T7 RNA polymerase variants
The present invention provides engineered RNA polymerase variants and compositions comprising these variants. The present invention further provides engineered T7 RNA polymerase variants and compositions comprising these variants. These variants have been evolved for selective incorporation of the m7G(5′)ppp(5′)m7G cap analog over GTP at the initiation of in vitro transcription. The present invention also provides methods for selective capping of RNA transcripts.
1. A method for producing capped mRNA, comprising performing an in vitro transcription reaction utilizing an engineered RNA polymerase, wherein said engineered RNA polymerase comprises a polypeptide sequence having at least 95% or more sequence identity to the reference sequence of SEQ ID NO:4, wherein said engineered RNA polymerase comprises substitution in said polypeptide sequence at position 664, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:4.
2. The method of claim 1 , wherein said engineered RNA polymerase further comprises at least one substitution or substitution set at one or more amino acid position selected from 397/513/635, 397/513/635/660, 513/635/660, 513/635/664, 513/660/664, and 660/664, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4.
3. The method of claim 1 , wherein said engineered RNA polymerase further comprises at least one substitution or substitution set at one or more amino acid position selected from 397, 397/513, 397/513/635, 397/513/635, 397/513/635/656, 397/513/635/656/660, 397/513/635/656/660/664, 397/513/635/656/664, 397/513/635/660, 397/513/635/660/664, 397/513/635/664, 397/513/656/660, 397/513/660, 397/513/660/664, 397/513/664, 397/513, 397/635, 397/635/656/660/664, 397/635/656/664, 397/635/660, 397/635/664, 397/635/664/850, 397/660, 397/664, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4.
4. The method of claim 1 , wherein said engineered RNA polymerase further comprises at least one substitution or substitution set at one or more amino acid position selected from 113/137/513, 136/357/404/514, 136/357/514, 136/394/404/446, 136/401, 136/401/404, 136/404/446, 136/404/514, 136/446, 136/514, 137, 137/401, 137/401/513, 137/401/513, 137/513, 137/513/621, 137/635, 137/656, 357/394/401/404/514, 357/394/446/514, 357/514, 394/446/514, 401/404, 401/404/514, 401/513/635, 401/635, 513/635, 513/635/656, 513/660, 635/656, 635/660, and 660, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4.
5. The method of claim 1 , wherein said engineered RNA polymerase comprises a polypeptide sequence that is at least 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered RNA polymerase variant set forth in SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 35, or 37.
6. The method of claim 1 , wherein said engineered RNA polymerase comprises a polypeptide sequence set forth in SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 35, or 37.
7. The method of claim 1 , wherein said engineered RNA polymerase generates greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more capped RNA transcripts relative to uncapped transcripts.
8. The method of claim 7 , wherein said engineered RNA polymerase generates greater than 90% or more capped RNA transcripts relative to uncapped transcripts.
9. The method of claim 8 , wherein said engineered RNA polymerase generates greater than 95% or more capped RNA transcripts relative to uncapped transcripts.
10. The method of claim 1 , wherein said engineered RNA polymerase comprises a polypeptide sequence having at least 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NO:4.