T7 RNA polymerase variants

Abstract

The present application relates to T7 RNA polymerase variants. The present invention provides engineered T7 RNA polymerase variants and compositions thereof. These variants have been evolved for selective incorporation of symmetrically capped GTP analogs relative to GTP at the initiation of in vitro transcription. The invention also provides methods of using the variants provided herein. The invention also provides an application of the composition provided by the invention.

Description

[0001] This application is a divisional application of an application filed on June 19, 2018, with application number 201880056274.9 and invention name “T7 RNA polymerase variant”.

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 527,764, filed on June 30, 2017, and U.S. Provisional Patent Application Serial No. 62 / 528,846, filed on July 5, 2017, both of which are incorporated by reference in their entirety for all purposes. Field of the Invention

[0003] The present invention provides engineered T7 RNA polymerase variants and compositions thereof. These variants have been evolved for selective incorporation of m7G(5')ppp(5')m7G cap analogs relative to GTP at the initiation of in vitro transcription. The present invention also provides methods of using the variants provided herein. The present invention also provides uses of the compositions provided herein. Reference to a sequence listing, table or computer program

[0004] An official copy of the sequence listing is submitted with the specification as an ASCII formatted text file via EFS-Web, with the file name "CX8-168CUSP1_ST25.txt", creation date July 5, 2017, and size of 184,320 bytes. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background of the Invention

[0005] T7 RNA polymerase (EC2.7.7.6) is a monomeric phage-encoded DNA-guided RNA polymerase that catalyzes the formation of RNA in a 5' to 3' direction. During transcription initiation, T7 RNA polymerase recognizes a specific promoter sequence (i.e., T7 promoter). The naturally occurring T7 RNA polymerase contains 883 amino acids. It is highly homologous to T3 RNA polymerase and to a certain extent homologous to SP6 RNA polymerase. T7 contains multiple domains, including an N-terminal domain, a "thumb", a "palm", and a "fingers" (see, e.g., Sousa and Mukherjee, Prog. Nucl. Acid Res. Mol. Biol., 73: 1-41

[2003] , and U.S. Patent No. 9,193,959). Between the initiation phase and the extension phase of the functional enzyme, the conformation of the N-terminal domain changes.

[0006] The cloning and expression of the gene encoding T7 RNA polymerase has been described (see, e.g., U.S. Patent No. 4,952,496). Due to its promoter specificity and high RNA polymerase activity, T7 has been used in a variety of applications. It can also be used for high-level expression of recombinant genes in Escherichia coli (E. coli) (see, Studier and Moffat, J. Mol. Biol., 18: 113-130

[1986] ). T7 is also used in a variety of nucleic acid amplification methods, including those for diagnostic methods. Since stability and thermostability are usually important considerations in the development of diagnostic method components, work on improving the thermostability and stability of T7 has been reported (see, e.g., U.S. Patent Nos. 9,193,959, 8,551,752 and 7,507,567, etc.). However, there is still a demand in the art for variant T7 enzymes that exhibit improved properties compared to naturally occurring enzymes. SUMMARY OF THE INVENTION

[0007] The present invention provides engineered T7 RNA polymerase variants and compositions thereof. These variants have been evolved for selective incorporation of symmetrically capped GTP analogs relative to GTP at the initiation of in vitro transcription. The present invention also provides methods for using the variants provided herein. The present invention also provides uses of the compositions provided herein.

[0008] The present invention provides an engineered RNA polymerase comprising a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence SEQ ID NO:4 and / or SEQ ID NO:15 or a functional fragment thereof, wherein the engineered RNA polymerase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4 or SEQ ID NO:15.

[0009] In some embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of 32 / 357, 49 / 642, 97 / 357, 136, 137, 160 / 643, 167 / 514, 250, 302 / 513, 314 / 401, 357, 392, 393, 394, 397, 401, 404, 444, 446, 478, 513, 514, 582, 635, 636, 637, 639, 642, 643, 645, 653, 656, 660, 660 / 806, 661, and 664, and / or any combination thereof, wherein amino acid positions are numbered with reference to SEQ ID NO:4. In some additional embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from: 32V / 357I, 49G / 642L, 97D / 357G, 136E / I, 137W, 160L / 643S, 167N / 514L, 250D, 302V / 513G, 314C / 401V, 357G / K / L / M / N / Q / R / S / T / V / W, 392D, 393L / Y, 394A / L / R, 397F / M / Q / W, 401A / I / L / S / V, 404E / Y, 444F / H / I / V, 446W / Y, 478F / M / W, 513C / F / K / L / R / T / W, 514F / I / L / Y, 582N, 635W, 636L, 637G / P / S, 639H, 642L, 643A, 645V, 653C, 656F / W, 660A / C / M / S / T / W, 660N / 806Y, 661E / Y, and 664W, and / or any combination thereof, wherein amino acid positions are numbered with reference to SEQ ID NO:4.In some additional embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of A32V / E357I, E49G / M642L, E97D / E357G, A136E / I, D137W, R160L / T643S, K167N / S514L, T250D, A302V / D513G, R314C / R401V, E357G / K / L / M / N / Q / R / S / T / V / W, Y392D, R393L / Y, K394A / L / R, A397F / M / Q / W, R401

[00136] The invention relates to proteins comprising the following: A / I / L / S / V, S404E / Y, N444F / H / I / V, M446W / Y, D478F / M / W, D513C / F / K / L / R / T / W, S514F / I / L / Y, A582N, S635W, V636L, T637G / P / S, R639H, M642L, T643A, A645V, F653C, Q656F / W, D660A / C / M / S / T / W, D660N / H806Y, T661E / Y, and P664W, and / or any combination thereof, wherein amino acid positions are numbered with reference to SEQ ID NO:4.

[0010] In some embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of 397 / 513 / 635, 397 / 513 / 635 / 660, 513 / 660 / 664, 513 / 635 / 660, 513 / 635 / 664, 513 / 660 / 664 and 660 / 664, and / or any combination thereof, wherein amino acid positions are numbered with reference to SEQ ID NO: 4. In some additional embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of 397F / 513W / 635W, 397F / 513Y / 635W, 397W / 513Y / 635W, 397W / 513W / 635W, 397W / 513Y / 635W / 660F, 397W / 513W / 635W / 660W, 397Y / 513W / 6 35W / 660F, 475V / 513W / 635W / 660Y, 513F / 660W / 664Y, 513W / 635W / 660F, 513W / 635W / 664W, 513Y / 635W / 660F, 513Y / 635W / 660Y, 513Y / 660W / 664W and 660Y / 664W, and / or any combination thereof, wherein amino acid positions are numbered with reference to SEQ ID NO:4. In some additional embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of A397F / D513W / S635W, A397F / D513Y / S635W, A397W / D513Y / S635W, A397W / D513W / S635W, A397W / D513Y / S635W / D660F, A397W / D513W / S635W / D660W, A397Y / D513W / S635W 5W / D660F, A475V / D513W / S635W / D660Y, D513F / D660W / P664Y, D513W / S635W / D660F, D513W / S635W / P664W, D513Y / S635W / D660F, D513Y / S635W / D660Y, D513Y / D660W / P664W and D660Y / P664W, and / or any combination thereof, wherein amino acid positions are numbered with reference to SEQ ID NO:4.

[0011] In some embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of: 397, 397 / 513, 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 / 66 4. 397 / 635 / 664 / 850, 397 / 660, 397 / 664, 397 / 660 / 664, 397 / 837, 399 / 635 / 660, 475 / 513 / 635 / 660, 513, 513 / 635, 513 / 635 / 656, 513 / 635 / 656 / 660, 513 / 635 / 656 / 664, 513 / 635 / 660, 513 / 635 / 660 / 664, 5 13 / 635 / 664, 513 / 656 / 660, 513 / 656 / 664, 513 / 660, 513 / 660 / 664, 513 / 664, 635, 635 / 656, 635 / 656 / 664, 635 / 660, 635 / 660 / 664, 635 / 664, 656 / 660 / 664, 658, 660, 660 / 664 and 664, and / or any combination thereof wherein amino acid positions are numbered with reference to SEQ ID NO:4. In some additional embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of 397F, 397F / 513F, 397F / 513F / 635F, 397F / 513F / 635W, 397F / 513F / 635W / 660W, 397F / 513W / 635W, 397F / 513W / 635W / 656F / 664F, 397F / 513F / 635W 3W / 664W, 397F / 513Y, 397F / 513Y / 635W, 397F / 513Y / 635W / 656W, 397F / 513Y / 635W / 664F, 37F / 513Y / 656H / 660W, 397F / 635F / 660W, 397F / 635W, 397F / 660W, 397F / 664W, 397W, 397W / 513F,<h2 style=";text-align:left;direction:ltr">397W / 513F / 635F / 656F / 660F / 664W, 397W / 513F / 635F / 660W, 397W / 513F / 635W, 397W / 513F / 660F, 397W / 513F / 660W, 397W / 513F / 660Y / 664F, 397W / 513 W、397W / 513W / 635F、397W / 513W / 635F / 656W / 660F、397W / 513W / 635W、397W / 513W / 635W / 656Y、397W / 513W / 635W / 660W、397W / 513W / 635W / 660W / 664Y、39 7W / 513W / 635W / 660Y / 664Y、397W / 513W / 660W、397W / 513W / 660W / 664Y、397W / 513W / 664F、397W / 513W / 664W、397W / 513Y / 635F、397W / 513Y / 635W / 656Y / 6 60W / 664W, 397W / 513Y / 635W, 397W / 513Y / 635W / 656Y / 664Y, 397W / 513Y / 635W / 660F, 397W / 635F, 397W / 635F / 664F, 397W / 635W, 397W / 635W / 656F / 664F 397W / 635W / 656F / 664W, 397W / 635W / 656F / 664Y, 397W / 635W / 660W, 397W / 635W / 660Y, 397W / 635W / 664F, 397W / 635W / 664W / 850T, 397W / 660F / 664F, 397 W / 660F / 664Y、397W / 660W、397W / 837K、397Y、397Y / 513F / 635W / 656F / 660W / 664F、397Y / 513F / 635W / 664W、397Y / 513W / 635F / 664Y、397Y / 513W / 635W / 66 0F、397Y / 513W / 656W / 660W、397Y / 513Y、397Y / 513Y / 635W、397Y / 635F、397Y / 635F / 660W、397Y / 635W、397Y / 635W / 656F / 660Y / 664W、397Y / 635W / 660F、3 97Y / 660F / 664W, 397Y / 664F, 399E / 635F / 660W, 475V / 513W / 635W / 660Y, 513F, 513F / 635F, 513F / 635W, 513F / 635W / 656W, 513F / 635W / 664W, 513F / 660W513F / 660W / 664F, 513F / 660W / 664Y, 513W, 513W / 635F, 513W / 635W, 513Y / 635F / 6 60F / 664Y, 513W / 635W / 656W / 660F, 513W / 635W / 660F, 513W / 635W / 664W, 513W / 65 6W / 664W, 513W / 656Y / 660W, 513W / 660F, 513W / 660W, 513Y / 635F, 513Y / 635F / 664 W、513Y / 635R / 656F / 664Y、513Y / 635W、513Y / 635W / 660F、513Y / 635W / 660Y、513Y / 635W / 660Y / 664F, 513Y / 660W, 513Y / 660W / 664W, 513Y / 660Y / 664F, 513Y / 664Y, 6 35F / 656F / 664Y, 635F / 656Y / 664W, 635F / 660W / 664F, 635W, 635W / 656W, 635W / 660 F, 635W / 660W, 635W / 664W, 656W / 660F / 664Y, 656W / 660W / 664Y, 658P, 660F, 660F / 664F, 660F / 664Y, 660W / 664F, 660Y / 664W, 664F and 664W, and / or any combination thereof, wherein amino acid positions are numbered with reference to SEQ ID NO:4. In some additional embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of A397F, A397F / D513F, A397F / D513F / S635F, A397F / D513F / S635W, A397F / D513F / S635W / D660W, A397F / D513W / S635W, A397F / D513W / S635W / Q656F / P664F, A397F / D513W / P664W, A397F / D513Y, A397F / D513Y / S635W, A397F / D513Y / S635W. 635W / Q656W, A397F / D513Y / S635W / P664F, A397F / D513Y / Q656H / D660W, A397F / S635F / D660W, A397F / S635W, A397F / D660W, A397F / P664W, A397W, A397W / D513F, A397W / D513F / S635F / Q656F / D660F / P664W, A397W / D513F / S635F / D660W, A397W / D513F / S635W, A397W / D513F / D660F,<h2 style=";text-align:left;direction:ltr">A397W / D513F / D660W, A397W / D513F / D660Y / P664F, A397W / D513W, A397W / D513W / S635F, A397W / D513W / S635F / Q656W / D660F, A397W / D513W / S635W, A39 7W / D513W / S635W / Q656Y、A397W / D513W / S635W / D660W、A397W / D513W / S635W / D660W / P664Y、A397W / D513W / S635W / D660Y / P664Y、A397W / D513W / D660W、A 397W / D513W / P664F, A397W / D513W / D660W / P664Y, A397W / D513W / P664W, A397W / D513Y / S635F, A397W / D513Y / S635W / Q656Y / D660W / P664W, A397W / D513Y / S635W, A397W / D513Y / S635W / Q656Y / P664Y, A397W / D513Y / S635W / D660F, A397W / S635F, A397W / S635F / P664F, A397W / S635W, A397W / S635W / Q656F / P66 4F、A397W / S635W / Q656F / P664W、A397W / S635W / Q656F / P664Y、A397W / S635W / D660W、A397W / S635W / D660Y、A397W / S635W / P664F、A397W / S635W / P664W / A850T, A397W / D660F / P664F, A397W / D660F / P664Y, A397W / D660W, A397W / E837K, A397Y, A397Y / D513F / S635W / Q656F / D660W / P664F, A397Y / D513F / S635 W / P664W, A397Y / D513W / S635F / P664Y, A397Y / D513W / S635W / D660F, A397Y / D513W / Q656W / D660W, A397Y / D513Y, A397Y / D513Y / S635W, A397Y / S635F, A3 97Y / S635F / D660W, A397Y / S635W, A397Y / S635W / Q656F / D660Y / P664W, A397Y / S635W / D660F, A397Y / D660F / P664W, A397Y / P664F, K399E / S635F / D660WA475V / D513W / S635W / D660Y, D513F, D513F / S635F, D513F / S635W, D513F / S635W / Q656W, D513F / S635W / P664W, D 513F / D660W, D513F / D660W / P664F, D513F / D660W / P664Y, D513W, D513W / S635F, D513W / S635W, D513Y / S635F / D6 60F / P664Y, D513W / S635W / Q656W / D660F, D513W / S635W / D660F, D513W / S635W / P664W, D513W / Q656W / P664W, D51 3W / Q656Y / D660W, D513W / D660F, D513W / D660W, D513Y / S635F, D513Y / S635F / P664W, D513Y / S635R / Q656F / P664Y , D513Y / S635W, D513Y / S635W / D660F, D513Y / S635W / D660Y, D513Y / S635W / D660Y / P664F, D513Y / D660W, D513Y / D660W / P664W, D513Y / D660Y / P664F, D513Y / P664Y, S635F / Q656F / P664Y, S635F / Q656Y / P664W, S635F / D660W / P6 64F, S635W, S635W / Q656W, S635W / D660F, S635W / D660W, S635W / P664W, Q656W / D660F / P664Y, Q656W / D660W / P664Y, L658P, D660F, D660F / P664F, D660F / P664Y, D660W / P664F, D660Y / P664W, P664F / W, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:4. ,

[0012] In some embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of: 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 / 51 3, 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 combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:15. In some additional embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of 113M / 137W / 513R, 136E / 357I / 404Y / 514I, 136I / 357I / 514F, 136I / 357K / 514F, 136I / 394R / 404Y / 446W, 136I / 401V, 136I / 401V / 404Y, 136E / 404Y / 446W, 136E / 404Y / 514F, 136I / 446W, 136E / 514F, 136I / 514I, 137W, 137W / 401I, 137W / 401S, 137W / 513R. 3W, 137W / 401V, 137W / 513W, 137W / 513R / 621R, 137W / 635W, 137W / 656F, 357N / 39 4R / 446W / 514I, 357R / 394R / 401V / 404Y / 514L, 357R / 514F, 394R / 446W / 514I, 40 1S / 513R / 635W, 401S / 635W, 401V / 404Y, 401V / 404Y / 514L, 513L / 635W, 513L / 660W, 513R / 635W / 656F, 635W / 656F, 635W / 660T and 660S / T, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:15.In some additional embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from the group consisting of L113M / D137W / D513R, A136E / E357I / S404Y / S514I, A136E / S404Y / M446W, A136E / S404Y / S514F, A136E / S514F, A136I / E357I / S514F, A136I / E 357K / S514F, A136I / K394R / S404Y / M446W, A136I / R401V, A136I / R401V / S404Y, A136I / M446W, A136I / S514I, D137W, D137W / R401I, D137W / R401S, D137W / R401S / D513R, D137W / R401S / D513W , D137W / R401V, D137W / D513W, D137W / D513R / K621R, D137W / S635W, D137W / Q656F, E357N / K394 R / M446W / S514I, E357R / K394R / R401V / S404Y / S514L, E357R / S514F, K394R / M446W / S514I, R40 1S / D513R / S635W, R401S / S635W, R401V / S404Y, R401V / S404Y / S514L, D513L / S635W, D513L / D660W, D513R / S635W / Q656F, S635W / Q656F, S635W / D660T and D660S / T, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 15.

[0013] In some other embodiments, the engineered RNA polymerase comprises a polypeptide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of the engineered RNA polymerase variant listed in at least one Table 5.3, Table 5.4, Table 5.5 and / or Table 5.6. In some other embodiments, the engineered RNA polymerase is a variant engineered polymerase provided in Table 5.3, Table 5.4, Table 5.5 and / or Table 5.6.

[0014] In still other embodiments, the engineered RNA polymerase comprises a polypeptide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 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: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39. In some other embodiments, the engineered RNA polymerase is a variant engineered polymerase set forth in SEQ ID NO: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39.

[0015] In some other embodiments, compared with wild-type T7 RNA polymerase, the engineered polymerase comprises at least one improved property. In some other embodiments, the engineered RNA polymerase exhibits at least one improved property, and the at least one improved property is selected from the improved selectivity of the cap analog relative to GTP during transcription initiation, improved protein expression, improved stability in storage buffer and improved stability under reaction conditions. In some other embodiments, the engineered RNA polymerase maintains RNA yield, transcription fidelity, thermal stability, protein expression, stability at -20 ° C or stability under reaction conditions comparable to wild-type T7 RNA polymerase. In some other embodiments, the engineered RNA polymerase is purified. In still some other embodiments, the invention provides a composition comprising at least one engineered RNA polymerase provided herein.

[0016] The present invention also provides a polynucleotide sequence encoding at least one engineered RNA polymerase provided herein. In some embodiments, the polynucleotide sequence encodes at least one engineered RNA polymerase, the engineered RNA polymerase comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence SEQ ID NO: 4 and / or SEQ ID NO: 15 or its functional fragment, wherein the engineered RNA polymerase is included in at least one substitution at one or more amino acid positions. In some other embodiments, the polynucleotide sequence encodes an engineered RNA polymerase provided herein, the engineered RNA polymerase comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39 or its functional fragment. In some other embodiments, the polynucleotide sequence encoding at least one engineered RNA polymerase comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and / or 38. In some other embodiments, the polynucleotide sequence is operably linked to a control sequence. In still some other embodiments, the polynucleotide sequence is codon optimized.

[0017] The present invention also provides an expression vector comprising at least one polynucleotide sequence provided herein. The present invention also provides a host cell comprising at least one expression vector provided herein. The present invention also provides a method for producing an engineered RNA polymerase in a host cell, the method comprising culturing a host cell provided herein under suitable culture conditions, thereby producing at least one engineered RNA polymerase. In some embodiments, the method further comprises reclaiming at least one engineered RNA polymerase from the culture and / or host cell. In some other embodiments, the method further comprises the step of purifying at least one engineered RNA polymerase.

[0018] The present invention also provides a method for producing a capped RNA transcript, the method comprising providing i) a composition comprising at least one engineered RNA polymerase provided herein and a dinucleotide cap analog, and ii) a DNA template; exposing the DNA template to the composition under conditions such that the engineered RNA polymerase produces a capped RNA transcript. In some embodiments of the method, the dinucleotide cap analog is α,γ-bis(N7-methylguanosine) triphosphate (m7G(5')ppp(5')m7G) or an anti-reverse cap analog 3'-O-Me-m 7 G(5')ppp(5')G. In some additional embodiments of the method, the dinucleotide cap analog is α,γ-bis(N7-methylguanosine)triphosphate. In some additional embodiments of the method, an inorganic pyrophosphatase is added to the reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The chemical structure of "m7G(5')ppp(5')m7G", also known as "α,γ-bis(N7-methylguanosine) triphosphate", "capped GTP", "symmetric cap analog" or "cap" is provided. Description of the invention

[0020] The present invention provides engineered T7 RNA polymerase variants and compositions thereof. These variants have been evolved for selective incorporation of m7G(5')ppp(5')m7G analogs relative to GTP at the initiation of in vitro transcription. The present invention also provides methods for using the variants provided herein. The present invention also provides uses of the compositions provided herein.

[0021] The present invention provides compositions and methods for effectively producing capped RNA. Most eukaryotic cell mRNA transcripts and most eukaryotic viral mRNA transcripts, as well as other forms of eukaryotic RNA (e.g., small nuclear RNA [snRNA] and pre-micro RNA [pre-miRNA]) are capped or "capped" at their 5' ends (see, e.g., U.S. Patent No. 8,846,348, which is incorporated herein by reference in its entirety). The cap is a guanine nucleoside connected to a triphosphate group via its 5' carbon, which is connected to the 5' carbon of the most 5' end nucleotide of the primary mRNA transcript. In most eukaryotic cells, the nitrogen at position 7 of the guanine in the cap nucleotide is methylated. The 5' caps of these RNA molecules play an important role in RNA stability and processing. The cap plays a very important role in mRNA metabolism and is required for the processing and maturation of RNA transcripts in the nucleus, the transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and the efficient translation of mRNA into protein (see, e.g., Lewis et al., Eur. J. Biochem., 247:461-9

[1997] .). The 5' cap structure is involved in the initiation of protein synthesis of eukaryotic mRNA and viral mRNA. The 5' cap also provides resistance to 5'-exonuclease activity, and its absence leads to rapid degradation of mRNA (see, e.g., U.S. Patent No. 8,836,348; and Furiichi et al., Nat., 266:235-9

[1997] ). Due to the advantages provided by capping, the efficient synthesis of capped RNA transcripts provides considerable value for a variety of functions, including but not limited to in vitro and in vivo protein synthesis.

[0022] In vitro synthesis of capped RNA can actually be accomplished by one of two enzymatic pathways. Vaccinia virus capping enzyme has two subunits and three enzymatic activities, adding a 7-methylguanosine cap structure to the 5' phosphate of in vitro transcribed RNA (see, Mao and Shuman, J. Biol. Chem., 269:24472-9

[1994] ).

[0023] An alternative method for in vitro synthesis of capped RNA is to incorporate the cap structure using cap analogs during in vitro transcription. These 7-methylguanosine-containing dinucleotides contain a 5'-5' triphosphate bond and are incorporated at the transcription start site to produce a 7-methylguanosine capped RNA product. T7 RNA polymerase naturally initiates transcription with the incorporation of guanosine relative to the cytosine residues on the template. In order to achieve high efficiency of capping, guanosine cap analogs must be present in the in vitro transcription reaction at a high concentration compared to GTP, and guanosine cap analogs compete with GTP for incorporation at the first position of mRNA. A useful feature of in vitro co-transcription capping is that because cap incorporation is achieved during initiation, the final sequence or secondary structure of the full-length mRNA does not affect the extent of capping. In addition, the process only requires the use of one enzyme (RNA polymerase) in a single-step reaction. The use of chemically synthesized cap analogs also increases the flexibility of using selectable nucleotide cap structures that are not well recognized by vaccinia capping enzymes for methylation. However, cap analogs are expensive relative to nucleotides and other reaction components and must be present in the reaction at high concentrations. The present invention provides T7 RNA polymerase variants that are selective for incorporating cap analogs relative to GTP at the start of transcription, thereby enabling efficient capping using reduced concentrations of cap analogs, providing a more cost-effective and scalable method for the production of capped RNA.

[0024] In some embodiments of the invention, an inorganic pyrophosphatase is present during the in vitro transcription reaction to degrade pyrophosphate, a product inhibitor of RNA polymerase transcription, into orthophosphate.

[0025] Abbreviations and definitions: Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, biochemistry, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly used in the art. Such technology is well-known and described in many textbooks and references well known to those skilled in the art. For chemical synthesis and chemical analysis, standard techniques or their modified forms are used. All patents, patent applications, articles and publications mentioned herein (both above and below) are expressly incorporated herein by reference.

[0026] Although any suitable method and material similar to or equal to those methods and materials described herein can be used in the practice of the present invention, some methods and materials are described herein. It should be understood that the present invention is not limited to the specific methodology, scheme and reagent described, because these can depend on the environment in which those skilled in the art use it and change. Therefore, the terms defined below are more fully described by reference to the application as a whole. All patents, patent applications, articles and publications mentioned herein (both above and below) are expressly incorporated herein by reference.

[0027] Furthermore, as used herein, the singular "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0028] Numerical ranges include the numbers that define the ranges. Therefore, each numerical range disclosed herein is intended to encompass each narrower numerical range that falls within such a wider numerical range, as if such narrower numerical ranges were all explicitly written out herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein includes each lower (or higher) numerical limit, as if such lower (or higher) numerical limits were explicitly written out herein.

[0029] The term "about" means an acceptable error for a particular value. In some cases, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some cases, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0030] Additionally, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the application as a whole.

[0031] Accordingly, the terms defined immediately below are more fully defined by reference to the application as a whole. However, to aid in the understanding of the present invention, a number of terms are defined below.

[0032] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0033] As used herein, the term "comprising" and its cognates are used in their inclusive sense (ie, equivalent to the term "including" and its corresponding cognates).

[0034] "EC" number refers to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reaction that the enzyme catalyzes.

[0035] "ATCC" refers to the American Type Culture Collection, whose biological collections include genes and strains.

[0036] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0037] As used herein, "T7 RNA polymerase" refers to a monomeric T7 bacteriophage-encoded DNA-directed RNA polymerase that catalyzes the formation of RNA in the 5' to 3' direction.

[0038] As used herein, the term "cap" refers to a guanosine nucleoside that is linked via its 5' carbon to a triphosphate group that is in turn linked to the 5' carbon of the 5'-most nucleotide of the mRNA transcript. In some embodiments, the nitrogen at position 7 of the guanine in the cap is methylated.

[0039] As used herein, the terms "capped RNA," "5'-capped RNA," and "capped mRNA" refer to RNA and mRNA, respectively, that comprise a cap.

[0040] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleosides covalently linked together. Polynucleotides can completely contain ribonucleotides (i.e., RNA), completely contain 2' deoxyribonucleotides (i.e., DNA) or contain a mixture of ribonucleotides and 2' deoxyribonucleotides. Although nucleosides are typically linked together via standard phosphodiester bonds, polynucleotides can include one or more non-standard bonds. Polynucleotides can be single-stranded or double-stranded, or can include both single-stranded regions and double-stranded regions. In addition, although polynucleotides typically contain naturally occurring coding nucleoside bases (i.e., adenine, guanine, uracil, thymine and cytosine), it can include one or more modified and / or synthetic nucleoside bases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleoside bases are nucleoside bases encoding amino acid sequences.

[0041] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (eg, glycosylation or phosphorylation).

[0042] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0043] The abbreviations for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Are or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0044] When a three-letter abbreviation is used, unless explicitly preceded by "L" or "D" or clear from the context in which the abbreviation is used, the amino acid may be referred to about the α-carbon (C α ) is L-configuration or D-configuration. For example, "Ala" represents alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When single-letter abbreviations are used, capital letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter abbreviations or three-letter abbreviations (or mixtures thereof), the sequence is presented in the amino (N) to carboxyl (C) direction according to conventional conventions.

[0045] Abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically stated otherwise, abbreviated nucleosides may be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides may be designated as ribonucleosides or 2'-deoxyribonucleosides on an individual or aggregate basis. When a nucleic acid sequence is presented as a string of single-letter abbreviations, the sequence is presented in a 5' to 3' direction according to conventional practice, and the phosphate is not shown.

[0046] The terms "engineered," "recombinant," "non-naturally occurring," and "variant" when used in reference to cells, polynucleotides or polypeptides refer to materials that have been modified or are identical to native or natural forms of that material that do not originally exist in nature but that are produced or obtained by synthetic materials and / or manipulation using recombinant techniques.

[0047] As used herein, "wild-type" and "naturally occurring" refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0048] "Coding sequence" refers to that portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0049] The term "sequence identity percentage (%)" is used herein to refer to the comparison between polynucleotides and polypeptides, and is determined by comparing two optimally aligned sequences across a comparison window, wherein for optimal alignment of the two sequences, the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., spaces) compared to the reference sequence. The percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences or the number of positions where the nucleic acid base or amino acid residue is aligned with a space to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. It is understood by those skilled in the art that there are many established algorithms that can be used to compare two sequences. Optimal alignment of sequences for comparison can be performed by, for example, the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443 [1970), the search by similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection, as is known in the art. Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (see, respectively, Altschul et al., J. Mol. Biol., 215: 403-410

[1990] ; and Altschul et al., Nucleic Acids Res., 25: 3389-3402

[1977] ). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information website. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (see, Altschul et al., supra).These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence to the extent that the cumulative alignment score cannot be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hit in each direction is terminated when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score reaches 0 or below 0 due to the accumulation of one or more negative scoring residue alignments; or when either sequence end is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as default values: a word length (W) of 11, an expectation value (E) of 10, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses the following as defaults: a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary determinations of sequence alignments and % sequence identity can be made using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided.

[0050] "Reference sequence" refers to a designated sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, a length of at least 100 residues, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) contain a similar sequence (i.e., a portion of a complete sequence) between the two sequences, and (2) can also contain a different sequence between the two sequences, a sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides on a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein a reference sequence is a sequence that can have one or more changes in the primary sequence.

[0051] "Comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window can contain 20% or less additions or deletions (i.e., gaps) for optimal alignment of the two sequences compared to the reference sequence (which does not contain additions or deletions). The comparison window can be longer than 20 consecutive residues, and optionally includes windows of 30, 40, 50, 100 or more.

[0052] In the context of numbering for a given amino acid or polynucleotide sequence, "corresponding to," "referenced to," or "relative to" refers to the numbering of the residues of the reference sequence as specified when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to the reference sequence, rather than being specified by the actual numerical positions of the residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered T7 RNA polymerase, can optimize the residue matching between the two sequences by introducing gaps to align with the reference sequence. In these cases, despite the presence of gaps, the numbering of the residues in a given amino acid or polynucleotide sequence is made according to the reference sequence to which it is aligned.

[0053] "Amino acid difference" or "residue difference" refers to the difference of the amino acid residue at one position of a polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. The position of the amino acid difference is usually referred to as "Xn" in this article, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "the residue difference at position X93 compared to SEQ ID NO:2" refers to the difference of the amino acid residue at the polypeptide position corresponding to position 93 of SEQ ID NO:2. Therefore, if the reference polypeptide SEQ ID NO:2 has serine at position 93, "the residue difference at position X93 compared to SEQ ID NO:2" refers to the amino acid substitution of any residue except serine at the polypeptide position corresponding to position 93 of SEQ ID NO:2. In most cases herein, the specific amino acid residue difference at one position is indicated as "XnY", where "Xn" specifies the corresponding position as described above, and "Y" is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue different from the reference polypeptide). In some cases (e.g., in the tables provided in the Examples herein), the disclosure also provides specific amino acid differences represented by the conventional symbol "AnB", where A is a single-letter identifier for a residue in a reference sequence, "n" is the number of a residue position in a reference sequence, and B is a single-letter identifier for a residue substitution in the sequence of an engineered polypeptide. In some cases, a polypeptide of the disclosure may include one or more amino acid residue differences relative to a reference sequence, indicated by a column of designated positions where residue differences exist relative to the reference sequence. In some embodiments, in the case where more than one amino acid can be used in a specific residue position of a polypeptide, a variety of amino acid residues that can be used are separated by " / " (e.g., X10H / X10P or X10H / P). In some embodiments, an enzyme variant includes more than one substitution. For ease of reading, these substitutions are separated by slashes (e.g., C14A / K122A). The present application includes engineered polypeptide sequences containing one or more amino acid differences, and the one or more amino acid differences include either / or both of conservative and non-conservative amino acid substitutions.

[0054] "Conservative amino acid substitutions" refer to replacement of a residue with a different residue having a similar side chain, and thus generally include replacement of an amino acid in a polypeptide with an amino acid from the same or similar defined class of amino acids. For example, but not limited to, an amino acid with an aliphatic side chain may be replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain may be replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain may be replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain may be replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain may be replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic amino acid or a hydrophilic amino acid may be replaced by another hydrophobic amino acid or a hydrophilic amino acid, respectively.

[0055] "Non-conservative substitutions" refer to substitutions of amino acids in a polypeptide with amino acids having significantly different side chain properties. Non-conservative substitutions may use amino acids between defined groups rather than within defined groups and affect: (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., substitution of glycine with proline), (b) charge or hydrophobicity, or (c) side chain bulk. For example, but not limited to, exemplary non-conservative substitutions may be substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

[0056] "Deletion" refers to a polypeptide modification by removing one or more amino acids from a reference polypeptide. Deletion can include removing 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total amino acids that make up the reference enzyme, or up to 20% of the total amino acids that make up the reference enzyme, while retaining enzymatic activity and / or retaining the improved properties of engineered enzymes. Deletion can involve the interior and / or ends of a polypeptide. In various embodiments, deletion can include continuous segments or can be discontinuous.

[0057] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. Insertions can be internal to a polypeptide or inserted at the carboxyl or amino terminus. Insertions as used herein include fusion proteins known in the art. Insertions can be continuous stretches of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0058] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that are naturally associated with it. The term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). The recombinant T7 RNA polymerase polypeptide can be present in a cell, in a cell culture medium, or prepared in a variety of forms (such as a lysate or an isolated preparation). Therefore, in some embodiments, the recombinant T7 RNA polymerase polypeptide can be an isolated polypeptide.

[0059] "Substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis, the polypeptide species is more abundant than any other individual macromolecular species in the composition), and is generally a substantially pure composition when the target species constitutes at least about 50% of the macromolecular species present by mole or % weight. Typically, a substantially pure T7 RNA polymerase composition constitutes about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species present in the composition by mole or % weight. In some embodiments, the target species is purified to substantial homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species. In some embodiments, the isolated recombinant T7 RNA polymerase polypeptide is a substantially pure polypeptide composition.

[0060] "Improved enzymatic properties" of a T7 RNA polymerase refers to an engineered T7 RNA polymerase polypeptide that exhibits an improvement in any enzymatic property compared to a reference T7 RNA polymerase polypeptide and / or a wild-type T7 RNA polymerase polypeptide and / or another engineered T7 RNA polymerase polypeptide. Improved properties include, but are not limited to, properties such as: increased selectivity for cap analogs compared to GTP, increased replication fidelity, increased RNA yield, increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzyme activity, increased substrate specificity or affinity, increased specific activity, increased tolerance to substrate or end-product inhibition (including pyrophosphate), increased chemical stability, improved solvent stability, increased tolerance to acidic or alkaline pH, increased tolerance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, and altered temperature profile.

[0061] "Increased enzyme activity" or "enhanced catalytic activity" refers to an improved property of an engineered T7 RNA polymerase, which can be expressed as an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the percentage conversion of substrate to product (e.g., the percentage conversion of a starting amount of substrate to product using a specified amount of variant T7 RNA polymerase over a specified period of time) compared to a reference T7 RNA polymerase. Exemplary methods for determining enzyme activity are provided in the Examples. Any property associated with enzyme activity may be affected.

[0062] "Hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washes of different but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity, preferably about 75% identity, about 85% identity, and greater than about 90% identity with the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS, followed by washing at 42°C in 0.2×SSPE, 0.2% SDS. "High stringency hybridization" generally refers to a thermal melting temperature T determined under solution conditions with a defined polynucleotide sequence. m Conditions that differ by about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow only the hybridization of those nucleic acid sequences that form stable hybrids at 65°C in 0.018 M NaCl (i.e., if the hybrid is unstable at 65°C in 0.018 M NaCl, it will be unstable under high stringency conditions as considered herein). For example, high stringency conditions can be provided by hybridizing in conditions equivalent to 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS at 42°C, followed by washing in 0.1×SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization in conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v) SDS at 65°C and washing in 0.1×SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions and moderate stringency conditions are described in the references cited above.

[0063] "Codon optimized" refers to changing the codons of the polynucleotide encoding the protein to those codons preferentially used in a specific organism so that the encoded protein is more effectively expressed in the organism of interest. Although the genetic code is degenerate, i.e., most amino acids are represented by several codons referred to as "synonyms" or "synonymous" codons, it is well known that the codon usage of a specific organism is non-random and has a preference for specific codon triplets. With respect to a given gene, a gene with a common function or ancestral origin, a highly expressed protein relative to a low copy number protein and an aggregated protein coding region of an organism's genome, this codon usage preference may be higher. In some embodiments, the polynucleotide encoding T7 RNA polymerase can be codon optimized for optimal production from a host organism selected for expression.

[0064] "Control sequences" herein refer to all components that are necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present application. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, start sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. Control sequences may be provided together with linkers for the purpose of introducing specific restriction sites that facilitate the connection of control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0065] "Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide of interest.

[0066] "Promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence comprises a transcriptional control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0067] "Suitable reaction conditions" refer to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, cosolvent, etc.), under which the T7 RNA polymerase polypeptide of the present application can convert the substrate into the desired product compound.

[0068] In the context of an enzymatic conversion reaction, "substrate" refers to the compound or molecule that is acted upon by the T7 RNA polymerase polypeptide.

[0069] In the context of an enzymatic conversion process, "product" refers to the compound or molecule resulting from the action of the T7 RNA polymerase polypeptide on the substrate.

[0070] As used herein, the term "culturing" refers to the growth of a population of microbial cells under any suitable conditions (eg, using liquid, gel, or solid culture media).

[0071] Recombinant polypeptide can be produced using any suitable method known in the art. The gene encoding the wild-type polypeptide of interest can be cloned in a vector such as a plasmid, and expressed in a desired host such as Escherichia coli, Saccharomyces cerevisiae (S.cerevisiae) etc. The variant of the recombinant polypeptide can be produced by a variety of methods known in the art. In fact, there are a variety of different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. It is available to produce specific substitutions at the defined amino acid place (site-specific), produce specific or random mutations in the local region of the gene (region-specific), or produce random mutagenesis (for example, saturation mutagenesis) in the whole gene. Many suitable methods known to those skilled in the art produce enzyme variants, including but not limited to site-directed mutagenesis, cassette mutagenesis, gene synthesis, error-prone PCR, rearrangement and chemical saturation mutagenesis using PCR to single-stranded DNA or double-stranded DNA, or any other suitable method known in the art. Non-limiting examples of methods for DNA and protein engineering are provided in the following patents: U.S. Patent No. 6,117,679; U.S. Patent No. 6,420,175; U.S. Patent No. 6,376,246; U.S. Patent No. 6,586,182; U.S. Patent No. 7,747,391; U.S. Patent No. 7,747,393; U.S. Patent No. 7,783,428; and U.S. Patent No. 8,383,346. After variants are generated, they can be screened for any desired properties (e.g., high or increased activity or low or decreased activity, increased thermal activity, increased thermal stability and / or acidic pH stability, etc.).

[0072] In some embodiments, a "recombinant T7 RNA polymerase polypeptide" (also referred to herein as an "engineered T7 RNA polymerase polypeptide," "variant T7 RNA polymerase," and "T7 RNA polymerase variant") may be used.

[0073] As used herein, "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, a vector is an expression vector operably linked to a suitable control sequence capable of achieving expression of a polypeptide encoded in a DNA sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgenic) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

[0074] As used herein, the term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0075] As used herein, the term "production" refers to the production of proteins and / or other compounds from a cell. It is intended that the term encompasses any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of a polypeptide from a cell.

[0076] As used herein, an amino acid or nucleotide sequence (eg, a promoter sequence, signal peptide, terminator sequence, etc.) is heterologous to another sequence to which it is operably linked if the two sequences are not associated in nature.

[0077] As used herein, the terms "host cell" and "host strain" refer to suitable hosts for expression vectors containing DNA provided herein (e.g., polynucleotides encoding T7 RNA polymerase variants). In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques as known in the art.

[0078] The term "analogue" when used in reference to a polypeptide means a polypeptide having greater than 70% sequence identity but less than 100% sequence identity (e.g., greater than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to a reference polypeptide. In some embodiments, an analogue means a polypeptide comprising one or more non-naturally occurring amino acid residues and naturally occurring amino acids, wherein the one or more non-naturally occurring amino acid residues include, but are not limited to, homoarginine, ornithine, and norvaline. In some embodiments, an analogue further comprises one or more D-amino acid residues and non-peptide bonds between two or more amino acid residues.

[0079] The term "effective amount" means an amount sufficient to produce the desired result. One of ordinary skill in the art can determine what the effective amount is by using routine experimentation.

[0080] The terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0081] As used herein, "composition" and "formulation" encompass products comprising at least one engineered T7 RNA polymerase of the invention, which products are intended for any suitable use (eg, research, diagnostics, etc.).

[0082] The term "transcription" is used to refer to the process by which a portion of a DNA template is copied into RNA by the action of RNA polymerase.

[0083] The term "DNA template" is used to refer to a double-stranded or single-stranded DNA molecule that comprises a promoter sequence and a sequence encoding a transcribed RNA product.

[0084] The term "promoter" is used to refer to a DNA sequence recognized by RNA polymerase as a transcription start site. The promoter recruits RNA polymerase and, in the case of T7 RNA polymerase, determines the start site of transcription.

[0085] The term "RNA polymerase" is used to refer to a DNA-directed RNA polymerase that copies a DNA template into RNA polynucleotides by stepwise incorporation of nucleotide triphosphates into the growing RNA polymer.

[0086] The terms "messenger RNA" and "mRNA" are used to refer to RNA molecules that encode proteins. The protein is decoded by translation.

[0087] The terms "7-methylguanosine cap", "7meG", "five-prime cap" and "5' cap" are used to refer to a specific modified nucleotide structure present at the 5' end of a eukaryotic mRNA. The 7-methylguanosine cap structure is attached to the first nucleotide in the mRNA via a 5' to 5' triphosphate bond. In vivo, this cap structure is added to the 5' end of the nascent mRNA by the sequential action of a variety of enzymes. In vitro, the cap can be incorporated directly at the initiation of transcription by RNA polymerase by using a cap analog.

[0088] The term "cap analog" refers to a dinucleotide containing a 5'-5' di-, tri- or tetraphosphate bond. One end of the dinucleotide terminates in a guanosine or substituted guanosine residue; from this end, RNA polymerase will initiate transcription by extending from the 3' hydroxyl. The other end of the dinucleotide is a guanosine that mimics the eukaryotic cap structure and will typically have a 7-methyl, 7-benzyl or 7-ethyl substitution and / or a 7-aminomethyl or 7-aminoethyl substitution. In some cases, the nucleotide is also substituted with a methoxy group at the 3' hydroxyl group to prevent transcription from initiating from the cap end of the molecule.

[0089] The terms "ARCA" and "antiretrograde cap analog" refer to chemically modified forms of cap analogs that are designed to maximize the efficiency of in vitro translation by ensuring that the cap analog is properly incorporated into the transcript in the correct orientation. These analogs can be used to enhance translation. In some embodiments, ARCA, which is known in the art, can be used (e.g., Peng et al., Org. Lett., 4:161-164

[2002] ).

[0090] The term "riboswitch" is used to refer to an autocatalytic RNA enzyme that cleaves itself or another RNA in the presence of a ligand.

[0091] The term "fidelity" is used to refer to the accuracy with which an RNA polymerase transcribes or copies a DNA template into an RNA polynucleotide. Inaccurate transcription can result in single nucleotide polymorphisms (SNPs) or insertions and deletions (Indels).

[0092] The term "single nucleotide polymorphism" or "SNP" refers to a change in a nucleotide present at a single position in a polynucleotide. In the case of transcription, a SNP can be caused by the misincorporation of a non-complementary ribonucleotide (A, C, G or U) by RNA polymerase at a position on the DNA template.

[0093] The term "indel" is used to refer to the insertion or deletion of one or more polynucleotides. In the case of transcription by RNA polymerase, an indel error can be caused by the addition of one or more extra ribonucleotides or the failure to incorporate one or more nucleotides at a position on the DNA template.

[0094] The term "selectivity" is used to refer to the property of an enzyme to have higher activity towards one substrate as compared to another during a catalytic reaction. In the case of co-transcriptional capping, RNA polymerases can have high or low selectivity for cap analogs compared to GTP.

[0095] The term "inorganic pyrophosphatase" is used to refer to an enzyme that degrades inorganic pyrophosphate to orthophosphate.

[0096] Engineered T7 RNA polymerase activity: In some embodiments, the engineered T7 RNA polymerase exhibiting improved properties has at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% amino acid sequence identity to SEQ ID NO: 4 and / or SEQ ID NO: 15, and has an amino acid residue difference at one or more amino acid positions compared to SEQ ID NO: 4 and / or SEQ ID NO: 15 (such as a difference from SEQ ID NO: 4 and / or SEQ ID NO: 15 or a difference from SEQ ID NO: 4 and / or SEQ ID NO: 15). NO:15 has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more amino acid sequence identity, with amino acid residue differences at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20 or more amino acid positions. In some embodiments, the residue differences at one or more positions compared to SEQ ID NO:4 will include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the engineered T7 RNA polymerase polypeptide is a polypeptide listed in Table 5.3, Table 5.5 and / or Table 5.6. In some embodiments, the engineered T7 RNA polymerase polypeptide is selected from SEQ ID NO: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39. In some embodiments, the residue difference at one or more positions compared to SEQ ID NO: 15 will include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the engineered T7 RNA polymerase polypeptide is a polypeptide listed in Table 5.4. In some embodiments, the engineered T7 RNA polymerase polypeptide is selected from SEQ ID NO: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39.

[0097] In some embodiments, the engineered T7 RNA polymerase polypeptide comprises a functional fragment of the engineered T7 RNA polymerase polypeptide encompassed by the present invention. The functional fragment has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the activity of the engineered T7 RNA polymerase polypeptide (i.e., parental engineered T7 RNA polymerase) from which it is derived. The functional fragment comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% of the parental sequence of the engineered T7 RNA polymerase. In some embodiments, the functional fragment is truncated by less than 5, less than 10, less than 15, less than 20, less than 25, less than 30, less than 35, less than 40, less than 45, and less than 50 amino acids.

[0098] Polynucleotides encoding engineered polypeptides, expression vectors and host cells: The invention provides polynucleotides encoding engineered T7 RNA polymerase polypeptides described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing a polypeptide. An expression construct comprising a heterologous polynucleotide encoding an engineered T7 RNA polymerase polypeptide can be introduced into an appropriate host cell to express the corresponding T7 RNA polymerase polypeptide.

[0099] As will be apparent to the skilled person, the availability of protein sequences and knowledge of codons corresponding to a variety of amino acids provide a description of all polynucleotides capable of encoding a target polypeptide. The degeneracy of the genetic code, in which the same amino acid is encoded by selectable or synonymous codons, allows the preparation of a very large number of nucleic acids, all of which encode engineered T7 RNA polymerase polypeptides. Therefore, knowing a specific amino acid sequence, one skilled in the art can prepare any number of different nucleic acids by simply changing the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates each possible change of a polynucleotide that can be prepared to encode a polypeptide described herein by selecting a combination based on possible codon selections, and for any polypeptide described herein, including variants provided in Table 5.3, Table 5.4, Table 5.5 and / or Table 5.6, all such changes are considered to be specifically disclosed.

[0100] In various embodiments, codons are preferably selected to conform to the host cell in which the protein is produced. For example, the preferred codons used in bacteria are used for expression in bacteria. Therefore, the codon-optimized polynucleotides encoding the engineered T7 RNA polymerase polypeptides comprise preferred codons at about 40%, 50%, 60%, 70%, 80% or greater than 90% of the codon positions in the full-length coding region.

[0101] In some embodiments, as described above, the polynucleotide encodes an engineered polypeptide having T7 RNA polymerase activity having the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 15, or an amino acid sequence of any variant disclosed in Table 5.3, Table 5.4, Table 5.5 and / or Table 5.6 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity, and an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity, and a polypeptide having SEQ ID NO: 15. NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39 or the amino acid sequence of any variant disclosed in Table 5.3, Table 5.4, Table 5.5 and / or Table 5.6 has one or more residue differences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions). In some embodiments, the reference sequence is selected from SEQ ID NO: 4 and / or SEQ ID NO: 15. In some embodiments, the polynucleotide encodes an engineered polypeptide having T7 RNA polymerase activity having properties disclosed herein, wherein the polypeptide comprises an amino acid sequence that, when optimally aligned with the polypeptide of SEQ ID NO: 4 and / or SEQ ID NO: 15, has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4 and / or SEQ ID NO: 15, and has one or more residue differences compared to SEQ ID NO: 4 and / or SEQ ID NO: 15 at a residue position selected from those provided in Table 5.3, Table 5.4, Table 5.5 and / or Table 5.6.

[0102] In some embodiments, the polynucleotide encoding the engineered T7 RNA polymerase polypeptide comprises a polynucleotide sequence selected from the group consisting of polynucleotide sequences encoding SEQ ID NOs: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39. In some embodiments, the polynucleotide encoding the engineered T7 RNA polymerase polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 93%, 95%, 96%, 97%, 98%, 99% nucleotide residue identity to SEQ ID NOs: 3, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38. In some embodiments, the polynucleotide encoding the engineered T7 RNA polymerase polypeptide comprises a polynucleotide sequence selected from SEQ ID NO: 3, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.

[0103] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from SEQ ID NO: 3 and / or SEQ ID NO: 14, or a complementary sequence thereof, or a polynucleotide sequence encoding any variant T7 RNA polymerase polypeptide provided herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a T7 RNA polymerase polypeptide comprising an amino acid sequence having one or more residue differences compared to SEQ ID NO: 4 and / or SEQ ID NO: 15 at a residue position selected from any of the positions listed in Table 5.3, Table 5.4, Table 5.5, and / or Table 5.6.

[0104] In some embodiments, the polynucleotides encoding the separation of any engineered T7 RNA polymerase polypeptide provided herein are operated in a variety of ways to provide expression of the polypeptide. In some embodiments, the polynucleotides encoding the polypeptide are provided as expression vectors, wherein there are one or more control sequences to regulate the expression of the polynucleotides and / or polypeptides. Depending on the expression vector, the operation of the polynucleotides of the separation before the polynucleotides of the separation are inserted into the vector can be desired or necessary. The technology of utilizing recombinant DNA methods to modify polynucleotides and nucleotide sequences is well known in the art.

[0105] In some embodiments, control sequences include, among other sequences, a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. As known in the art, suitable promoters can be selected based on the host cell used. Exemplary promoters for filamentous fungal host cells include promoters obtained from the following genes: Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 99 / 0545 ... 96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast 8:423-488

[1992] ). Exemplary promoters for mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), Homo sapiens phosphoglycerate kinase, β-actin, elongation factor-1a, or glyceraldehyde-3-phosphate dehydrogenase, or Gallus gallus β-actin.

[0106] In some embodiments, the control sequence is a suitable transcription terminator sequence, which is a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably connected to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the following genes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., supra). Exemplary terminators for mammalian cells include, but are not limited to, those terminators from cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), or Homo sapiens growth hormone.

[0107] In some embodiments, the control sequence is a suitable leader sequence, and the leader sequence is a non-translated region of mRNA that is very important to being translated by the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence of the coded polypeptide. Any leader sequence that has function in the host cell of selection can be used. The exemplary leader sequence for filamentous fungal host cells is obtained from the following gene: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leader sequences for yeast host cells include, but are not limited to, those obtained from the following gene: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0108] The control sequence can also be a polyadenylation sequence, which is a sequence operably linked to the 3' end of the nucleic acid sequence, and which, when transcribed, is recognized by the host cell as a signal for adding polyadenosine residues to the mRNA transcribed. Any polyadenylation sequence that is functional in the host cell of selection can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to those from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthetase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (see, e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990

[1995] ).

[0109] In some embodiments, control sequence is a signal peptide coding region, which encodes the amino acid sequence connected to the amino terminal of the polypeptide and directs the encoded polypeptide to the secretory pathway of the cell. The 5' end of the coding sequence of the nucleic acid sequence can inherently include a signal peptide coding region, which conforms to the translation reading frame and is naturally connected to the segment of the coding region of the secretory polypeptide of the coding sequence. Alternatively, the 5' end of the coding sequence can include a signal peptide coding region foreign to the coding sequence. Any signal peptide coding region in the secretory pathway of the host cell selected by directing the expressed polypeptide can be used for the expression of the engineered T7 RNA polymerase polypeptide provided herein. The effective signal peptide coding region for filamentous fungal host cells includes but is not limited to the signal peptide coding region obtained from the following gene: Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include, but are not limited to, those from the gene for immunoglobulin gamma (IgG).

[0110] In some embodiments, the control sequence is a propeptide coding region, which encodes an amino acid sequence positioned at the amino terminus of a polypeptide. The polypeptide produced is referred to as a "proenzyme," "propolypeptide," or in some cases, a "zymogen." The propolypeptide can be converted into a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

[0111] In another aspect, the present invention also provides recombinant expression vectors, which include polynucleotides encoding engineered T7 RNA polymerase polypeptides and one or more expression regulatory regions, such as promoters and terminators, replication origins, etc., depending on the type of host into which the polynucleotides are introduced. In some embodiments, the various nucleic acids described above and control sequences are linked together to produce a recombinant expression vector, which includes one or more convenient restriction sites to allow the nucleic acid sequence encoding variant T7 RNA polymerase polypeptides to be inserted or replaced at such sites. Alternatively, the polynucleotide sequence of the present invention is expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the polynucleotide sequence into an appropriate vector for expression. When creating an expression vector, the coding sequence is located in the vector so that the coding sequence is operably connected to an appropriate control sequence for expression.

[0112] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and can result in the expression of variant T7 RNA polymerase polynucleotide sequences. The selection of the vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear plasmid or a closed circular plasmid.

[0113] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, and its replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some selectable embodiments, the vector may be a vector that is integrated into the genome and replicated with the chromosome into which it is integrated when introduced into the host cell. In addition, a single vector or plasmid or two or more vectors or plasmids or transposons may be used, and the two or more vectors or plasmids together contain the total DNA to be introduced into the genome of the host cell.

[0114] In some embodiments, the expression vector preferably comprises one or more selectable markers that allow easy selection of transformed cells. "Selectable markers" are genes whose products provide biocide or virus tolerance, tolerance to heavy metals, prototrophy to auxotrophic types, etc. Suitable markers for yeast host cells include but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include but are not limited to amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase) and trpC (anthranilic acid synthetase), and equivalents thereof. In another aspect, the present invention provides a host cell comprising a polynucleotide encoding at least one engineered T7 RNA polymerase polypeptide of the present application, the polynucleotide being operably linked to one or more control sequences for expressing the engineered T7 RNA polymerase in the host cell. Host cells for expressing polypeptides encoded by the expression vector of the present invention are well known in the art, and include but are not limited to fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [e.g., ATCC deposit number 201178]); insect cells (e.g., Drosophila S2 cells and Spodoptera Sf9 cells), plant cells, animal cells (e.g., CHO, COS and BHK) and human cells (e.g., HEK293T, human fibroblasts, THP-1, Jurkat and Bowes melanoma cell lines).

[0115] Therefore, in another aspect, the present invention provides a method for producing an engineered T7 RNA polymerase polypeptide, wherein the method comprises culturing a host cell capable of expressing a polynucleotide encoding an engineered T7 RNA polymerase polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises the step of isolating and / or purifying the T7 RNA polymerase polypeptide as described herein.

[0116] Suitable culture media and growth conditions for the host cells described above are well known in the art. Polynucleotides for expressing the T7 RNA polymerase polypeptide can be introduced into cells by a variety of methods known in the art. Techniques include, in particular, electroporation, bioparticle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0117] Engineered T7 RNA polymerases having properties disclosed herein can be obtained by subjecting polynucleotides encoding naturally occurring or engineered T7 RNA polymerase polypeptides to mutagenesis and / or directed evolution methods known in the art and as described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747-10751

[1994] ; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Pat. No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258–261

[1998] ), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140

[1994] ), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529

[1996] ).

[0118] For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate libraries of variants that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,3 44, No. 6,309,883, No. 6,319,713, No. 6,319,714, No. 6,323,030, No. 6,326,204, No. 6,335,160, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355,484, No. 6,358,740, No. 6,358,742, No. 6,365,377, No. 6,365,408, No. 6,368,861, No. 6,372,497, No. 6,376,246, No. 6,379,964, No. 6,387,702, No. 6,391 ,552, No. 6,391,640, No. 6,395,547, No. 6,406,855, No. 6,406,910, No. 6,413,745, No. 6,413,774, No. 6,420,175, No. 6,423,542, No. 6,426,224, No. 6,436,675, No. 6,444,468, No. 6,455,253, No. 6,479,652, No. 6,482,647, No. 6,489,146, No. 6,506,602, No. 6,506,603, No. 6,519,065, No. 6,521,453, No. 6,5 No. 28,311, No. 6,537,746, No. 6,573,098, No. 6,576,467, No. 6,579,678, No. 6,586,182, No. 6,602,986, No. 6,613,514, No. 6,653,072, No. 6,716,631, No. 6,777,218, No. 6,917,882, No. 6,946,296, No. 6,961,664, No. 6,995,017, No. 7,024,312, No. 7,058,515, No. 7,105,297, No. 7,148,054, No. 7,288,375, No. 7,No. 421,347, No. 7,430,477, No. 7,534,564, No. 7,620,500, No. 7,620,502, No. 7,629,170, No. 7,702,464, No. 7,747,391, No. 7,747,393, No. 7,751,986, No. 7,776,598, No. 7,783,428, No. 7,795,030, No. 7,853,410, No. 7,868,138, No. 7,873,477, No. 7,873,499, No. 7,904,249, No. 7,957,912, No. 8,014,9 61, 8,029,988, 8,058,001, 8,076,138, 8,018,150, 8,170,806, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,849,575, 8,876,066, 8,768,871, 9,593,326, 9,665,694, and all related U.S. and non-U.S. counterparts; Ling et al., Anal. Biochem., 254(2):157-78

[1997] ;Dale et al., Meth. Mol. Biol., 57:369-74

[1996] ;Smith, Ann. Rev. Genet., 19:423-462

[1985] ;Botstein et al., Science, 229:1193-1201

[1985] ;Carter, Biochem. J., 237:1-7

[1986] ;Kramer et al., Cell, 38:879-887

[1984] ;Wells et al., Gene, 34:315-323

[1985] ;Minshull et al., Curr. Op. Chem. Biol., 3:284-290

[1999] ;Christians et al., Nat. Biotechnol., 17:259-264

[1999] ; Crameri et al., Nature, 391:288-291

[1998] ; Crameri, et al., Nat. Biotechnol., 15:436-438

[1997] ; Zhang et al., Proc. Nat. Acad. Sci. USA, 94:4504-4509

[1997] ; Crameri et al.,Nat. Biotechnol., 14:315-319

[1996] ;Stemmer, Nature, 370:389-391

[1994] ;Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751

[1994] ;U.S. Patent Application Publication Nos. 2008 / 0220990, US 2009 / 0312196, US2014 / 0005057, US2014 / 0214391, US2014 / 0221216, US2015 / 0050658, US2015 / 0133307, US2015 / 0134315 and all related U.S. and non-U.S. counterparts; WO 95 / 22625, WO 97 / 0078, WO 97 / 35966, WO 98 / 27230, WO 00 / 42651, WO 01 / 75767 and WO 2009 / 152336; all of which are incorporated herein by reference).

[0119] In some embodiments, the enzyme variants obtained after mutagenesis are screened by subjecting the enzyme variants to a specified temperature (or other assay conditions), and measuring the amount of enzyme activity remaining after heat treatment or other assay conditions. The DNA comprising the polynucleotide encoding the T7 RNA polymerase polypeptide is then isolated from the host cell, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in different or identical host cells. Measuring the enzyme activity from the expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques, such as HPLC analysis).

[0120] For the engineered polypeptide of known sequence, the polynucleotide of encoding enzyme can be prepared by the solid phase method of standard according to known synthesis method.In some embodiments, the fragment of up to about 100 bases can be synthesized individually, then connected (for example, by enzyme connection method or chemical connection method or polymerase-mediated method) to form any desired continuous sequence.For example, polynucleotide disclosed herein and oligonucleotide can use classical phosphoramidite method (see for example, Beaucage et al., Tetra.Lett., 22:1859-69

[1981] and Matthes et al., EMBO J., 3:801-05

[1984] ) to prepare by chemical synthesis, as usually practiced in automatic synthesis method.According to phosphoramidite method, oligonucleotide is synthesized (for example, in automatic DNA synthesizer), purified, annealed, connected and cloned into suitable carrier.

[0121] Thus, in some embodiments, a method for preparing an engineered T7 RNA polymerase polypeptide may comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences of any variants provided in Table 5.3, Table 5.4, Table 5.5, and / or Table 5.6, and SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, and (b) expressing a T7 RNA polymerase polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide may optionally have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residues deleted, inserted, and / or substituted. In some embodiments, the amino acid sequence optionally has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45 or 1-50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45 or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions may be conservative substitutions or non-conservative substitutions.

[0122] Any desired improved property (e.g., activity, selectivity, stability, acid tolerance, protease sensitivity, etc.) of the expressed engineered T7 RNA polymerase polypeptide can be evaluated using any suitable assay known in the art, including but not limited to the assays and conditions described herein.

[0123] In some embodiments, any engineered T7 RNA polymerase polypeptide expressed in host cells is recovered from the cells and / or culture medium using any one or more of the well-known techniques for protein purification including, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography.

[0124] Chromatographic techniques for separating T7 RNA polymerase polypeptides include, in particular, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel electrophoresis and affinity chromatography. The conditions for purifying a particular enzyme depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to separate improved variant T7 RNA polymerases. In some embodiments utilizing affinity chromatography purification, any antibody specifically binding to variant T7 RNA polymerase polypeptides can be used. In some embodiments utilizing affinity chromatography purification, proteins bound to polysaccharides covalently attached to T7 RNA polymerase can be used. In still other embodiments utilizing affinity chromatography purification, any small molecule bound to the active site of T7 RNA polymerase can be used. In order to produce antibodies, a variety of host animals, including but not limited to rabbits, mice, rats, etc., are immunized by injecting T7 RNA polymerase polypeptides (e.g., T7 RNA polymerase variants) or fragments thereof. In some embodiments, the T7 RNA polymerase polypeptide or fragment is attached to a suitable carrier, such as BSA, via a side chain functional group or a linker attached to a side chain functional group.

[0125] In some embodiments, the engineered T7 RNA polymerase polypeptide is produced in a host cell by a method comprising culturing a host cell (e.g., Saccharomyces cerevisiae, Daucus carota, Nicotiana tabacum, H. sapiens (e.g., HEK293T), or Cricetulus griseus (e.g., CHO)) under conditions favorable for production of the engineered T7 RNA polymerase polypeptide, the host cell comprising a polynucleotide sequence encoding an engineered T7 RNA polymerase polypeptide described herein, and recovering the engineered T7 RNA polymerase polypeptide from the cells and / or culture medium.

[0126] In some embodiments, the invention encompasses a method of producing an engineered T7 RNA polymerase polypeptide, the method comprising culturing a recombinant eukaryotic cell under suitable culture conditions to allow production of the engineered T7 RNA polymerase polypeptide, and optionally recovering the engineered T7 RNA polymerase polypeptide from the culture and / or cultured bacterial cells, wherein the recombinant eukaryotic cell comprises a polynucleotide sequence encoding an engineered T7 RNA polymerase polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference sequence SEQ ID NO: 4 and / or SEQ ID NO: 15 when optimally aligned with the amino acid sequence of SEQ ID NO: 4 and / or SEQ ID NO: 15, and having a sequence identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 4 and / or SEQ ID NO: 15. NO:15 and / or a combination thereof, has one or more amino acid residue differences selected from those provided in Table 5.3, Table 5.4, Table 5.5 and / or Table 5.6.

[0127] In some embodiments, once the engineered T7 RNA polymerase polypeptides are recovered from recombinant host cells or cell culture media, they are further purified by any suitable method known in the art. In some other embodiments, the purified T7 RNA polymerase polypeptides are combined with other ingredients and compounds to provide compositions and preparations (e.g., pharmaceutical compositions) that appropriately contain engineered T7 RNA polymerase polypeptides for different applications and uses. In some other embodiments, the purified T7 RNA polymerase polypeptides or formulated T7 RNA polymerase polypeptides are lyophilized.

[0128] Composition: The present invention provides a variety of compositions and formats, including but not limited to those described below.In some embodiments, the present invention provides engineered T7 RNA polymerase polypeptides suitable for use in compositions for diagnostic purposes.

[0129] experiment The following examples, including the experiments and results obtained, are provided for illustrative purposes only and should not be construed as limiting the present invention.

[0130] In the experimental disclosure below, the following abbreviations are used: ppm (parts per million); M (mole / liter); mM (millimoles / liter); uM and µM (micromoles / liter); nM (nanomoles / liter); mol (mole); gm and g (grams); mg (milligrams); ug and µg (micrograms); L and l (liter); ml and mL (milliliters); cm (centimeter); mm (millimeter); um and µm (micrometers); sec. (second); min(s) (minute); h(s) and hr(s) (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); rcf (relative centrifugal force); °C (degrees Celsius); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); Escherichia coli W3110 (E. coli W3110) (a common laboratory strain of E. coli available from Coli Genetic Stock Center [CGSC], New Haven, CT); HPLC (high pressure liquid chromatography); MWCO (molecular weight cutoff); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); T7RNAP (T7 RNA polymerase); PES (polyethersulfone); CFSE (carboxyfluorescein succinimidyl ester); IPTG (isopropyl β-D-1-thiogalactoside); PMBS (polymyxin B sulfate); NADPH (nicotinamide adenine dinucleotide phosphate); GIDH (glutamate dehydrogenase); FIOPC (fold improvement over positive control); LB (Luria broth); MeOH (methanol); Athens Research (Athens Research Technology, Athens, GA); NEB (New England Biolabs, Ipswich, MA); IonTorrent (Ion Torrent, Gilford, NH); ProSpec (ProSpec Tany Technogene, East Brunswick, NJ); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Ram Scientific (Ram Scientific, Inc., Yonkers, NY); Pall Corp. (Pall, Corp., Pt. Washington, NY); Millipore (Millipore, Corp., Billerica MA); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Molecular Devices (Molecular Devices, LLC, Sunnyvale, CA); Kuhner (Adolf Kuhner, AG, Basel, Switzerland); Axygen (Axygen, Inc., Union City, CA); Toronto Research Chemicals (Toronto Research Chemicals Inc., Toronto, Ontario, Canada); Cambridge Isotope Laboratories, (Cambridge Isotope Laboratories, Inc., Tewksbury, MA); Applied Biosystems (Applied Biosystems, a part of Life Technologies, Corp., Grand Island, NY); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Thermo Scientific (a part of Thermo Fisher Scientific, Waltham, MA); Ion Torrent NGS (Ion Torrent Next Generation Sequencing); Li-COR (Li-COR, Lincoln, NE); UVP (UVP, Upland, CA); Biotium (Biotium, Inc., Fremont, CA); Corning (Corning, Inc., Palo Alto, CA); Megazyme (Megazyme International, Wicklow, Ireland); Enzo (Enzo Life Sciences, Inc., Farmingdale, NY); GE Healthcare (GE Healthcare Bio-Sciences, Piscataway, NJ); Pierce (Pierce Biotechnology (now part of Thermo Fisher Scientific), Rockford, IL); LI-COR (LI-COR Biotechnology, Lincoln, NE); Amicus (Amicus Therapeutics, Cranbury, NJ); Phenomenex (Phenomenex, Inc., Torrance, CA); Optimal (Optimal Biotech Group, Belmont, CA); and Bio-Rad (Bio-Rad Laboratories, Hercules, CA). .

[0131] The following polynucleotide and polypeptide sequences can be used in the present invention. In some cases (as shown below), the polynucleotide sequence is followed by the encoded polypeptide.

[0132] Example 1 Obtaining T7 RNA polymerase gene and constructing its expression vector Wild-type (WT) T7 RNA polymerase (SEQ ID NO: 2) is encoded by the genome (SEQ ID NO: 1) of bacteriophage T7. A synthetic gene (SEQ ID NO: 3) encoding a 6-histidine tag version (SEQ ID NO: 4) of T7 RNA polymerase was constructed and subcloned into an Escherichia coli expression vector pCK100900i (see, e.g., U.S. Pat. No. 7,629,157 and U.S. Patent Application Publication No. 2016 / 0244787, both of which are hereby incorporated by reference). These plasmid constructs were transformed into an Escherichia coli strain derived from W3110. A library of gene variants was generated from these plasmids using directed evolution techniques commonly known to those skilled in the art (see, e.g., U.S. Pat. No. 8,383,346 and WO 2010 / 144103, both of which are hereby incorporated by reference). Substitutions in the enzyme variants described herein are indicated with reference to WT T7 RNA polymerase (ie, SEQ ID NO: 4) or variants thereof, as indicated.

[0133] Example 2 High-throughput (HTP) expression and purification of T7 RNA polymerase In this example, experiments performed on T7 RNA polymerase expression and purification of T7 RNA polymerase variants are described.

[0134] High-throughput (HTP) growth of T7 RNA polymerase and variants Transformed E. coli cells were selected by plating on LB agar plates containing 1% glucose and 30 µg / ml chloramphenicol. After incubation overnight at 37°C, colonies were plated into wells of 96-well shallow flat-bottom NUNC™ (Thermo-Scientific) plates filled with 180 µl / well of LB medium supplemented with 1% glucose and 30 µg / ml chloramphenicol. The cultures were allowed to grow overnight in a shaker (200 rpm, 30°C, and 85% relative humidity; Kuhner) for 18-20 hours. Overnight growth samples (20 µL) were transferred to Costar 96-well deep plates filled with 380 µL of Terrific broth supplemented with 30 µg / ml chloramphenicol. The plates were incubated on a shaker (250 rpm, 30°C, and 85% relative humidity; Kuhner) for 120 min until the OD 600 reached between 0.4-0.8. The cells were then induced with 40 µL of 10 mM IPTG in sterile water and incubated overnight in a shaker (250 rpm, 30°C, and 85% relative humidity; Kuhner) for 18-20 hours. The cells were pelleted (4000 rpm×20 min), the supernatant discarded, and the cells frozen at -80°C prior to analysis.

[0135] Lysis of HTP precipitate The cell pellet was resuspended in 400 µL of lysis buffer (50 mM sodium phosphate pH 7.5, 10 mM imidazole, 0.1% Tween 20 ® , 1 mg / ml lysozyme and 0.5 mg / ml polymyxin B sulfate), and the mixture was stirred at room temperature for 2 h. The lysate was then pelleted (4000 rpm×5 min), and the clear supernatant was retained for purification.

[0136] HTP purification of T7 RNAP from crude lysate According to the manufacturer's instructions, HisPur TM T7 RNAP was purified from clarified E. coli lysate by metal affinity chromatography using Ni-NTA spin plates (Thermo Fisher). A total of 800 µl of binding buffer (50 mM sodium phosphate, pH 7.5, 300 mM NaCl, 10 mM imidazole, 0.1% v / v TWEEN-20) was used per well. ® Reagent) Balance HisPurTM Ni-NTA plates. Then, 100 µl of binding buffer was added to each well, followed by 100 ul of HTP lysate containing T7 RNAP. The plates were shaken briefly, centrifuged at 1100 relative centrifugal force (rcf) and 4°C for 1 min, and then incubated at room temperature for 15 min. The plates were washed with 600 µl of wash buffer (50 mM sodium phosphate, pH 7.5, 300 mM NaCl, 25 mM imidazole, 0.1% v / v TWEEN-20 ® The enzyme samples were washed twice with 105 µl of elution buffer (50 mM sodium phosphate, pH 7.5, 300 mM NaCl, 250 mM imidazole, 0.1% v / v TWEEN ® -20 reagent) and eluted by centrifugation at 1100 rcf for 1 min at 4°C.

[0137] The eluate was buffer exchanged using a Zeba™ spin desalting plate (Thermo Fisher). Briefly, the plate was equilibrated twice with 375 µl per well of 2× T7RNAP storage buffer (100 mM Tris.HCl, pH 7.9, 200 mM NaCl, 2 mM DTT, 2 mM EDTA, 0.2% w / v Triton X-100) and centrifuged at 1100 xg for 2 min at 4°C. The desalted plate was loaded with 90 µl of HisPur TM Ni-NTA spin plate eluate and centrifuge at 1100 x g for 2 min at 4° C. The eluate from the desalting plate was retained and mixed with an equal volume of glycerol to give a final storage buffer concentration of 50 mM Tris HCl, pH 7.9, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.1% v / v Triton X-100, and 50% glycerol (v / v).

[0138] The absence of RNase contamination in the purified preforms was confirmed using an RNase Alert Assay (IDT, Life Technologies).SDS-PAGE analysis of T7 RNAP samples showed that most samples had no detectable contaminating bands.

[0139] For in vitro transcription reactions using HTP-purified T7 RNAP, purified polymerase was added to a final concentration of 10% of the total reaction volume (5% for enzyme from shake flask cultures).

[0140] Example 3 Shake flask expression and purification of T7 RNA polymerase In this example, experiments involving shake flask expression and purification of T7 RNAP are described.

[0141] Shake flask expression Selected HTP cultures grown as described in Example 2 were plated on LB agar plates with 1% glucose and 30 μg / ml chloramphenicol and grown overnight at 37° C. A single colony from each culture was transferred to 6 ml of LB broth containing 1% glucose and 30 μg / ml chloramphenicol. The cultures were grown at 30° C., 250 rpm for 18 h, and subcultured to a final OD of 0.2 at a dilution of approximately 1:10 into 250 ml of Terrific broth containing 30 μg / ml chloramphenicol. 600 The culture was incubated at 30°C and 250 rpm for approximately 3 hours to an OD of 0.6 – 0.8. 600 , and then induced by adding IPTG at a final concentration of 1 mM. The induced culture was incubated at 30°C and 250 rpm for 20 h. After this incubation period, the culture was centrifuged at 4000 rpm×10 min. The culture supernatant was discarded, and the pellet was resuspended in 35 ml lysis buffer (50 mM NaH2PO4, pH 7.5, 500 mM NaCl, 0.1% Tween-20, 10 mM imidazole). The cell suspension was cooled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M-110L). The crude lysate was precipitated by centrifugation (at 4°C at 16,000 rpm for 60 min), and the supernatant was then filtered through a 0.2 µm PES membrane to further clarify the lysate.

[0142] Purification of T7 RNA polymerase from shake flask lysates T7RNAP lysate was purified using the AKTA Start purification system and a 5 ml HisTrap FF column (GE Healthcare) using the AC Step HiF setup (run parameters are provided below). The SF wash buffer contained 50 mM sodium phosphate, pH 7.5, 500 mM NaCl, 0.1% v / v TWEEN-20 ® Reagent (Sigma), and 25 mM imidazole. SF elution buffer contained 50 mM sodium phosphate, pH 7.5, 500 mM NaCl, 0.1% v / v TWEEN-20 ® reagent (Sigma) and 300 mM imidazole.

[0143]

[0144] The five most concentrated 3 ml fractions were identified by UV absorption (A280) and dialyzed overnight in 10K Slide-A-Lyzer™ dialysis cassettes (Thermo Fisher) in 2×T7 RNAP storage buffer (100 mM Tris HCl pH 7.9, 200 mM NaCl, 2 mM DTT, 2 mM EDTA, 0.2% v / v Triton X-100) for 16 hours, followed by a second buffer exchange for 24 hours. An equal volume of glycerol was added to the dialyzed material. The enzyme concentration in the preparation was measured by gel densitometry and absorbance at 280 nm.

[0145] Example 4 Transcriptional response The transcription reaction is assembled with the cap analog α,γ-bis(N7-methylguanosine) triphosphate (also referred to herein as "m7G(5')ppp(5')m7G", "capped GTP" or "cap") (see, Grudzien et al., RNA, 10:1479-87

[2004] ). The engineered transcription DNA template GlmS-16A (SEQ ID NO: 5) contains the T7RNAP promoter sequence coupled to the coding sequence of the Bacillus anthracis GlmS riboswitch (SEQ ID NO: 6). Under the induction of its ligand glucosamine-6-phosphate, the GlmS riboswitch self-cleaves and releases a 16-mer RNA nucleotide (SEQ ID NO: 7) from the 5' end of the transcript, which includes either a cap structure or an uncapped 5' phosphate. This small 16-mer RNA oligonucleotide cleavage product can be ionized and analyzed by LC-MS to distinguish between capped and uncapped species.

[0146] Reactions (30 ul) were assembled with a total of 30 ul per well with final concentrations of: 50 mM Tris HCl pH 7.9, 30 mM MgCl2, 10 mM DTT, 6 mM ATP, 6 mM CTP, 6 mM UTP, 4.8 mM GTP, 1.2 mM m7G(5')ppp(5')m7G, 50 ng / µl GlmS-16A transcription template, 1 U / µl RNasin inhibitor and 6.4 mM glucosamine-6-phosphate, and contained 10% (v / v) (3 ul) of purified and desalted T7RNAP from Example 3. For HTP screening, reactions were incubated at 37°C for 4 h and quenched with an equal (30 µl) volume of 40 mM EDTA. The sum of m7G(5')ppp(5')m7G and GTP was kept at 6 mM. Glucosamine-6-phosphate was added at the beginning of the reaction, and cleavage of the riboswitch was allowed to proceed during 4 hours of in vitro transcription.

[0147] Example 5 LC-MS activity assay This example describes an LC-MS method developed for the separation of smaller RNA fragments using the Thermo LTQ MS system for the analysis of shorter capped 5' triphosphate cleavage products and uncapped 5' triphosphate cleavage products.

[0148] The 7meG-capped and 5' triphosphate-uncapped 16-mer cleavage products (SEQ ID NO: 7) were chromatographically separated from the reaction and higher MW RNA using a reverse phase ion pairing HPLC method (see, Figure 1 ), the reverse phase ion pairing HPLC method used a C18 column and HFIP mobile phase commonly used for mass spectrometry oligonucleotide analysis (see, Table 5.1).

[0149]

[0150] Base-peak extraction was performed on capped and uncapped 16-mer fragmentation products using seven ions per species, including various negatively charged ions and metal adducts. The peak intensities of these seven ions were used to derive the signals for each capped and uncapped 16-mer fragmentation product. These ions included various charge states as well as sodium and potassium adducts (see, Table 5.2). All ions were observed within 1 mass unit of the expected value.

[0151]

[0152] Capping performance relative to WT T7RNAP (i.e., "fold improvement relative to parent" or "FIOP") is calculated by dividing the capped / uncapped peak intensity ratio of each sample by the ratio calculated for the WT parent control present on each plate (n=6 to 10). FIOP values ​​are used to rank variant performance relative to WT T7RNAP or library parent, which is included on each plate as a control. By using this relative quantitative method, inherent ionization differences between capped and uncapped substances, as well as signal changes caused by batch and EDTA concentrations of HFIP mobile phases, can be eliminated. In vitro transcription reactions were performed as described in Example 4, and reaction times and m7G(5')ppp(5')m7G concentrations are shown in the table below. Tables 5.3, 5.4, and 5.5 report average activity improvements for 3 to 6 replicates. Table 5.6 reports average activity improvements for variants for which replicate information is available.

[0154]

[0155]

[0156]

[0157]

[0158] Note that variant 85 was undetectable at levels of uncapped mRNA in the LC-MS assay.

[0159]

[0160]

[0161]

[0162]

[0163] Example 6 Determination of RNA yield In vitro transcription reactions were performed as described in Example 4, but using a luciferase template (SEQ ID NO: 10). The mRNA yield from the in vitro transcription reaction was obtained according to the manufacturer's protocol for the Quant-iT RNA assay kit (wide range, Q-33140, Thermo Fisher). mRNA abundance was calculated using a standard curve derived from the mRNA standards provided with the assay kit.

[0164]

[0165] Example 7 Determination of transcription fidelity of T7 RNAP variants Polymerase fidelity is measured based on direct sequencing of a large number of RT-PCR clones derived from mRNA transcribed from variant polymerases. In vitro transcription reactions were performed as described in Example 4, using 0.5 mM m7G (5') ppp (5') m7G, 5.5 mM GTP, and omitting glucosamine-6-phosphate. The inclusion of m7G (5') ppp (5') m7G allows measurement of the contribution of the cap analog (if present) to the error rate of WT polymerase and variant polymerase under process-related conditions. 1.7 kb luciferase template DNA (SEQ ID NO: 8) is used as a DNA template transcribed using wild-type T7 RNAP and variant T7 RNAP to produce full-length mRNA transcripts. RNA is separated using Zymo RNA Clean and concentrator-25 kits (Zymo Research), and residual DNA is removed from RNA samples by treating with DNA-free DNAase I kits (Ambion / Thermo Fisher) twice in a row. The samples were transcribed using Accuprime reverse transcriptase (Agilent) using oligo-(dT) annealed to the poly(A) tail of the luciferase template. 25 The RT reaction was then PCR using gene-specific primers annealed to the luciferase coding sequence (SEQ ID NO: 12, SEQ ID NO: 13), using HF buffer (New England Biolabs), using PHUSION ® Amplification was performed using a high-fidelity DNA polymerase to generate a 1675-bp amplicon. The amplified fragment was digested with BglI (New England Biolabs), ligated into a cloning vector, and transformed into E. coli to generate a single clone.

[0166] Individual clones were picked and sequenced using a multiplex barcoding strategy on the Ion Torrent PGM platform (Thermo Fisher). The barcoded reads were deconvoluted, and the sequences of individual clones were then assembled for the expected template sequence of the 1632-bp region between SEQ ID NO: 12 and SEQ ID NO: 13. Mutations including small insertions, deletions (i.e., indels), and single nucleotide polymorphisms were recorded. Most of the mutations observed were substitutions, although insertions and deletions were also observed. The total number of mutations per base for the sequenced mRNA-derived clones was calculated and reported in Tables 7.1 and 7.2. The expected total mutation rate per base based on the literature is due to T7RNAP (1×10-4 ) (Huang et al., Biochem., 39:11571-11580

[2000] ), Accuscript reverse transcriptase (6×10 -5 ) (Agilent; see product literature) and Phusion DNA polymerase (1.2 × 10 -5 ) (20 cycles; see NEB product literature) or overall 1.72×10 -4 The sum of the errors caused by the . Most variant polymerases showed a total error rate close to the literature value reported for T7 RNAP, even in the presence of 0.5 mMm7G(5')ppp(5')m7G.

[0167] If the actual error rate in the experiment is equal to the total error rate observed for T7RNAP-WT in the separate experiments presented in Table 7.1 and Table 7.2, a one-tailed (right-hand) binomial test is used to calculate the sampling probability of the observed number of errors (or more) for a given number of sequenced bases. For p values ​​greater than 0.05, the fidelity of a given variant is considered indistinguishable from WTT7RNAP in this assay. In Table 7.1, a "+" in the Observed Error Rate Result column is less than 1.7*10 -4 , and “-” is greater than 1.7*10 -4 , while the "+" in the binomial test result column is p > 0.05, and the "-" is p < 0.05. In Table 7.2, the "+" in the observed error rate result column is less than 1.5×10 -4 , and “-” is greater than 1.5×10 -4 , while the “+” in the binomial test result column is p > 0.05, and “-” is p < 0.05.

[0168]

[0169]

[0170] Although the present invention has been described with reference to specific embodiments, various changes may be made and equivalents may be substituted to adapt a particular situation, material, composition of matter, process, process step or steps so as to achieve the benefits of the present invention without departing from the scope of what is claimed.

[0171] Each and every publication and patent document cited in this application is incorporated herein by reference for all purposes in the United States to the same extent as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not to be construed as an indication that any such document is relevant prior art nor constitutes an admission as to its contents or date.

[0172] This application also provides the following items: 1. An engineered RNA polymerase comprising a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a reference sequence SEQ ID NO:4 and / or SEQ ID NO:15 or a functional fragment thereof, wherein the engineered RNA polymerase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4 or SEQ ID NO:15.

[0173] 2. The engineered RNA polymerase of claim 1, wherein at least one substitution or set of substitutions is selected from 397 / 513 / 635, 397 / 513 / 635 / 660, 513 / 660 / 664, 513 / 635 / 660, 513 / 635 / 664, 513 / 660 / 664 and 660 / 664, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:4.

[0174] 3. The engineered RNA polymerase of item 1, wherein at least one substitution or set of substitutions is 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 / 635 / 656 / 660, 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 combination thereof wherein amino acid positions are numbered with reference to SEQ ID NO:4.

[0175] 4. The engineered RNA polymerase of item 1, wherein at least one substitution or set of substitutions is 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, 1 635, 635 / 656, 635 / 660, and 660, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 15.

[0176] 5. An engineered RNA polymerase as described in item 1, wherein the engineered RNA polymerase comprises a polypeptide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered RNA polymerase variant listed in Table 5.3, Table 5.4, Table 5.5 and / or Table 5.6.

[0177] 6. An engineered RNA polymerase as described in item 1, wherein the engineered RNA polymerase comprises a variant engineered polymerase provided in Table 5.3, Table 5.4, Table 5.5 and / or Table 5.6.

[0178] 7. An engineered RNA polymerase as described in claim 1, wherein the engineered RNA polymerase comprises a polypeptide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered RNA polymerase variant listed in SEQ ID NO: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39.

[0179] 8. An engineered RNA polymerase as described in item 1, wherein the engineered RNA polymerase comprises a variant engineered polymerase listed in SEQ ID NO:4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39.

[0180] 9. An engineered RNA polymerase as described in any of items 1-8, wherein the engineered polymerase exhibits at least one improved property compared to wild-type T7 RNA polymerase.

[0181] 10. An engineered RNA polymerase as described in item 9, wherein the at least one improved property is selected from improved selectivity for cap analogs relative to GTP during transcription initiation, improved protein expression, improved stability in storage buffer, and improved stability under reaction conditions.

[0182] 11. An engineered RNA polymerase as described in any of items 1-10, wherein the polymerase maintains RNA yield, transcription fidelity, thermal stability, protein expression, stability at -20°C, or stability under reaction conditions comparable to wild-type T7 RNA polymerase.

[0183] 12. An engineered RNA polymerase as described in any of items 1-11, wherein the engineered polymerase is purified.

[0184] 13. A polynucleotide sequence encoding at least one engineered RNA polymerase according to any one of items 1-12.

[0185] 14. A polynucleotide sequence encoding at least one engineered RNA polymerase comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:4 and / or SEQ ID NO:15 or a functional fragment thereof, wherein the engineered RNA polymerase comprises at least one substitution at one or more amino acid positions.

[0186] 15. A polynucleotide sequence encoding at least one engineered RNA polymerase comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39 or a functional fragment thereof.

[0187] 16. A polynucleotide sequence encoding at least one engineered RNA polymerase, wherein the polynucleotide sequence comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and / or 38.

[0188] 17. A polynucleotide sequence as described in any one of items 13 to 16, wherein the polynucleotide sequence is operably linked to a control sequence.

[0189] 18. A polynucleotide sequence as described in any of items 13-16, wherein the polynucleotide sequence is codon optimized.

[0190] 19. An expression vector comprising at least one polynucleotide sequence according to any one of items 13-18.

[0191] 20. A host cell comprising at least one expression vector according to item 19.

[0192] 21. A method for producing an engineered RNA polymerase in a host cell, the method comprising culturing the host cell of item 20 under suitable culture conditions, thereby producing at least one engineered RNA polymerase.

[0193] 22. The method of claim 21, further comprising recovering at least one engineered RNA polymerase from the culture and / or host cell.

[0194] 23. The method as described in item 21 and / or 22 further comprises a step of purifying the at least one engineered RNA polymerase.

[0195] 24. A composition comprising at least one engineered RNA polymerase as described in any one of items 1-12.

[0196] 25. A method for producing a capped RNA transcript, the method comprising providing a composition comprising: i) at least one engineered RNA polymerase of any one of items 1-12, a dinucleotide cap analog, and ii) a DNA template; exposing the DNA template to the composition under conditions such that the engineered RNA polymerase produces a capped RNA transcript.

[0197] 26. A method as described in claim 25, wherein the dinucleotide cap analog is α,γ-bis(N7-methylguanosine) triphosphate (m7G(5')ppp(5')m7G) or the anti-reversal cap analog 3'-O-Me-m 7 G(5')ppp(5')G.

[0198] 27. A method as described in item 25 and / or 26, wherein the dinucleotide cap analog is α,γ-bis(N7-methylguanosine) triphosphate.

[0199] 28. A method as described in any one of items 25 to 27, further comprising adding inorganic pyrophosphatase.

Claims

1. An engineered RNA polymerase comprising a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a reference sequence SEQ ID NO:4 and / or SEQ ID NO:15 or a functional fragment thereof, wherein the engineered RNA polymerase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4 or SEQ ID NO:

15.

2. The engineered RNA polymerase of claim 1, wherein the at least one substitution or set of substitutions is selected from 397 / 513 / 635, 397 / 513 / 635 / 660, 513 / 660 / 664, 513 / 635 / 660, 513 / 635 / 664, 513 / 660 / 664 and 660 / 664, and / or any combination thereof, wherein amino acid positions are numbered with reference to SEQ ID NO:

4.

3. The engineered RNA polymerase of claim 1, wherein the at least one substitution or set of substitutions is selected from 397, 397 / 513, 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 / 6 64, 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 combination thereof wherein amino acid positions are numbered with reference to SEQ ID NO:

4.

4. The engineered RNA polymerase of claim 1, wherein the at least one substitution or set of substitutions is 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 / 513, 137 / 5 13 / 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 combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

15.

5. An engineered RNA polymerase as described in claim 1, wherein the engineered RNA polymerase comprises a polypeptide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered RNA polymerase variant listed in Table 5.3, Table 5.4, Table 5.5 and / or Table 5.

6.

6. An engineered RNA polymerase as described in claim 1, wherein the engineered RNA polymerase comprises an engineered polymerase variant provided in Table 5.3, Table 5.4, Table 5.5 and / or Table 5.

6.

7. An engineered RNA polymerase as described in claim 1, wherein the engineered RNA polymerase comprises a polypeptide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered RNA polymerase variant listed in SEQ ID NO: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39.

8. The engineered RNA polymerase of claim 1, wherein the engineered RNA polymerase comprises an engineered polymerase variant listed in SEQ ID NO: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39.

9. The engineered RNA polymerase of any one of claims 1-8, wherein the engineered RNA polymerase exhibits at least one improved property compared to wild-type T7 RNA polymerase.

10. The engineered RNA polymerase of claim 9, wherein the at least one improved property is selected from improved selectivity for cap analogs relative to GTP during transcription initiation, improved protein expression, improved stability in storage buffers, and improved stability under reaction conditions.

11. The engineered RNA polymerase of any one of claims 1-10, wherein the engineered RNA polymerase maintains RNA yield, transcription fidelity, thermal stability, protein expression, stability at -20°C, or stability under reaction conditions comparable to wild-type T7 RNA polymerase.

12. The engineered RNA polymerase of any one of claims 1-11, wherein the engineered RNA polymerase is purified.

13. A polynucleotide encoding at least one engineered RNA polymerase according to any one of claims 1-12.

14. A polynucleotide encoding at least one engineered RNA polymerase comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:4 and / or SEQ ID NO:15 or a functional fragment thereof, wherein the engineered RNA polymerase comprises at least one substitution at one or more amino acid positions.

15. A polynucleotide encoding at least one engineered RNA polymerase comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39 or a functional fragment thereof.

16. A polynucleotide encoding at least one engineered RNA polymerase, wherein the polynucleotide comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and / or 38.

17. The polynucleotide of any one of claims 13 to 16, wherein the polynucleotide is operably linked to a control sequence.

18. The polynucleotide of any one of claims 13-16, wherein the polynucleotide is codon optimized.

19. An expression vector comprising at least one polynucleotide according to any one of claims 13 to 18.

20. A host cell comprising at least one expression vector according to claim 19.

21. A method for producing an engineered RNA polymerase in a host cell, the method comprising culturing the host cell of claim 20 under suitable culture conditions, thereby producing at least one engineered RNA polymerase.

22. The method of claim 21, further comprising recovering at least one engineered RNA polymerase from the culture and / or host cell.

23. The method of claim 21 or 22, further comprising the step of purifying the at least one engineered RNA polymerase.

24. A composition comprising at least one engineered RNA polymerase according to any one of claims 1-12.

25. A method for producing a capped RNA transcript, the method comprising providing i) a composition comprising at least one engineered RNA polymerase according to any one of claims 1-12 and a dinucleotide cap analog, and ii) a DNA template; exposing the DNA template to the composition under conditions such that the engineered RNA polymerase produces a capped RNA transcript.

26. The method of claim 25, wherein the dinucleotide cap analog is α,γ-bis(N7-methylguanosine) triphosphate (m7G(5')ppp(5')m7G) or the anti-reversal cap analog 3'-O-Me-m 7 G(5')ppp(5')G.

27. The method of claim 25 or 26, wherein the dinucleotide cap analog is α,γ-bis(N7-methylguanosine) triphosphate.

28. The method of any one of claims 25 to 27, further comprising adding an inorganic pyrophosphatase.

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