Back to results
US 10,378,035 B2Granted
Synthesis of transcripts using Syn5 RNA polymerase
AssigneePresident and Fellows of Harvard College
InventorsBin Zhu, Charles C. Richardson, Stanley Tabor
Life of this patent
Estimating the term…
Abstract
Methods of in vitro transcription using cyanophage Syn5 RNA polymerase (RNAP) or mutants thereof and transcription conditions are provided.
Claims61
A method of performing in vitro transcription comprising the steps of:
- combining a double stranded nucleic acid including a template strand and a coding strand, a cyanophage Syn5 RNA polymerase (RNAP) or cyanophage Syn5 RNAP Y564F mutant, one or more nucleotides, one or more modified nucleotides and one or more of 3′-fluoro-dCTP, or 2′-fluoro-dUTP, and
- producing a transcript,
- wherein the double stranded nucleic acid includes a cyanophage Syn5 RNAP promoter sequence comprising SEQ ID NO:
- 1,
- wherein the coding strand includes an initiation sequence of 12 nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9-nt10-nt11-nt12 after the promoter sequence,
- wherein nt is dCMP, dGMP, dAMP or dTMP and
- wherein nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9 include no more than 2 dTMPs or no more than 2 dCMPs with the remaining nucleotides being one or more of dGMP or dAMP with the proviso that nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9 cannot include 2 dTMPs and 2 dCMPs.
4 more elements
A method of performing in vitro transcription comprising the steps of:
- combining a double stranded nucleic acid including a template strand and a coding strand, a cyanophage Syn5 RNAP or cyanophage Syn5 RNAP mutant Y564F, one or more nucleotides, one or more modified nucleotides and one or more of 2′-fluoro-dCTP, or 2′-fluoro-dUTP, and
- producing a transcript,
- wherein the double stranded nucleic acid includes a cyanophage Syn5 RNAP promoter sequence comprising SEQ ID NO:
- 1,
- wherein the coding strand includes an initiation sequence of 12 nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9-nt10-nt11-nt12 after the promoter sequence,
- wherein the first 9 nucleotides are altered to include beginning at nt1 one of dGMP, dGMP-dCMP, dGMP-dCMP-dAMP, dGMP-dCMP-dAMP-dGMP, dGMP-dCMP-dAMP-dGMP-dAMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP-dAMP or dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP-dAMP-dAMP.
3 more elements
A method of performing in vitro transcription comprising the steps of:
- combining a double stranded nucleic acid including a template strand and a coding strand, a cyanophage Syn5 RNAP or cyanophage Syn5 RNAP mutant Y564F, one or more nucleotides, one or more modified nucleotides and one or more of 2′-fluoro-dCTP, or 2′-fluoro-dUTP,
- wherein the double stranded nucleic acid includes a cyanophage Syn5 RNAP promoter sequence comprising SEQ ID NO:
- 1,
- wherein the coding strand includes an initiation sequence of 12 nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9-nt10-nt11-nt12 after the promoter sequence,
- wherein one or more of dGMP, dGMP-dCMP, dGMP-dCMP-dAMP, dGMP-dCMP-dAMP-dGMP, dGMP-dCMP-dAMP-dGMP-dAMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP-dAMP or dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP-dAMP-dAMP is added to the coding strand after the promoter sequence and before the initiation sequence to create an altered initiation sequence of 12 nucleotides after the promoter sequence with complementary nucleotides added to the template strand, and
- producing a transcript.
3 more elements
A method of performing in vitro transcription comprising the steps of:
- combining a double stranded nucleic acid including a template strand and a coding strand, a cyanophage Syn5 RNAP or cyanophage Syn5 RNAP mutant Y564F, one or more nucleotides, one or more modified nucleotides and one or more of 2′-fluoro-dCTP, or 2′-fluoro-dUTP,
- wherein the double stranded nucleic acid includes a cyanophage Syn5 RNAP promoter sequence comprising SEQ ID NO:
- 1,
- wherein the coding strand includes an initiation sequence of 12 nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9-nt10-nt11-nt12 after the promoter sequence,
- wherein one or more of sequence dGMP, dGMP-dCMP, dGMP-dCMP-dAMP, dGMP-dCMP-dAMP-dGMP, dGMP-dCMP-dAMP-dGMP-dAMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP-dAMP or dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP-dAMP-dAMP coupled to a removable RNA element is added to the coding strand after the promoter sequence and before the initiation sequence to create an altered initiation sequence of 12 nucleotides after the promoter sequence with complementary nucleotides added to the template strand, and
- producing a transcript.
3 more elements
A method of performing in vitro transcription comprising the steps of:
- combining a double stranded nucleic acid including a template strand and a coding strand, a cyanophage Syn5 RNAP or cyanophage Syn5 RNAP mutant Y564F, one or more of 2′-fluoro-dNTP, where N is A, G, C, T or U,
- wherein the double stranded nucleic acid includes a cyanophage Syn5 RNAP promoter sequence comprising SEQ ID NO:
- 1,
- wherein the coding strand includes an initiation sequence of 12 nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9-nt10-nt11-nt12 after the promoter sequence including 3 or more of the same rXMP where X is A, G, C, T or U within the first 9 nt,
- wherein one or more of sequence dGMP, dGMP-dCMP, dGMP-dCMP-dAMP, dGMP-dCMP-dAMP-dGMP, dGMP-dCMP-dAMP-dGMP-dAMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP, dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP-dAMP or dGMP-dCMP-dAMP-dGMP-dAMP-dAMP-dGMP-dAMP-dAMP is added to the coding strand after the promoter sequence and before the initiation sequence to create an altered initiation sequence of 12 nucleotides after the promoter sequence with complementary nucleotides added to the template strand, and
- producing a transcript.
3 more elements
A method of performing in vitro transcription comprising the steps of:
- combining a double stranded nucleic acid including a template strand and a coding strand, a cyanophage Syn5 RNAP or cyanophage Syn5 RNAP mutant Y564F, one or more of 2′-fluoro-dNTP, where N is A, G, C, T or U,
- wherein the double stranded nucleic acid includes a cyanophage Syn5 RNAP promoter sequence comprising SEQ ID NO:
- 1,
- wherein the coding strand includes an initiation sequence of 12 nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9-nt10-nt11-nt12 after the promoter sequence including 3 or more of the same dXMP where X is A, G, C, T or U within the first 9 nt,
- wherein the coding strand is modified to include 2 or fewer of the dXMP, where X is A, G, C, or T, within the first 9 nucleotides to improve the incorporation of 2′-fluoro-dXMP, where X is A, G, C or U, respectively, into the entire transcript, and
- producing a transcript.
3 more elements
A method of performing in vitro transcription comprising the steps of:
- combining a double stranded nucleic acid including a template strand and a coding strand, a cyanophage Syn5 RNAP or cyanophage Syn5 RNAP mutant Y564F, one or more of 2′-fluoro-dNTP, where N is A, G, C, T or U,
- wherein the double stranded nucleic acid includes a cyanophage Syn5 RNAP promoter sequence comprising SEQ ID NO:
- 1,
- wherein the coding strand includes an initiation sequence of 12 nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9-nt10-nt11-nt12 after the promoter sequence including 3 or more of the same rXMP where X is A, G, C, T or U within the first 9 nt,
- altering the promoter sequence to include 2 or fewer of the same dXMP where X is A, G, C, T or U within the first 9 nt.
2 more elements
A method of performing in vitro transcription comprising the steps of:
- combining a double stranded nucleic acid including a template strand and a coding strand, a cyanophage Syn5 RNAP or cyanophage Syn5 RNAP mutant Y564F, one or more of 2′-fluoro-dNTP, where N is A, G, C, T or U,
- wherein the double stranded nucleic acid includes a cyanophage Syn5 RNAP promoter sequence comprising SEQ ID NO:
- 1,
- wherein the coding strand includes an initiation sequence of 12 nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9-nt10-nt11-nt12 after the promoter sequence including 3 or more of the same dXMP where X is A, G, C, or T within the first 9 nt,
- wherein one or more dNMP is added to the coding strand after the promoter sequence with complementary nucleotides added to the template strand so that the coding strand is modified to include 2 or fewer of the dXMP (where X is A, G, C, or T) within the first 9 nt to facilitate the incorporation of 2′-fluoro-dXMP (where X is A, G, C, or U, respectively) into the entire transcript, and
- producing a transcript.
3 more elements
A method of performing in vitro transcription comprising the steps of:
- combining a double stranded nucleic acid including a template strand and a coding strand, a cyanophage Syn5 RNAP or cyanophage Syn5 RNAP mutant Y564F, one or more of 2′-fluoro-dNTP, where N is A, G, C, T or U,
- wherein the double stranded nucleic acid includes a cyanophage Syn5 RNAP promoter sequence comprising SEQ ID NO:
- 1,
- wherein the coding strand includes an initiation sequence of 12 nucleotides nt1-nt2-nt3-nt4-nt5-nt6-nt7-nt8-nt9-nt10-nt11-nt12 after the promoter sequence including 3 or more of the same dXMP where X is A, G, C, or T within the first 9 nt,
- wherein one or more dNMP coupled to a removable RNA element is added to the coding strand after the promoter sequence with complementary nucleotides added to the template strand so that the coding strand is modified to include 2 or fewer of the dXMP (where X is A, G, C, or T) within the first 9 nt to facilitate the incorporation of 2′-fluoro-dXMP (where X is A, G, C, or U, respectively) into the entire transcript, and
- producing a transcript.
3 more elements
Dependencies are inferred from the claim text.