Compositions and methods relating to synthetic RNA polynucleotides created from synthetic DNA oligonucleotides
Compositions and methods are provided for forming a single RNA polynucleotide from a plurality of DNA oligonucleotides in a single reaction chamber using combined reagents in a single step reaction. DNA polymerase, RNA polymerase and single stranded (ss) DNA oligonucleotides are combined where each DNA oligonucleotide has one or more sequence modules, wherein one sequence module in the first ss DNA oligonucleotide is complementary to a sequence module at the 3′ end of the second ss DNA oligonucleotide; and wherein a second module on the first ss DNA oligonucleotide is an RNA polymerase promoter sequence; and forming a single RNA polynucleotide, excluding the RNA promoter sequence, derived from the first and second DNA oligonucleotides.
1 . A method, the method comprising forming an RNA chimera from a plurality of DNA oligonucleotides in a single reaction chamber in a single reaction step, wherein the RNA chimera is encoded by a chimeric coding sequence and wherein the single step consists of contacting in the reaction chamber:
(a) the plurality of DNA oligonucleotides, wherein the plurality of DNA oligonucleotides comprises:
a first synthetic single-stranded DNA oligonucleotide having a size of up to 200 nucleotides and comprising, in a 5′ to 3′ direction, a 5′ end, a non-hybridizing sequence, a first hybridizing sequence, and a 3′ end, wherein the non-hybridizing sequence comprises, in a 5′ to 3′ direction, an RNA polymerase promoter sequence and at least a portion of the chimeric coding sequence; and
a second synthetic single-stranded DNA oligonucleotide having a size of up to 150 nucleotides and comprising, in a 5′ to 3′ direction, a 5′ end, a second hybridizing sequence, and a 3′ end, wherein the second hybridizing sequence comprises at least a portion of the chimeric coding sequence and is complementary to the first hybridizing sequence; and
(b) dNTPs; and
(c) rNTPs; and
(d) a DNA polymerase; and
(e) an RNA polymerase,
wherein
the first and second hybridizing sequences hybridize; and
the 3′ end of the first synthetic oligonucleotide is extended by the DNA polymerase to form a coding strand comprising, in a 5′ to 3′ direction, the RNA promoter and the chimeric coding sequence; and
the 3′ end of the second synthetic oligonucleotide is extended by the DNA polymerase to form a template strand comprising, in a 5′ to 3′ direction, a sequence complementary to the chimeric coding sequence and a sequence complementary to the RNA promoter; and
the chimeric RNA is formed by templated synthesis of RNA by the RNA polymerase using the template strand.
2 . A method according to claim 1 , wherein the plurality of DNA oligonucleotides further comprises:
a third synthetic oligonucleotide comprising, in a 5′ to 3′ direction, a 5′ end, a sequence module that hybridizes to the 5′ end of the second synthetic oligonucleotide, a third hybridizing sequence, and a 3′ end, and
a fourth synthetic oligonucleotide comprising, in a 5′ to 3′ direction, a 5′ end, a fourth hybridizing sequence, and a 3′ end, wherein the fourth hybridizing sequence is complementary to the third hybridizing sequence.
3 . A method according to claim 1 , wherein the first non-hybridizing sequence further comprises a variable region 3′ of the RNA polymerase promoter.
4 . A method according to claim 3 , wherein the variable region comprises a crispr sequence.
5 . A method according to claim 1 , wherein the second synthetic oligonucleotide further comprises, in a 5′ to 3′ direction, the 5′ end, a constant region, the second hybridizing sequence, and the 3′ end.
6 . A method according to claim 5 , wherein the constant region comprises a sequence complementary to a trans-activating crispr RNA sequence.
7 . A method according to claim 6 , wherein the trans-activating crispr RNA sequence is a N. meningitidis tracrRNA sequence or a S. aureus tracrRNA sequence or a S. pyogenes tracrRNA sequence.
8 . A method according to claim 1 , wherein the second synthetic oligonucleotide comprises, in a 5′ to 3′ direction, the 5′ end, a constant region, a sequence complementary to a GNRA tetraloop sequence, the second hybridizing sequence, and the 3′ end.
9 . A method according to claim 1 , wherein the RNA polymerase promoter is a T7 RNA polymerase promoter, a T7 Class III RNA polymerase promoter, a T7 phi 2.5 RNA polymerase promoter, an SP6 RNA polymerase promoter, a T3 RNA polymerase promoter, a Syn5 RNA polymerase promoter, a T5 RNA polymerase promoter, or an E. coli RNA polymerase promoter.
10 . A method according to claim 1 , wherein the extending the 3′ ends of each oligonucleotide further comprises extending the 3′ ends with a polymerase, wherein the polymerase is T4 DNA polymerase, E. coli DNA polymerase I Klenow fragment 5 exo minus, E. coli DNA polymerase I Klenow fragment, Pfu DNA polymerase, E. coli DNA polymerase I, Taq polymerase, Bst DNA polymerase, Sulphololobus polymerase, or Phi29 polymerase.
11 . A method according to claim 1 , wherein the first hybridizing sequence is 5-50 nucleotides in length and the second hybridizing sequence is 5-50 nucleotides in length.
12 . A method according to claim 1 , wherein the first synthetic oligonucleotide is 20-150 nucleotides in length and the second synthetic oligonucleotide is 20-150 in length.
13 . A method according to claim 2 , wherein the third synthetic oligonucleotide is 20-500 nucleotides in length and the fourth synthetic oligonucleotide is 20-500 in length.
14 . A method according to claim 1 , wherein the RNA chimera is a guide RNA, an aptamer, a mRNA, a tRNA, a microRNA, a shRNA, an snRNA, a short non-coding RNA, a long non-coding RNA, an RNA probe, or a ribozyme.
15 . A method according to claim 14 , wherein the aptamer is an RNA mango aptamer or an RNA broccoli aptamer.
16 . A method according to claim 1 , wherein the first and second hybridizing sequences hybridize to form a dsDNA, wherein the dsDNA comprises:
a coding strand comprising a coding sequence corresponding to all or part of an RNA chimera, wherein the coding sequence comprises in a 5′ to 3′ direction or a 3′ to 5′ direction, either i) the first non-hybridizing sequence, the first hybridizing sequence, and a sequence complementary to the second non-hybridized sequence, or ii) a sequence complementary to the first non-hybridizing sequence, the second hybridizing sequence, and the second non-hybridizing sequence, and, optionally, one or more sequences comprised in the third and/or fourth synthetic oligonucleotides, or sequences complementary thereto; and/or
a template strand comprising a template sequence complimentary to all or part of the RNA chimera, wherein the template sequence comprises in a 5′ to 3′ direction or a 3′ to 5′ direction, either i) the first non-hybridizing sequence, the first hybridizing sequence, and a sequence complementary to the second non-hybridized sequence, or ii) a sequence complementary to the first non-hybridizing sequence, the second hybridizing sequence, and the second non-hybridizing sequence, and, optionally, one or more sequences comprised in the third and/or fourth synthetic oligonucleotides, or sequences complementary thereto.
17 . A method, the method comprising forming an RNA chimera from a plurality of DNA oligonucleotides in a single reaction chamber in a single reaction step, wherein the RNA chimera is encoded by a chimeric coding sequence and wherein the single step consists of contacting in the reaction chamber:
(a) the plurality of DNA oligonucleotides, wherein the plurality of DNA oligonucleotides comprises:
a first synthetic single-stranded DNA oligonucleotide comprising, in a 5′ to 3′ direction, a 5′ end, a non-hybridizing sequence, a first hybridizing sequence, and a 3′ end, wherein the non-hybridizing sequence comprises, in a 5′ to 3′ direction, an RNA polymerase promoter sequence and at least a portion of the chimeric coding sequence; and
a second synthetic single-stranded DNA oligonucleotide comprising, in a 5′ to 3′ direction, a 5′ end, a second hybridizing sequence, and a 3′ end, wherein the second hybridizing sequence comprises at least a portion of the chimeric coding sequence and is complementary to the first hybridizing sequence; and
(b) dNTPs; and
(c) rNTPs; and
(d) a DNA polymerase; and
(e) an RNA polymerase,
wherein
the first and second hybridizing sequences hybridize; and
the 3′ end of the first synthetic oligonucleotide is extended by the DNA polymerase to form a coding strand comprising, in a 5′ to 3′ direction, the RNA promoter and the chimeric coding sequence; and
the 3′ end of the second synthetic oligonucleotide is extended by the DNA polymerase to form a template strand comprising, in a 5′ to 3′ direction, a sequence complementary to the chimeric coding sequence and a sequence complementary to the RNA promoter; and
the chimeric RNA is formed by templated synthesis of RNA by the RNA polymerase using the template strand.
18 . A method according to claim 17 , wherein
the first synthetic single-stranded DNA oligonucleotide has a size of up to 200 nucleotides; or
the second synthetic single-stranded DNA oligonucleotide has a size of up to 150 nucleotides; or
the first synthetic single-stranded DNA oligonucleotide has a size of up to 200 nucleotides and the second synthetic single-stranded DNA oligonucleotide has a size of up to 150 nucleotides.
19 . A method according to claim 17 , wherein the first and second hybridizing sequences hybridize to form a dsDNA, wherein the dsDNA comprises:
a coding strand comprising a coding sequence corresponding to all or part of an RNA chimera, wherein the coding sequence comprises in a 5′ to 3′ direction or a 3′ to 5′ direction, either i) the first non-hybridizing sequence, the first hybridizing sequence, and a sequence complementary to the second non-hybridized sequence, or ii) a sequence complementary to the first non-hybridizing sequence, the second hybridizing sequence, and the second non-hybridizing sequence, and, optionally, one or more sequences comprised in the third and/or fourth synthetic oligonucleotides, or sequences complementary thereto; and/or
a template strand comprising a template sequence complimentary to all or part of the RNA chimera, wherein the template sequence comprises in a 5′ to 3′ direction or a 3′ to 5′ direction, either i) the first non-hybridizing sequence, the first hybridizing sequence, and a sequence complementary to the second non-hybridized sequence, or ii) a sequence complementary to the first non-hybridizing sequence, the second hybridizing sequence, and the second non-hybridizing sequence, and, optionally, one or more sequences comprised in the third and/or fourth synthetic oligonucleotides, or sequences complementary thereto.