was in the composition.
25 . A method according to claim 21 , wherein the reaction mixture further comprises a DNase I buffer or a high magnesium buffer.
26 . A method according to claim 21 , wherein the composition comprises one or more DNA:DNA duplexes, one or more RNA:DNA duplexes, or both one or more DNA:DNA duplexes and one or more RNA:DNA duplexes.
27 . A method according to claim 26 , wherein the duplex DNA comprises no more than one mismatch per 10 paired nucleotides.
28 . A method according to claim 26 , wherein the duplex DNA comprises repetitive DNA sequences.
29 . A composition comprising a dsDNase having an amino acid sequence that is at least 85% identical to SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, or SEQ ID NO: 18, wherein the composition is free of one or more of a DNA polymerase, an RNA polymerase, and a protease.
30 . A method for depleting a target sequence from a population of duplex DNA molecules, the population comprising at least one copy of a DNA strand having the target sequence, the method comprising:
(a) hybridizing to the at least one strand a polynucleotide having a sequence complementary to the target sequence to form a target duplex, and
(b) contacting the target duplex with a dsDNase having an amino acid sequence that is at least 85% identical to SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO: 18 to form a target sequence depleted population of duplex DNA molecules and DNA hydrolysis products.
31 . A method according to claim 30 , wherein the polynucleotide having a sequence complementary to the target sequence is an oligonucleotide probe.
32 . A method according to claim 30 , wherein the polynucleotide having a sequence complementary to the target sequence comprises RNA.
33 . A method according to claim 30 , wherein the polynucleotide having a sequence complementary to the target sequence comprises DNA.
34 . A method according to claim 33 , wherein the polynucleotide having a sequence complementary to the target sequence is a hairpin probe comprising, in a 5′ to 3′ direction, a fluorophore, a first hairpin stem sequence, the sequence complementary to the target sequence, a second hairpin stem sequence complementary to the first hairpin stem sequence, and a quencher corresponding to the fluorophore.
35 . A method according to claim 34 , wherein the DNA hydrolysis products comprise the fluorophore and the quencher, wherein the fluorophore is sufficiently separated from the quencher to be fluorescently active.
36 . A method according to claim 30 , wherein the target sequence is a repetitive sequence present in the population of duplex DNA molecules.
37 . A method according to claim 36 , wherein (a) further comprises denaturing and reannealing the population of duplex DNA molecules and each target duplex comprises hybridized repetitive sequences.
38 . A method for hydrolyzing DNA comprising contacting (a) a composition comprising DNA and optionally RNA, and (b) a dsDNase having an amino acid sequence that is at least 85% identical to SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18 to form a reaction mixture comprising DNA hydrolysis products.
39 . A method according to claim 38 , wherein the reaction mixture has a total salt concentration of 0 mM to 150 mM.
40 . A method according to claim 38 , wherein the composition comprises RNA and the reaction mixture comprises at least 90% of the RNA that was in the composition.
41 . A method according to claim 38 , wherein the reaction mixture comprises less than 10% of the DNA that was in the composition.
42 . A method according to claim 38 , wherein the reaction mixture further comprises a DNase I buffer or a high magnesium buffer.
43 . A method according to claim 38 , wherein the composition comprises one or more DNA:DNA duplexes, one or more RNA:DNA duplexes, or both one or more DNA:DNA duplexes and one or more RNA:DNA duplexes.
44 . A method according to claim 43 , wherein the duplex DNA comprises no more than one mismatch per 10 paired nucleotides.
45 . A method according to claim 43 , wherein the duplex DNA comprises repetitive DNA sequences.
46 . A composition comprising:
a means for selectively cleaving a DNA strand of a DNA:RNA duplex; and
a non-naturally occurring buffer,
wherein the selective cleavage comprises cleaving ≥90% of the DNA strands of a DNA:RNA duplex at least once and cleaving ≤10% of the RNA strands even once.
47 . A composition according to claim 46 , wherein the means for selectively cleaving a DNA strand of a DNA:RNA duplex comprises an engineered dsDNase having an amino acid sequence that is (a) at least 95% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, or SEQ ID NO: 13, and (b) optionally 100% identical to SEQ ID NO:32.
48 . A composition according to claim 46 , wherein the means for selectively cleaving a DNA strand of a DNA:RNA duplex comprises an engineered dsDNase having an amino acid sequence that is (a) at least 95% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, or SEQ ID NO:13, and (b) optionally 100% identical to SEQ ID NO:31.
49 . A composition according to claim 46 , further comprising one or more enzymes other than the dsDNase, one or more detergents, one or more glass-forming agents, one or more salts, one or more stabilizers, one or more DNA:DNA duplexes, one or more DNA:RNA duplexes, or combinations thereof.
50 . A composition according to claim 46 , wherein the non-naturally occurring buffer is a storage buffer or a reaction buffer.