T7 RNA polymerase variants

Abstract

To provide T7 RNA polymerase variants.SOLUTION: The present invention provides engineered T7 RNA polymerase variants and a composition thereof. The variants have been devised in order to selectively incorporate a symmetrically capped GTP analogue exceeding GTP in starting in-vitro transcription. The present invention further provides a method for using a variant provided in the specification. The present invention further provides a use of a composition provided in the specification. The engineered RNA polymerase variant can include at least one substitution or substitution set in a polypeptide sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 527,764, filed June 30, 2017, and U.S. Provisional Patent Application No. 62 / 528,846, filed July 5, 2017. No. 6,239,999, both of which are incorporated by reference in their entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention provides engineered T7 RNA polymerase variants and compositions thereof. These variants are designed to selectively incorporate the m7G(5')ppp(5')m7G cap analog over GTP during initiation of in vitro transcription. The present invention also provides methods for using the variants provided herein. The present invention further provides uses of the compositions provided herein. Reference to a sequence listing, table, or computer program An official copy of the Sequence Listing will be submitted contemporaneously with the present specification via EFS-Web as an ASCII formatted text file (filename "CX8-168CUSP1_S T25.txt, created July 5, 2017, and 184,320 bytes in size). The Sequence Listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Background of the Invention T7 RNA polymerase (EC 2.7.7.6) is a monomeric, bacteriophage-encoded, DNA-directed RNA polymerase that catalyzes the formation of RNA in the 5' to 3' direction. During the process of transcription initiation, T7 RNA polymerase recognizes a specific promoter sequence (i.e., the T7 promoter). Naturally occurring T7 RNA polymerase contains 883 amino acids. It is highly homologous to T3 RNA polymerase and somewhat homologous to SP6 RNA polymerase. T7 is composed of multiple domains, including the N-terminal domain, "thumb," "palm," and "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). The conformation of the N-terminal domain changes between the initiation and elongation phases of functioning of the enzyme. The cloning and expression of the gene encoding T7 RNA polymerase has been described (see, e.g., U.S. Pat. No. 4,952,496). Due to its promoter specificity and high RNA polymerase activity, T7 has been used in a variety of applications. It is also useful for high-level expression of recombinant genes in E. coli (see, Studier and Moffat, J. Mol. Biol., 18:113-130

[1986] ). T7 is also used in various nucleic acid amplification methods, including those used in diagnostics. Because stability and thermostability are often important considerations in the development of diagnostic components, studies have been reported on improving the thermostability and stability of T7 (see, e.g., U.S. Pat. Nos. 9,193,959, 8,551,752, and 7,507,567). Nevertheless, there remains a need in the art for variant T7 enzymes that exhibit improved properties compared to the naturally occurring enzyme. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 4,952,496 [Patent Document 2] U.S. Patent No. 9,193,959 [Patent Document 3] U.S. Patent No. 8,551,752 [Patent Document 4] U.S. Patent No. 7,507,567 [Non-patent literature]

[0005] [Non-Patent Document 1] Sousa and Mukherjee, Prog. Nucl. Acid Res. Mol. Biol., 73:1-41

[2003] [Non-patent document 2] Studier and Moffat, J. Mol. Biol., 18:113-130

[1986] Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention The present invention provides engineered T7 RNA polymerase variants and compositions thereof.These variants are designed to selectively incorporate symmetrically capped GTP analogs over GTP in the initiation of in vitro transcription.The present invention also provides methods for using the variants provided herein.The present invention further provides the use of the compositions provided herein.

[0007] The present invention provides engineered RNA polymerases comprising a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 4 and / or 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 15.

[0008] In some embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from 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 the amino acid positions are numbered with reference to SEQ ID NO:4. In some further embodiments, the engineered RNA polymerase is 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 said amino acid positions are numbered with reference to SEQ ID NO:4.In some additional embodiments, the engineered RNA polymerase is 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, R401A / I / L / S / V, S404E / Y, N444F / H / I / V, M ... and / or any combination thereof, wherein said amino acid positions are numbered with reference to SEQ ID NO:4.

[0009] In some embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions 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. In some further embodiments, the engineered RNA polymerase is 397F / 513W / 635W, 397F / 513Y / 635W, 397W / 513Y / 635W, 397W / 513W / 635W, 397W / 513Y / 635W / 660F, 397W / 513W / 635W / 660W, 397Y / 513W / 635W / 660F, 475V / 513W / 635W / 660Y, 51 and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:4. In some additional embodiments, the engineered RNA polymerase is A397F / D513W / S635W, A397F / D513Y / S635W, A397W / D513Y / S635W, A397W / D513W / S635W, A397W / D513Y / S635W / D660F, A397W / D513W / S635W / D660W, A397Y / D513W / S635W / D660F, A475V / D513W / S635W / D660 Y, 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 the amino acid positions are numbered with reference to SEQ ID NO:4.

[0010] In some embodiments, the engineered RNA polymerase is 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 64, 397 / 513 / 635 / 664, 397 / 513 / 656 / 660, 397 / 513 / 660, 397 / 513 / 660 / 664, 397 / 513 / 664, 397 / 513, 397 / 635, 397 / 635 / 656 / 660 / 664, 397 / 635 / 656 / 664, 397 / 635 / 660, 397 / 635 / 664, 397 / 635 / 664 / 850, 397 / 660, 397 / 66 4, 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, 513 / 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 the amino acid positions are numbered with reference to SEQ ID NO: 4. In some further embodiments, the engineered RNA polymerase comprises at least one substitution or set of substitutions selected from 397F, 397F / 513F, 397F / 513F / 635F, 397F / 513F / 635W, 397F / 513F / 635W / 660W, 397F / 513W / 635W, 397F / 513W / 635W, 397F / 513W / 635W / 656F / 6 64F, 397F / 513W / 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, 397W / 513F / 635F / 656F / 660F / 664W, 397W / 513F / 635F / 660W, 397W / 513F / 635W, 397W / 513F / 660F, 397W / 513F / 660W, 397W / 513F / 660Y / 664F, 397W / 513W, 397W / 513W / 635F, 397W / 513W / 635F / 656W / 660F 397W / 513W / 635W, 397W / 513W / 635W / 656Y, 397W / 513W / 635W / 660W, 397W / 513W / 635W / 660W / 664Y, 397W / 513W / 635W / 660Y / 664Y, 397W / 513W / 660W, 397W / 513W / 660W / 664Y, 397W / 513W / 664F, 397W / 513W / 664W, 397W / 513Y / 635F, 397W / 513Y / 635W / 656Y / 660W / 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, 397W / 660F / 664Y, 397W / 660W, 397W / 837K, 397Y, 397Y / 513F / 635W / 656F / 660W / 664F, 397Y / 513F / 635W / 664W, 397Y / 513W / 635F / 664Y, 397Y / 513W / 635W / 660F, 397Y / 513W / 656W / 660W 397Y / 513Y, 397Y / 513Y / 635W, 397Y / 635F, 397Y / 635F / 660W, 397Y / 635W, 397Y / 635W / 656F / 660Y / 664W, 397Y / 635W / 660F, 397Y / 660F / 664W, 397Y / 664F, 399E / 635F / 660W, 475V / 513W / 635W / 660Y, 513F, 513F / 635F, 513F / 635W, 513F / 635W / 656W513F / 635W / 664W, 513F / 660W, 513F / 660W / 664F, 513F / 660W / 664Y, 513W, 513W / 635F, 513W / 635W, 513Y / 635F / 660F / 664Y, 513W / 6 35W / 656W / 660F, 513W / 635W / 660F, 513W / 635W / 664W, 513W / 656W / 664W, 513W / 656Y / 660W, 513W / 660F, 513W / 660W, 513Y / 635F, 51 3Y / 635F / 664W, 513Y / 635R / 656F / 664Y, 513Y / 635W, 513Y / 635W / 660F, 513Y / 635W / 660Y, 513Y / 635W / 660Y / 664F, 513Y / 660W, 513 Y / 660W / 664W, 513Y / 660Y / 664F, 513Y / 664Y, 635F / 656F / 664Y, 635F / 656Y / 664W, 635F / 660W / 664F, 635W, 635W / 656W, 635W / 660F, and at least one substitution or set of substitutions selected from 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 the amino acid positions are numbered with reference to SEQ ID NO:4. In some additional embodiments, the engineered RNA polymerase is A397F, A397F / D513F, A397F / D513F / S635F, A397F / D513F / S635W, A397F / D513F / S635W / D660W, A397F / D513W / S635W, A397F / D513W / S635W / Q656F / P664F, A397F / D513W / P66 4W, A397F / D513Y, A397F / D513Y / S635W, A397F / D513Y / S635W / Q656W, A397F / D513Y / S635W / P664F, A397F / D 513Y / 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、A397W / D513F / D660W、A397W / D513F / D660Y / P664F、A397W / D513W、A397W / D513W / S635F, A397W / D513W / S635F / Q656W / D660F, A397W / D513W / S635W, A397W / D513W / S635W / Q656Y, A397W / D513W / S635W / D660W, A397W / D513W / S635W / D660W / P664Y, A397W / D513W / S635W / D660Y / P664Y A397W / D513W / D660W, A397W / D513W / P664F, A397W / D513W / D660W / P664Y, A397W / D513W / P664W, A397W / D513Y / S635F, A397W / D513Y / S635W / Q656Y / D660W / P664W, A397W / D513Y / S635W, A397W / D513Y / S635 W / Q656Y / P664Y, A397W / D513Y / S635W / D660F, A397W / S635F, A397W / S635F / P664F, A397W / S635W, A397W / S635W / Q656F / P664F, A397W / S635W / Q656F / P664W, A397W / S635W / Q656F / P664Y, A397W / S635W / D66 0W、A397W / S635W / D660Y、A397W / S635W / P664F、A397W / S635W / P664W / A850T、A397W / D660F / P664F、A3 97W / D660F / P664Y、A397W / D660W、A397W / E837K、A397Y、A397Y / D513F / S635W / Q656F / D660W / P664F、A 397Y / D513F / S635W / P664W, A397Y / D513W / S635F / P664Y, A397Y / D513W / S635W / D660F, A397Y / D513W / Q656W / D660W, A397Y / D513Y, A397Y / D513Y / S635W, A397Y / S635F, A397Y / S635F / D660W, A397Y / S635WA397Y / S635W / Q656F / D660Y / P664W, A397Y / S635W / D660F, A397Y / D660F / P664W, A397Y / P664F, K399E / S635F / D660W, A475V / D513W / S6 35W / D660Y, D513F, D513F / S635F, D513F / S635W, D513F / S635W / Q656W, D513F / S635W / P664W, D513F / D660W, D513F / D660W / P664F, D513 F / D660W / P664Y, D513W, D513W / S635F, D513W / S635W, D513Y / S635F / D660F / P664Y, D513W / S635W / Q656W / D660F, D513W / S635W / D660F, D513W / S635W / P664W, D513W / Q656W / P664W, D513W / Q656Y / D660W, D513W / D660F, D513W / D660W, D513Y / S635F, D513Y / S635F / P664W, D51 3Y / 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 / P664F, S635W, S635W / Q6 and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 4.

[0011] In some embodiments, the engineered RNA polymerase is 113 / 137 / 513, 136 / 357 / 404 / 514, 136 / 357 / 514, 136 / 394 / 404 / 446, 136 / 401, 136 / 401 / 404, 136 / 404 / 446, 136 / 404 / 514, 136 / 446, 136 / 514, 137, 137 / 401, 137 / 401 / 513, 137 / 401 / 513, 137 / 513, 137 / 513 / 621, 137 / 635, 137 / 656, 35 and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO: 15.In some further embodiments, the engineered RNA polymerase is 113M / 137W / 513R, 136E / 357I / 404Y / 514I, 136I / 357I / 514F, 136I / 357K / 514F, 136I / 394R / 404Y / 446W, 136I / 401V, 136I / 401V / 404 Y, 136E / 404Y / 446W, 136E / 404Y / 514F, 136I / 446W, 136E / 514F, 136I / 514I, 137W, 137W / 401I, 137W / 401S, 137W / 401S / 513R, 137W / 401S / 513W, 137W / 401V, 137W / 513W, 137W / 513 and / or any combination thereof, wherein said amino acid positions are numbered with reference to SEQ ID NO: 15.In some additional embodiments, the engineered RNA polymerase is L113M / D137W / D513R, A136E / E357I / S404Y / S514I, A136E / S404Y / M446W, A136E / S404Y / S514F, A136E / S514F, A136I / E357I / S514F, A136I / E357K / 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 / K394R / M446W / S514I, E357R / K394R / R401V / S404Y / S514L, E357R / S514F, K394R / M446W / S514I, R401S / 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.

[0012] In some additional 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 shown in Table 5.3, Table 5.4, Table 5.5, and / or Table 5.6. In some additional 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.

[0013] In yet some additional 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: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39. In some further embodiments, the engineered RNA polymerase is a variant engineered polymerase set forth in SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39.

[0014] In some further embodiments, the engineered polymerase comprises at least one improved property compared to wild-type T7 RNA polymerase. In some further embodiments, the engineered RNA polymerase exhibits at least one improved property selected from improved selectivity for a cap analog over GTP during transcription initiation, improved protein expression, improved stability in storage buffers, and improved stability under reaction conditions. In some additional embodiments, the engineered RNA polymerase maintains RNA yield, transcription fidelity, thermostability, protein expression, stability at -20°C, or stability under reaction conditions equivalent to wild-type T7 RNA polymerase. In yet some additional embodiments, the engineered RNA polymerase is purified. In yet some further embodiments, the present invention provides compositions comprising at least one engineered RNA polymerase provided herein.

[0015] The present invention also provides polynucleotide sequences encoding at least one engineered RNA polymerase provided herein. In some 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 the reference sequence of SEQ ID NO: 4 and / or 15, or a functional fragment thereof, wherein the engineered RNA polymerase comprises at least one substitution at one or more amino acid positions. In some further embodiments, the polynucleotide sequences encoding the engineered RNA polymerases provided herein comprise 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. In some further embodiments, the polynucleotide sequences encoding at least one engineered RNA polymerase comprise 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 still some additional embodiments, the polynucleotide sequence is operably linked to a regulatory sequence. In still some further embodiments, the polynucleotide sequence is codon optimized.

[0016] The present invention also provides expression vectors comprising at least one polynucleotide sequence provided herein. The present invention further provides host cells comprising at least one expression vector provided herein. The present invention also provides methods for producing an engineered RNA polymerase in a host cell, the method comprising culturing a host cell provided herein under suitable culture conditions such that at least one engineered RNA polymerase is produced. In some embodiments, the method comprises recovering the at least one engineered RNA polymerase from the culture and / or host cell. In yet some additional embodiments, the method further comprises purifying the at least one engineered RNA polymerase.

[0017] The present invention also provides methods for producing capped RNA transcripts, the methods comprising: i) providing a composition comprising at least one engineered RNA polymerase provided herein, a dinucleotide cap analog, and ii) a DNA template; and 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 methods, the dinucleotide cap analog is α,γ-bis(N7-methylguanosine)triphosphate (m7G(5')ppp(5')m7G) or the anti-reverse cap analog 3'-O-Me-m 7 In some further embodiments of the above methods, the dinucleotide cap analog is α,γ-bis(N7-methylguanosine)triphosphate. In some additional embodiments of the above methods, inorganic pyrophosphatase is added to the reaction. [Brief explanation of the drawings]

[0018] [Figure 1]Figure 1 provides the chemical structure of "m7G(5')ppp(5')m7G" (also referred to as "α,γ-bis(N7-methylguanosine) triphosphate," "capped GTP," "symmetrical cap analog," or "cap"). DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description of the Invention The present invention provides engineered T7 RNA polymerase variants and compositions thereof. These variants are designed to selectively incorporate the m7G(5')ppp(5')m7G cap analog over GTP during initiation of in vitro transcription. The present invention also provides methods of using the variants provided herein. The present invention further provides uses of the compositions provided herein.

[0020] The present invention provides compositions and methods for the efficient production of capped RNA. Most eukaryotic cell mRNA transcripts and most eukaryotic viral mRNA transcripts, as well as other forms of eukaryotic RNS (e.g., small nuclear RNAs [snRNAs] and pre-microRNAs [pre-miRNAs]), are blocked or "capped" at their 5' ends (see, e.g., U.S. Patent No. 8,846,348, incorporated herein by reference in its entirety). The cap is a guanine nucleoside linked via its 5'-carbon to a triphosphate group, which is linked to the 5'-carbon of the 5'-most nucleotide of the primary mRNA transcript. In most eukaryotes, the nitrogen at position 7 of the guanine in the cap nucleotide is methylated. The 5' cap of these RNA molecules plays an important role in RNA stability and processing. The cap plays a crucial role in mRNA metabolism and is required for the processing and maturation of RNA transcripts in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and 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 and viral mRNAs. The 5' cap also provides resistance to 5'-exonuclease activity, and its absence results in rapid degradation of the mRNA (see, e.g., U.S. Pat. No. 8,836,348; and Furiichi et al., Nat., 266:235-9

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

[0021] In vitro synthesis of capped RNA can actually be achieved through one of two enzymatic pathways: Vaccinia virus capping enzyme has two subunits and three enzymatic activities that add 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] ).

[0022] An alternative method for in vitro synthesis of capped RNA is to incorporate the cap structure during in vitro transcription with the use of cap analogs. These 7-methylguanosine-containing dinucleotides contain a 5'-5' triphosphate linkage and are incorporated at the initiation site of transcription, resulting in a 7-methylguanosine-capped RNA product. T7 RNA polymerase naturally initiates transcription with the incorporation of a guanosine opposite a cytosine residue on its template. To achieve high capping efficiency, the guanosine cap analog must be present in the in vitro transcription reaction at a high concentration relative to GTP, which competes for incorporation at the first position in the mRNA. One useful feature of co-transcriptional in vitro 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 degree of capping. Furthermore, the process requires the use of only one enzyme (RNA polymerase) in a single-step reaction. The use of chemically synthesized cap analogs also provides increased flexibility for using alternative nucleotide cap structures that are not well-recognized for methylation by vaccinia capping enzyme. However, cap analogs are expensive relative to nucleotides and other reaction components and must be present at high concentrations in the reaction. The present invention provides T7 RNA polymerase variants that are selective for incorporating cap analogs over GTP at the start of transcription, allowing for efficient capping using reduced cap analog concentrations, providing a more cost-effective and scalable process for producing capped RNA.

[0023] In some embodiments of the invention, inorganic pyrophosphatase is present during the in vitro transcription reaction to degrade pyrophosphate, a product inhibitor of transcription by RNA polymerase, to orthophosphate.

[0024] Abbreviations and definitions: Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures for 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 techniques are well known and described in many textbooks and reference works well known to those skilled in the art. Standard techniques, or modifications thereof, are used for chemical synthesis and chemical analysis. All patents, patent applications, literature, and publications mentioned herein (both supra and infra) are expressly incorporated herein by reference.

[0025] Although any suitable methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, several methods and materials are described herein. It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art. Thus, the terms defined immediately below are more fully described by reference to their application as a whole. All patents, patent applications, literature, and publications mentioned herein (both supra and infra) are expressly incorporated herein by reference.

[0026] Also, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0027] Numerical ranges are inclusive of the numerical endpoints defining the range. Accordingly, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Every maximum (or minimum) numerical limitation disclosed herein is also intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0028] The term "about" refers to 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 range. In some cases, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0029] Moreover, the headings provided herein are not limitations of the various aspects or embodiments of the invention that may be imparted by reference to the application as a whole.

[0030] Accordingly, the terms defined immediately below are more fully described by reference to the application as a whole. Nevertheless, to facilitate understanding of the invention, a number of terms are defined below.

[0031] 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.

[0032] As used herein, the terms "comprising" and cognates are used in their inclusive sense (i.e., equivalent to the terms "including" and their corresponding cognates).

[0033] The "EC" numbers refer 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 they catalyze.

[0034] "ATCC" refers to the American Type Culture Collection, whose biorepository collections include genes and strains.

[0035] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0036] As used herein, "T7 RNA polymerase" refers to a DNA-directed RNA polymerase encoded by the monomeric T7 bacteriophage that catalyzes the formation of RNA in the 5' to 3' direction.

[0037] As used herein, the term "cap" refers to a guanine nucleoside that is linked through its 5' carbon to a triphosphate group that is linked to the 5' carbon of the 5'-most nucleotide of an mRNA transcript. In some embodiments, the nitrogen at position 7 of the guanine in the cap is methylated.

[0038] As used herein, the terms "capped RNA," "5' capped RNA," and "capped mRNA" refer to RNA and mRNA, respectively, that include their cap.

[0039] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleosides covalently linked together. The polynucleotide may be composed entirely of ribonucleotides (i.e., RNA), entirely of 2' deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleotides and 2' deoxyribonucleotides. While the nucleosides are typically linked together via standard phosphodiester linkages, the polynucleotide may contain one or more non-standard linkages. The polynucleotide may be single- or double-stranded, or may contain both single- and double-stranded regions. Furthermore, while a polynucleotide is typically composed of naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may contain one or more modified and / or synthetic nucleobases (such as, for example, inosine, xanthine, hypoxanthine, etc.). In some embodiments, such modified or synthetic nucleobases are nucleobases that encode amino acid sequences.

[0040] "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 modification (e.g., glycosylation or phosphorylation).

[0041] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter symbols.

[0042] Abbreviations used 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).

[0043] When the three-letter abbreviation is used, the amino acid is the alpha carbon (C α ) can be in either the L- or D-configuration. For example, "Ala" refers to alanine without specifying the configuration about its alpha carbon, while "D-Ala" and "L-Ala" refer to D-alanine and L-alanine, respectively. When the single-letter abbreviations are used, an uppercase letter refers to the amino acid in the L-configuration about its alpha carbon, and a lowercase letter refers to the amino acid in the D-configuration about its alpha carbon. For example, "A" refers to L-alanine and "a" refers to D-alanine. When a polypeptide sequence is represented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequence is represented in the amino (N) to carboxy (C) direction, following common convention.

[0044] The abbreviations used for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically noted, the abbreviated nucleoside may be either a ribonucleoside or a 2'-deoxyribonucleoside. The nucleoside may be identified individually or collectively as either a ribonucleoside or a 2'-deoxyribonucleoside. When a nucleic acid sequence is represented as a string of single-letter abbreviations, the sequence is represented in the 5' to 3' direction according to common convention, and the phosphate is not indicated.

[0045] The terms "engineered," "recombinant," "non-naturally occurring," and "variant," when used in reference to a cell, polynucleotide, or polypeptide, refer to a substance or substance that does not occur in nature in other circumstances, or that corresponds to the natural or native form of the substance but is the same as, but is produced or derived from synthetic materials and / or has been modified by manipulation using recombinant techniques.

[0046] As used herein, "wild-type" and "naturally occurring" refer to forms 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 source in nature and has not been intentionally modified by human manipulation.

[0047] "Coding sequence" refers to that portion of a nucleic acid (e.g., a gene) that codes for the amino acid sequence of a protein.

[0048] The term "percent (%) sequence identity" is used herein to refer to comparisons among polynucleotides and polypeptides and is determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where either the same nucleic acid base or amino acid residue occurs in both sequences, or where the nucleic acid base or amino acid residue is aligned with a gap, to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms available to align two sequences.Optimal alignment of sequences for comparison can be carried out as known in the art, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), by the similarity search method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

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

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

[1977] , respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, either match or meet some positive-valued threshold score T. 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 for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score becomes 0 or less due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence 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 as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses 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 sequence alignments and determination of percent sequence identity are performed using the default parameters provided by GCG Wisconsin. The BESTFIT or GAP programs in the Software package (Accelrys, Madison Wis.) may be used.

[0049] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, e.g., a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the entire length of the nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each contain (1) sequences that are similar between the two sequences (i.e., a portion of the complete sequence), and (2) additional sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over 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, where the reference sequence can have one or more changes in its primary sequence.

[0050] A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues in which a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and where a portion of the sequence within the comparison window can contain 20% or fewer additions or deletions (i.e., gaps) when compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, including 30, 40, 50, 100, or longer windows, as appropriate.

[0051] "Corresponding to," "reference to," or "relative to," when used in the context of numbering a given amino acid or polynucleotide sequence, refers to the numbering of residues in a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, residue numbers or residue positions in a given polymer are specified with respect to the reference sequence, rather than by the actual numbered positions of residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence (e.g., that of an engineered T7 RNA polymerase) can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of residues in the given amino acid or polynucleotide sequence is done with respect to the reference sequence to which it is aligned.

[0052] An "amino acid difference" or "residue difference" refers to an amino acid residue difference at a position in a polypeptide sequence relative to the amino acid residue at the corresponding position in a reference sequence. The position of an amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, a "residue difference at position X93 as compared to SEQ ID NO:2" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 93 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has a serine at position 93, then a "residue difference at position X93 as compared to SEQ ID NO:2" includes an amino acid substitution of any residue other than serine at the polypeptide position corresponding to position 93 of SEQ ID NO:2. In most cases herein, a specific amino acid residue difference at a position is designated "XnY," where "Xn" identifies 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 that differs from that in the reference polypeptide). In some cases (e.g., in the tables provided in the Examples herein), the present disclosure also provides specific amino acid differences designated by the conventional annotation "AnB" (where A is the single-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the single-letter identifier of the residue substitution in the engineered polypeptide sequence). In some cases, a polypeptide of the present disclosure may contain one or more amino acid residue differences relative to a reference sequence, as indicated by a list of the identified positions at which a residue difference exists compared to the reference sequence. In some embodiments, when more than one amino acid can be used at a specific residue position in a polypeptide, the various amino acid residues that can be used can be separated by a " / " (e.g., X10H / X10P or X10H / P). In some embodiments, the enzyme variant contains more than one substitution.These substitutions are separated by a slash for ease of reading (e.g., C14A / K122A). The present application encompasses engineered polypeptide sequences containing one or more amino acid differences, including either or both conservative and non-conservative amino acid substitutions.

[0053] A "conservative amino acid substitution" refers to the replacement of a residue with a different residue having a similar side chain, and thus typically involves replacing an amino acid in a polypeptide with an amino acid within the same class or a similar defined amino acid class. By way of example and not limitation, an amino acid having an aliphatic side chain can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid having a hydroxyl side chain is substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain is substituted with another amino acid having a basic side chain (e.g., lysine and arginine); an amino acid having an acidic side chain is substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0054] A "non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. A non-conservative substitution may use an amino acid between, rather than within, its defined group and affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0055] "Deletion" refers to a modification to a reference polypeptide by removing one or more amino acids from the polypeptide. Deletions can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids comprising the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or improving the properties of the engineered enzyme. Deletions can be directed to internal and / or terminal portions of the polypeptide. In various embodiments, the deletions can include a contiguous segment or can be discontinuous.

[0056] An "insertion" refers to a modification to a polypeptide by the addition of one or more amino acids from the reference polypeptide. The insertion may be in an internal portion of the polypeptide, or at the carboxy or amino terminus. As used herein, an insertion includes fusion proteins, as known in the art. The insertion may be a contiguous segment of amino acids, or may be separated by one or more of the amino acids in a naturally occurring polypeptide.

[0057] An "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany the polypeptide. The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). The recombinant T7 RNA polymerase polypeptide may be present intracellularly, in cell culture medium, or prepared in various forms (e.g., a lysate or isolated preparation). Thus, in some embodiments, the recombinant T7 RNA polymerase polypeptide may be an isolated polypeptide.

[0058] A "substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., more abundant in amount than any other individual macromolecular species in the composition, on a molar or weight basis), generally constituting at least about 50% of the macromolecular species present, by molar or weight. Generally, a substantially pure T7 RNA polymerase composition comprises 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 the total macromolecular species present in the composition, by molar or weight. In some embodiments, the target species is purified to essential homogeneity (i.e., contaminants cannot be detected in the composition by conventional detection methods), where the composition essentially consists of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant T7 RNA polymerase polypeptide is a substantially pure polypeptide composition.

[0059] Improved enzyme properties of T7 RNA polymerase An "improved property" refers to an engineered T7 RNA polymerase polypeptide that exhibits an improvement in any enzymatic property when 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, increased selectivity for cap analogs over GTP, increased fidelity of replication, increased RNA yield, increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition (including pyrophosphate), increased chemical stability, improved solvent stability, increased tolerance to acidic or basic pH, increased tolerance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, and an altered temperature profile.

[0060] "Increased enzymatic activity" or "enhanced catalytic activity" refers to an improved property of an engineered T7 RNA polymerase, which can be expressed by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of substrate to product (e.g., percent conversion of a starting amount of substrate to product in a specified period of time using a specified amount of variant T7 RNA polymerase, as compared to a reference T7 RNA polymerase). Exemplary methods for determining enzymatic activity are provided in the Examples. Any property related to enzymatic activity can be affected.

[0061] "Hybridization stringency" refers to hybridization conditions (e.g., washing conditions) in nucleic acid hybridization. Generally, hybridization reactions are performed under lower stringency conditions, followed by washes of varying but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids with about 60% identity, preferably about 75% identity, about 85% identity, or greater than about 90% identity with the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally refers to hybridization that achieves a thermal melting temperature, T, as determined under solution conditions, of a defined polynucleotide sequence. mHigh stringency conditions refer to conditions of about 10°C or lower. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it is not stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization under conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, and 0.2% SDS at 42°C, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization under conditions equivalent to hybridization in 5x SSC containing 0.1% (w:v) SDS at 65°C, followed by washing in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions, and mildly stringent conditions, are described in the references cited above.

[0062] "Codon optimized" refers to changing the codons of a protein-encoding polynucleotide to those preferentially used in a target organism so that the encoded protein is more efficiently expressed in that organism. While the genetic code is degenerate in that most amino acids are represented by a few codons (referred to as "synonyms" or "synonymous codons"), it is well known that codon usage by a particular organism is not random but is biased toward certain codon triplets. This codon usage bias can be higher in certain genes, in reference to common functions or ancestral genes, highly expressed proteins versus low copy number proteins, and in the aggregate protein-coding regions of an organism's genome. In some embodiments, the polynucleotide encoding the T7 RNA polymerase enzyme can be codon optimized for optimal production from the host organism selected for expression.

[0063] The term "control sequences," as used herein, refers to all components necessary or advantageous for 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, a leader, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, initiation sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0064] "Operably linked" is defined herein as a configuration in which the control sequences are appropriately positioned (i.e., in a functional relationship) relative to a polynucleotide of interest so as to direct or regulate the expression of the polynucleotide and / or polypeptide of interest.

[0065] "Promoter sequence" refers to a nucleic acid sequence recognized by a host cell for expression of a polynucleotide of interest (e.g., a coding sequence). The promoter sequence contains transcriptional control sequences, which mediate expression of the polynucleotide of interest. The promoter may be any nucleic acid sequence that shows transcriptional activity in the host cell of choice, including mutated, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0066] "Suitable reaction conditions" refers to those conditions in an enzyme conversion reaction solution (e.g., range of enzyme loading, substrate loading, temperature, pH, buffer, co-solvent, etc.) under which the T7 RNA polymerase polypeptide of the present application can convert a substrate into a desired product compound.

[0067] "Substrate," in the context of an enzymatic conversion reaction process, refers to a compound or molecule upon which a T7 RNA polymerase polypeptide acts.

[0068] A "product" in the context of an enzymatic conversion reaction process refers to a compound or molecule that results from the action of a T7 RNA polymerase polypeptide on a substrate.

[0069] As used herein, the term "culturing" refers to growing a population of microbial cells under any suitable conditions (e.g., using liquid, gel, or solid media).

[0070] Recombinant polypeptides can be produced using any suitable method known in the art. A gene encoding the wild-type polypeptide of interest can be cloned into a vector (e.g., a plasmid) and expressed in a desired host (e.g., E. coli, S. cerevisiae, etc.). Variants of recombinant polypeptides can be produced by a variety of methods known in the art. Indeed, there are a wide 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. Methods are available for making specific substitutions at defined amino acids (site-directed), specific or random mutations in local regions of a gene (region-specific), or random mutagenesis throughout a gene (e.g., saturation mutagenesis). Many suitable methods are known to those skilled in the art for generating enzyme variants, including, but not limited to, site-directed mutagenesis of single- or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used 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 the variants are generated, they can be screened for any desired property (e.g., high or increased activity, or low or reduced activity, increased thermal activity, increased thermostability, and / or acidic pH stability, etc.).

[0071] In some embodiments, a "recombinant T7 RNA polymerase polypeptide" ("engineered T7 RNA polymerase polypeptide") is used. "T7 RNA polymerase polypeptide," "variant T7 RNA polymerase enzyme," and "T7 RNA polymerase variants," also referred to as "T7 RNA polymerase polypeptide," find use.

[0072] As used herein, a "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector operably linked to a suitable control sequence that can effect expression in a suitable host of a polypeptide encoded in the DNA sequence. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also contains a transcription terminator sequence.

[0073] As used herein, the term "expression" includes any step involved in producing the polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes secretion of the polypeptide from the cell.

[0074] As used herein, the term "produce" refers to the production of proteins and / or other compounds by a cell. The term is intended to encompass any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of the polypeptide from the cell.

[0075] As used herein, an amino acid or nucleotide sequence (e.g., 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.

[0076] As used herein, the terms "host cell" and "host strain" refer to a cell or organism in which the DNA provided herein (e.g., the T7 " refers to a host suitable for an expression vector containing a polynucleotide encoding an RNA polymerase variant. 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.

[0077] The term "analog," when used in reference to a polypeptide, refers to 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 analog refers to a polypeptide that includes one or more non-naturally occurring amino acid residues (including, but not limited to, homoarginine, ornithine, and norvaline) and naturally occurring amino acids. In some embodiments, an analog also includes one or more D-amino acid residues and a non-peptide linkage between two or more amino acid residues.

[0078] The term "effective amount" means an amount sufficient to produce a desired result. One of ordinary skill in the art can determine what an effective amount is by using routine experimentation.

[0079] 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 rather is intended as a relative definition.

[0080] As used herein, "composition" and "formulation" encompass products containing at least one engineered T7 RNA polymerase of the invention intended for any suitable use (e.g., research, diagnostics, etc.).

[0081] 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 an RNA polymerase enzyme.

[0082] The term "DNA template" is used to refer to a single- or double-stranded DNA molecule that contains a promoter sequence and a sequence that encodes the RNA product of transcription.

[0083] The term "promoter" is used to refer to a DNA sequence recognized by RNA polymerase as the start site of transcription. The promoter recruits RNA polymerase and, in the case of T7 RNA polymerase, determines the start site of transcription.

[0084] The term "RNA polymerase" is used to refer to a DNA-directed RNA polymerase that copies a DNA template into an RNA polynucleotide by stepwise incorporation of nucleotide triphosphates into the growing RNA polymer.

[0085] The terms "messenger RNA" and "mRNA" are used to refer to RNA molecules that encode proteins. The proteins are decoded through the process of translation.

[0086] 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 eukaryotic mRNA. The 7-methylguanosine cap structure is attached to the first nucleotide in the mRNA through a 5'-5' triphosphate linkage. In vivo, this cap structure is added to the 5' end of a nascent mRNA through the sequential action of multiple enzymes. In vitro, the cap can be incorporated directly at the initiation of transcription by RNA polymerase through the use of cap analogs.

[0087] The term "cap analog" refers to a dinucleotide containing a 5'-5' di-, tri-, or tetra-phosphate linkage. One end of the dinucleotide terminates in either a guanosine or a substituted guanosine residue; this is the end at which RNA polymerase begins transcription by extending from the 3' hydroxyl. The other end of the dinucleotide is a guanosine that mimics the eukaryotic cap structure, typically with a 7-methyl-, 7-benzyl-, or 7-ethyl-substitution and / or a 7-aminomethyl or 7-aminoethyl substitution. In some cases, this nucleotide is also methoxy substituted at the 3' hydroxyl group to prevent initiation of transcription from the capped end of the molecule.

[0088] The terms "ARCA" and "anti-reverse cap analog" refer to chemically modified forms of cap analogs designed to maximize the efficiency of in vitro translation by ensuring that the cap analog is properly incorporated into transcripts in the correct orientation. These analogs find use in enhancing translation. In some embodiments, ARCAs known in the art find use (e.g., Peng et al., Org. Lett., 4:161-164

[2002] ).

[0089] The term "riboswitch" is used to refer to an autocatalytic RNA enzyme that cleaves itself or another RNA in the presence of a ligand.

[0090] The term "fidelity" is used to refer to the accuracy of an RNA polymerase in transcribing or copying a DNA template into an RNA polynucleotide. Inaccurate transcription can result in single nucleotide polymorphisms (SNPs) or indels.

[0091] The term "single-nucleotide polymorphism" or "SNP" refers to a nucleotide change present at a single position in a polynucleotide. In the context of transcription, SNPs can arise from the misincorporation of a non-complementary ribonucleotide (A, C, G, or U) by an RNA polymerase at a single position on a DNA template.

[0092] The term "indel" is used to refer to the insertion or deletion of one or more polynucleotides. In the context of transcription by RNA polymerase, an indel error results from the addition of one or more extra ribonucleotides or the inability to incorporate one or more nucleotides at a position on a DNA template.

[0093] The term "selectivity" is used to refer to the trait of an enzyme having higher activity for one substrate compared to another during the catalyzed reaction. In the context of co-transcriptional capping, the RNA polymerase may have a higher or lower preference for the cap analog over GTP.

[0094] The term "inorganic pyrophosphatase" is used to refer to an enzyme that breaks down inorganic pyrophosphate to orthophosphate.

[0095] Engineered T7 RNA polymerase activity: In some embodiments, the engineered T7 RNA polymerase exhibiting improved properties has an affinity for SEQ ID NOs: 4 and / or 15 by 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 about 100%. Amino acid sequence identity, when compared to SEQ ID NO: 4 and / or 15, has amino acid residue differences at one or more amino acid positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20 or more amino acid positions compared to SEQ ID NO: 4 and / or 15), or a sequence having 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 SEQ ID NO: 4 and / or 15. In some embodiments, the residue differences at one or more positions when compared to SEQ ID NO: 4 comprise 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 NOs: 4, 15, 17, 19, 21, 23, 25, 27, 29, 32, 33, 35, 37, and / or 39. In some embodiments, the residue differences at one or more positions when compared to SEQ ID NO: 15 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 NOs: 4, 15, 17, 19, 21, 23, 25, 27, 29, 32, 33, 35, 37, and / or 39.

[0096] In some embodiments, the engineered T7 RNA polymerase polypeptide comprises a functional fragment of an engineered T7 RNA polymerase polypeptide encompassed by the present invention, wherein 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 resulting engineered T7 RNA polymerase polypeptide (i.e., the parent engineered T7 RNA polymerase). Functional fragments comprise at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and even 99% of the parent sequence of the engineered T7 RNA polymerase. In some embodiments, the functional fragment is truncated by fewer than 5, fewer than 10, fewer than 15, fewer than 10, fewer than 25, fewer than 30, fewer than 35, fewer than 40, fewer than 45, and fewer than 50 amino acids.

[0097] Polynucleotides encoding engineered polypeptides, expression vectors and host cells: The present invention provides polynucleotides encoding the 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 generate recombinant polynucleotides capable of expressing the polypeptides. An expression construct containing a heterologous polynucleotide encoding the engineered T7 RNA polymerase polypeptide can be introduced into a suitable host cell to express the corresponding T7 RNA polymerase polypeptide.

[0098] As will be apparent to those skilled in the art, the availability of a protein sequence and knowledge of the codons corresponding to its various amino acids provides a description of all polynucleotides that can encode the polypeptides of the invention. The degeneracy of the genetic code allows for a vast number of nucleic acids to be created when the same amino acid is coded for by alternative or synonymous codons, all of which will encode the engineered T7 RNA polymerase polypeptide. Thus, with knowledge of a particular amino acid sequence, one skilled in the art can create any number of different nucleic acids by simply modifying 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 and every possible variation of polynucleotides that can be made that encode the polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations should be considered specifically disclosed with respect to any polypeptide described herein (including the variants provided in Table 5.3, Table 5.4, Table 5.5, and / or Table 5.6).

[0099] In various embodiments, the codons are preferably selected to be compatible with the host cell in which the protein is produced. For example, preferred codons used in bacteria are used for expression in bacteria. Consequently, the codon-optimized polynucleotide encoding the engineered T7 RNA polymerase polypeptide contains preferred codons at more than about 40%, 50%, 60%, 70%, 80%, or 90% of the codon positions of the full-length coding region.

[0100] In some embodiments, as described above, the polynucleotide encodes an engineered polypeptide having T7 RNA polymerase activity with the properties disclosed herein, wherein the polypeptide has 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 to a reference sequence selected from SEQ ID NOs: 4 and 15, or a sequence disclosed in Table 5.3, Table 5.4, Table 5.5, and / or Table 5.6. and any variant amino acid sequence as set forth in SEQ ID NOs: 17, 19, 21, 23, 25, 27, 29, 32, 33, 35, 37, and / or 39, or any variant amino acid sequence as disclosed in Table 5.3, Table 5.4, Table 5.5, and / or Table 5.6 (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions) when compared to the reference polypeptide of SEQ ID NOs: 17, 19, 21, 23, 25, 27, 29, 32, 33, 35, 37, and / or 39. In some embodiments, the reference sequence is selected from SEQ ID NOs: 4 and / or 15. In some embodiments, the polynucleotide encodes an engineered polypeptide having T7 RNA polymerase activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence that 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 reference sequences SEQ ID NOs: 4 and / or 15 and one or more residue differences when compared to SEQ ID NOs: 4 and / or 15 at residue positions selected from those provided in Table 5.3, Table 5.4, Table 5.5, and / or Table 5.6 when optimally aligned with the polypeptide of SEQ ID NOs: 4 and / or 15.

[0101] In some embodiments, the polynucleotide encoding the engineered T7 RNA polymerase polypeptide comprises a polynucleotide sequence selected from the polynucleotide sequences encoding SEQ ID NOs: 4, 15, 17, 19, 21, 23, 25, 27, 29, 32, 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 NOs: 3, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.

[0102] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from SEQ ID NOs: 3 and / or 14, or its complement, or to a polynucleotide sequence encoding any of the variant T7 RNA polymerase polypeptides 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 at a residue position selected from any of the positions set forth in Table 5.3, Table 5.4, Table 5.5, and / or Table 5.6, when compared to SEQ ID NOs: 4 and / or 15.

[0103] In some embodiments, an isolated polynucleotide encoding any of the engineered T7 RNA polymerase polypeptides provided herein can be engineered in a variety of ways to provide for expression of the polypeptide. In some embodiments, the polynucleotide encoding the polypeptide is provided as an expression vector in which one or more regulatory sequences are present to regulate expression of the polynucleotide and / or polypeptide. Manipulation of the isolated polynucleotide prior to insertion into a vector may be desirable or necessary, depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0104] In some embodiments, the control sequences include, among other sequences, a promoter, leader sequence, polyadenylation sequence, propeptide sequence, signal peptide sequence, and transcription terminator. As is known in the art, appropriate promoters can be selected based on the host cell used. Exemplary promoters for filamentous fungal host cells include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha amylase, Aspergillus niger acid-stable alpha amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (Aspergillus niger neutral alpha amylase and Aspergillus Examples of promoters that can be used include promoters derived from the 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 genes. 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 use in mammalian cells include, but are not limited to, those derived from cytomegalovirus (CMV), Simian vacuolating virus 40 (SV40), Homo sapiens phosphoglycerate kinase, beta-actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, or from Gallus gallus beta-actin.

[0105] In some embodiments, the control sequence is a suitable transcription terminator sequence (a sequence recognized by the host cell) to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the peptide. Any terminator functional in the selected host cell finds use in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for 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 derived from cytomegalovirus (CMV), simian vacuolar virus 40 (SV40), or Homo sapiens growth hormone.

[0106] In some embodiments, the control sequence is a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used. Exemplary leaders for filamentous fungal host cells are obtained from the Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase genes. Suitable leaders for yeast host cells include, but are not limited to, those obtained from the 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) genes.

[0107] The control sequence may also be a polyadenylation sequence (a sequence operably linked to the 3' end of the nucleic acid sequence and recognized by the host cell as a signal for adding polyadenosine residues to the transcribed mRNA when transcribed). Any polyadenylation sequence that is functional in the selected host cell may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, those derived from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, 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] ).

[0108] In some embodiments, the control sequence is a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and that directs the encoded polypeptide into a cellular secretory pathway. The 5' end of the coding region of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a selected host cell may be incorporated into the engineered T7 nucleotide sequences provided herein. Useful signal peptides find use for the expression of RNA polymerase polypeptides. Useful signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those obtained from the genes for 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 derived from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include, but are not limited to, those derived from the gene for immunoglobulin gamma (IgG).

[0109] In some embodiments, the control sequence is a propeptide coding region that encodes an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide may be referred to as a "proenzyme," a "propolypeptide," or a "zymogen" (in some cases). A propolypeptide can be converted to a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

[0110] In another aspect, the present invention also provides recombinant expression vectors comprising a polynucleotide encoding an engineered T7 RNA polymerase polypeptide and, depending on the type of host into which it is to be introduced, one or more expression control regions (e.g., promoters and terminators, origins of replication, etc.). In some embodiments, the various nucleic acids and control sequences described above are joined together to generate a recombinant expression vector, including one or more conventional restriction sites, allowing for the insertion or substitution of a nucleic acid sequence encoding a variant T7 RNA polymerase polypeptide at such sites. Alternatively, a 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. In creating the expression vector, the coding sequence is positioned within the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0111] The recombinant expression vector may be conventionally subjected to recombinant DNA procedures, and variants thereof may be The vector may be any vector (e.g., a plasmid or virus) capable of causing expression of the T7 RNA polymerase polynucleotide sequence. The choice of vector typically depends on the compatibility of the vector with the host cell into which it is to be introduced. The vector may be a linear or closed circular plasmid.

[0112] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity), the replication of which is independent of chromosomal replication (e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some alternative embodiments, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome, becoming integrated into the chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, which together contain the total DNA to be inserted into the genome of the host cell, may be used.

[0113] In some embodiments, the expression vector preferably contains one or more selectable markers, which allow for easy selection of transformed cells. A "selectable marker" is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy for auxotrophs, 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 (anthranilate synthase), 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 expression of the engineered T7 RNA polymerase enzyme in the host cell. Host cells for use in expressing polypeptides encoded by the expression vectors 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 Accession No. 201178]); insect cells (e.g., Drosophila S2 and Spodoptera Sf9 cells), plant cells, animal cells (e.g., CHO, COS, and BHK), and human cells (e.g., HEK293T, human fibroblast, THP-1, Jurkat, and Bowes melanoma cell lines).

[0114] Thus, in another aspect, the present invention provides methods for producing an engineered T7 RNA polymerase polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered T7 RNA polymerase polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises isolating and / or purifying the T7 RNA polymerase polypeptide as described herein.

[0115] Suitable culture medium and growth conditions for the above-mentioned host cells are well known in the art.The polynucleotide for expressing the T7 RNA polymerase polypeptide can be introduced into cells by various methods known in the art.Techniques include, inter alia, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0116] The engineered T7 RNA polymerase having the properties disclosed herein can be obtained by subjecting the polynucleotide encoding naturally occurring or engineered T7 RNA polymerase polypeptide to mutagenesis and / or directed evolution methods as known in the art and as described herein.Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, for example, 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. Patent No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (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] ).

[0117] For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (e.g., U.S. Patent 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, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,376,246, 6,379,9 64, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,489,146, 6,506,602, 6,506,603, 6,506,604, 6,506,605, 6,506,606, 6,506,607, 6,506,608, 6,506,609 ... 19,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,613,514, 6,653,072, 6,716,631, 6,777,218, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,288,375, 7,421,347, 7,430,477, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,873,477, 7,873,499, 7,904,249, 7,957,912, 8,014,961, 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 US and non-US 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. U.S.A., 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] ; US Patent Application Publication Nos. 2008 / 0220990, US 2009 / 0312196, US2014 / 0005057, US2014 / 0214391, US2014 / 0221216; US2015 / 0050658, US2015 / 0133307, US2015 / 0134315 and related US and non-US 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.

[0118] In some embodiments, the enzyme variants obtained after the mutagenesis treatment are screened by subjecting the enzyme variants to a defined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after the heat treatment or other assay conditions. DNA comprising a polynucleotide encoding the T7 RNA polymerase polypeptide is then isolated from the host cell, sequenced to identify the nucleotide sequence changes, if any, and used to express the enzyme in a different or the same host cell. Measuring enzyme activity from the expression library can be performed using any suitable method known in the art, such as standard biochemical techniques (e.g., HPLC analysis).

[0119] For engineered polypeptides of known sequence, polynucleotides encoding the enzymes can be prepared by standard solid-phase methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method, as typically performed in automated synthesis methods (see, e.g., Beaucage et al., Tetra. Lett., 22:1859-69

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

[1984] ). According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer), purified, annealed, ligated, and cloned into an appropriate vector.

[0120] Thus, in some embodiments, the method for preparing the engineered T7 RNA polymerase polypeptide can include the steps of: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from any variant amino acid sequence 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 the T7 RNA polymerase polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or several (e.g., 3, 4, up to 5, or up to 10) amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence has, as appropriate, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 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 or non-conservative substitutions.

[0121] The expressed engineered T7 RNA polymerase polypeptide can be evaluated for any desired improved properties (e.g., activity, selectivity, stability, acid resistance, protease sensitivity, etc.) using any suitable assay known in the art, including, but not limited to, the assays and conditions described herein.

[0122] In some embodiments, any of the engineered T7 RNA polymerase polypeptides expressed in host cells are recovered from the cells and / or culture medium using any one or more of the well-known techniques of protein purification, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others.

[0123] Chromatographic techniques for isolating the T7 RNA polymerase polypeptide include, among others, reverse-phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and the like, and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to isolate the improved variant T7 RNA polymerase enzyme. In some embodiments utilizing affinity chromatography purification, the variant T7 Any antibody that specifically binds an RNA polymerase polypeptide finds use. In some embodiments utilizing affinity chromatography purification, T7 Proteins that bind to glycans covalently linked to RNA polymerase find use. In yet other embodiments utilizing affinity chromatography purification, any small molecule that binds to the T7 RNA polymerase active site finds use. For antibody production, various host animals (including, but not limited to, rabbits, mice, rats, etc.) are immunized by injecting a T7 RNA polymerase polypeptide (e.g., a T7 RNA polymerase variant) or a fragment thereof. In some embodiments, the T7 RNA polymerase polypeptide or fragment is bound to a suitable carrier (e.g., BSA) by a side chain functional group or a linker bound to the side chain functional group.

[0124] 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., S. cerevisiae, Daucus carota, Nicotiana tabacum, H. sapiens (e.g., HEK293T), or Cricetulus griseus (e.g., CHO)) comprising a polynucleotide sequence encoding an engineered T7 RNA polymerase polypeptide as described herein under conditions that result in the production of the engineered T7 RNA polymerase polypeptide, and recovering the engineered T7 RNA polymerase polypeptide from the cell and / or culture medium.

[0125] In some embodiments, the invention encompasses methods for producing an engineered T7 RNA polymerase polypeptide, the methods comprising culturing a recombinant eukaryotic cell comprising 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 with the reference sequence of SEQ ID NO:4 and / or 15 and one or more amino acid residue differences compared to SEQ ID NO:4 and / or 15 selected from those provided in Table 5.3, Table 5.4, Table 5.5, and / or Table 5.6, and / or combinations thereof, when optimally aligned with the amino acid sequence of SEQ ID NO:4 and / or 15, under suitable culture conditions that allow production of the engineered T7 RNA polymerase polypeptide, and optionally recovering the engineered T7 RNA polymerase polypeptide from the culture and / or the cultured bacterial cells.

[0126] In some embodiments, once the engineered T7 RNA polymerase polypeptides are recovered from recombinant host cells or cell culture medium, they are further purified by any suitable method known in the art. In some additional embodiments, the purified T7 RNA polymerase polypeptides are combined with other ingredients and compounds to provide compositions and formulations (e.g., pharmaceutical compositions) comprising the engineered T7 RNA polymerase polypeptides, as appropriate for various applications and uses. In some additional embodiments, the purified or formulated T7 RNA polymerase polypeptides are lyophilized.

[0127] 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.

[0128] experiment The following examples, including the experiments and results achieved, are provided for illustrative purposes only and should not be construed as limiting the invention.

[0129] In the experiments disclosed below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (mole); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min(s) (minutes); h(s) and hr(s) (hours); U (units); MW (molecular weight); rpm (revolutions per minute); rcf (relative centrifugal force); °C (temperature in degrees Celsius); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (a commonly used laboratory E. coli strain available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HPLC (high performance liquid chromatography); MWCO (molecular weight cut-off); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); T7RNAP (T7 RNA polymerase); PES (polyethersulfone); CFSE (carboxyfluorescein succinimidyl ester); IPTG (isopropyl β-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); NADPH (nicotinamide adenine dinucleotide phosphate); GIDH (glutamate dehydrogenase); FIOPC (fold improvement over positive control); LB (Luria Broth); MeOH(メタノール); Athens Research(Athens Research Technology, Athens, GA); NEB(New England Biolabs, Ipswich, MA); Ion Torrent(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, part of Life Technologies, Corp., Grand Island, NY), Agilent (Agilent Technologies, Inc., Santa Clara, CA); Thermo Scientific (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).

[0130] The following polynucleotide and polypeptide sequences find use in the present invention. In some cases (as noted below), the polynucleotide sequence is followed by the encoded polypeptide. [Example]

[0131] Example 1 Obtaining the T7 RNA polymerase gene and constructing an expression vector The wild-type (WT) T7 RNA polymerase enzyme (SEQ ID NO: 2) is a bacteriophage The T7 RNA polymerase is encoded by the genome of T7 (SEQ ID NO: 1). A synthetic gene (SEQ ID NO: 3) encoding a 6-histidine-tagged version of T7 RNA polymerase (SEQ ID NO: 4) was constructed and subcloned into the Escherichia coli expression vector pCK100900i (see, e.g., U.S. Patent No. 7,629,157 and U.S. Patent Application Publication No. 2016 / 0244787, both incorporated by reference). These plasmid constructs were transformed into E. coli strains derived from W3110. Directed evolution techniques commonly known by those skilled in the art were used to generate libraries of gene variants from these plasmids (see, e.g., U.S. Patent No. 8,383,346 and WO 2010 / 144103, both incorporated by reference). The substitutions in the enzyme variants described herein are 6His-targeted WT, as indicated. Indicated with reference to the T7 RNA polymerase enzyme (ie, SEQ ID NO: 4) or variants thereof.

[0132] Example 2 High-throughput (HTP) T7 RNA polymerase expression and purification This example describes experiments performed on T7 RNA polymerase expression and purification of T7 RNA polymerase variants.

[0133] High-Throughput (HTP) Amplification of T7 DNA Polymerase and Variants Transformed E. coli cells were selected by plating on LB agar plates containing 1% glucose and 30 μg / ml chloramphenicol. After overnight incubation at 37°C, colonies were plated onto 96-well shallow flat-bottom NUNC plates filled with 180 μl / well of LB medium (supplemented with 1% glucose and 30 μg / ml chloramphenicol). TM The cultures were grown overnight on a shaker (200 rpm, 30°C, and 85% relative humidity; Kuhner) for 18-20 hours. Samples (20 μL) of the overnight growth were transferred to a Costar 96-well deep plate filled with 380 μL of Terrific Broth (supplemented with 30 μg / ml chloramphenicol). The plates were incubated for 120 minutes on a shaker (250 rpm, 30°C, and 85% relative humidity; Kuhner) until OD 600 The cells were incubated until a pH of 0.4-0.8 was reached. The cells were then induced with 40 μL of 10 mM IPTG in sterile water and incubated overnight on a shaker (250 rpm, 30°C, and 85% relative humidity; Kuhner) for 18-20 hours. The cells were pelleted (4000 rpm x 20 minutes), the supernatant was discarded, and the cells were frozen at -80°C before analysis.

[0134] Dissolution of HTP pellets The cell pellet was lysed in 400 μL of lysis buffer (20 mM Tris pH 7.5, 1 mM The cells were resuspended in 0.5 mg / ml MgSO4, 1 mg / ml lysozyme, and 0.5 mg / ml polymyxin B sulfate, and the mixture was stirred for 1.5 hours at room temperature. The lysate was then pelleted (4000 rpm x 5 minutes), and the clarified supernatant was saved for purification.

[0135] HTP purification of T7RNAP from crude lysate T7RNAP was isolated from clarified E. coli lysate using His-Select ((登録商標)Purification was performed by metal-affinity chromatography using high-capacity (HC) nickel-coated plates (Sigma) according to the manufacturer's instructions. (登録商標) The plate was washed with a total of 800 μl of wash buffer per well (50 mM sodium phosphate pH 7.5, 300 mM NaCl, 25 mM imidazole, 0.1% v / v TWEEN-20 (登録商標) Reagents [Sig Then, 200 μl of HTP lysate containing T7RNAP was added to 200 μl of PBS. 1 μl of wash buffer was mixed, loaded onto the plate, and centrifuged for 1 minute at 2000 relative centrifugal force (rcf) and 4°C. The plate was washed twice with 600 μl of wash buffer / well (3 minutes of centrifugation at 3000 rcf and 4°C for each wash). The enzyme samples were eluted with 200 μl elution buffer (50 mM sodium phosphate pH 7.5, 300 mM NaCl, 250 mM imidazole, 0.1% v / v TWEEN) by centrifugation at 3000 rcf for 1 minute at 4°C. (登録商標) -20 reagent) was added and eluted.

[0136] The eluent was TM Buffer exchange was performed using a Spin Desalting Plate (Thermo Fisher). Briefly, plates were 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 for 2 minutes at 1100×g at 4°C. 100 μl of His-Select ATP was added to the desalting plate. (登録商標) The plate sample eluate was loaded and centrifuged for 2 minutes at 1100 x g at 4°C. The eluate from the desalting plate was retained and mixed with an equal volume of glycerol for a final storage buffer concentration of 50 mM Tris HCl pH 8.0, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.1% v / v Triton X-100, and 50% glycerol (v / v).

[0137] The absence of RNase contamination in the purified preparations was confirmed using the RNase Alert assay (IDT, Life Technologies). SDS-PAGE analysis of T7RNAP samples showed no detectable contaminating bands for the majority of samples.

[0138] For in vitro transcription reactions using HTP-purified T7RNAP, purified polymerase was added to a final concentration of 10% of the total reaction volume (5% for enzyme from shake-flask cultures).

[0139] Example 3 Shake flask expression and purification of T7 RNA polymerase This example describes experiments involving shake flask expression and purification of T7RNAP.

[0140] Shake flask expression Selected HTP cultures, grown as described in Example 2, were plated onto 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 with 1% glucose and 30 μg / ml chloramphenicol. The cultures were grown for 18 hours at 30° C., 250 rpm, and reached a final OD of 0.2 on an approximately 1:10 dilution into 250 ml of Terrific Broth with 30 μg / ml chloramphenicol. 600 The culture was grown at 30°C, 250 rpm for approximately 3 hours to an OD of 0.6-0.8. 600The cells were incubated at 30°C for 60 min and then induced by the addition of IPTG to a final concentration of 1 mM. The induced culture was incubated for 20 h at 30°C and 250 rpm. After the incubation period, the culture was centrifuged at 4000 rpm for 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 disrupter (Microfluidics M-110L). The crude lysate was pelleted by centrifugation (16,000 rpm for 60 min at 4°C), and the supernatant was then filtered through a 0.2 μm PES membrane to further clarify the lysate.

[0141] Purification of T7 RNA polymerase from shake flask lysates T7RNAP lysates were purified using the AKTA Start purification system and AC Step HiF settings (run parameters provided below) in a 5ml HisTrap™ filter. Purification was performed using an FF column (GE Healthcare). The SF wash buffer was 50 mM sodium phosphate pH 7.5, 500 mM NaCl, 0.1% v / v TWEEN-20. (登録商標) The SF elution buffer consisted of 50 mM sodium phosphate pH 7.5, 500 mM NaCl, 0.1% v / v TWEEN-20, and 25 mM imidazole. (登録商標) reagent (Sigma), and 300 mM imidazole. [Table 2.1]

[0142] The five single most concentrated 3 ml fractions were identified by UV absorbance (A280) and incubated overnight in 2× T7RNAP storage buffer (100 mM Tris HCl pH 8.0, 200 mM NaCl, 2 mM DTT, 2 mM EDTA, 0.2% v / v Triton X-100) on a 10K Slide-A-Lyzer. TM The material was dialyzed overnight in a dialysis cassette (Thermo Fisher) for 16 hours for buffer exchange, followed by 24 hours for a second buffer exchange. An equal volume of glycerol was added to the dialyzed material. The enzyme concentration in the preparation was determined by gel densitometry and absorbance at 280 nm.

[0143] Example 4 Transcription reaction Transcription reactions were assembled with the cap analog α,γ-bis(N7-methylguanosine)triphosphate (also referred to 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) was derived from Bacillus anthracis It contains a T7 RNAP promoter sequence linked to the coding sequence of the GlmS riboswitch (SEQ ID NO: 6). Upon induction with its ligand, glucosamine-6-phosphate, the GlmS riboswitch self-cleaves, releasing a 16-mer RNA nucleotide (SEQ ID NO: 7) from the 5' end of the transcript, which contains either a capped or uncapped 5' phosphate. This small 16-mer RNA oligonucleotide cleavage product was amenable to ionization and analysis by LC-MS, which was used to distinguish between capped and uncapped species.

[0144] Reactions (30 μl) were assembled with a final concentration of 50 mM Tris HCl pH 7.9, 30 mM MgCl, 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. The total of m7G(5')ppp(5')m7G and GTP was maintained at 6 mM. Glucosamine-6-phosphate was included at the start of the reaction, and riboswitch cleavage was allowed to proceed during 4 hours of in vitro transcription. For HTP screening, reactions were quenched with an equal (30 μl) volume of 40 mM EDTA. m7G(5')ppp(5')m7G and GTP in total 6 mM was maintained.

[0145] Example 5 LC-MS activity assay This example describes an LC-MS method developed for the separation of smaller RNA fragments using a Thermo LTQ MS system for the analysis of shorter capped 5'- and uncapped 5'-phosphate cleavage products.

[0146] The 7meG-capped and 5' triphosphate-uncapped 16-mer cleavage products (SEQ ID NO: 7) were chromatographically separated from the reactants and higher MW RNA by reversed-phase ion-pair HPLC (see Figure 1) using a C18 column and HFIP mobile phase, commonly used for the separation of oligonucleotides for mass spectrometry (see Table 5.1). [Table 5.1]

[0147] Base peak extraction for the capped and uncapped 16-mer cleavage products was performed using seven ions for each species, including multiple negatively charged ions and metal adducts. The peak intensities of these seven ions were used to derive the signal for each of the capped and uncapped 16-mer cleavage products. These ions included multiple charge states as well as sodium and potassium adducts (see Table 5.2). All ions were observed within one mass unit of their expected values. [Table 5.2]

[0148] Capping performance relative to WT T7RNAP (i.e., "fold improvement over parent," or "FIOP") was calculated by dividing the capped / uncapped peak intensity ratio for each sample by the ratio calculated for the WT parent control present on each plate (n = 6-10). The FIOP values ​​were used to rank variant performance relative to WT T7RNAP or the library parent (which was included as a control on every plate). By using this relative quantification method, it was possible to compensate for differences in ionization that were inherent between the capped and uncapped species, as well as signal variations caused by the batch and EDTA concentration of the HFIP mobile phase. In vitro transcription reactions were performed as described in Example 4, with reaction times and m7G(5')ppp(5')m7G concentrations as shown in the tables below. Tables 5.3, 5.4, and 5.5 report the average activity improvement for three to six replicates. Table 5.6 reports the average activity improvement for variants for which replicate information was available. [Table 5.3-1] [Table 5.3-2] [Table 5.4-1] [Table 5.4-2]

[0149] Variant 85 was noted to have undetectable levels of uncapped mRNA in the LC-MS assay. [Table 5.5] [Table 5.6-1] [Table 5.6-2] [Table 5.6-3] [Table 5.6-4]

[0150] Example 6 Determination of RNA yield In vitro transcription reactions were performed as described in Example 4, except for using a luciferase template (SEQ ID NO: 10). mRNA yields from in vitro transcription reactions were performed according to the manufacturer's protocol for the Quant-iT RNA Assay kit (broad range, Q-33140, Thermo Fisher). mRNA abundance was calculated using a standard curve derived for the mRNA standards provided with the assay kit. [Table 6.1]

[0151] Example 7 Determination of T7RNAP variant transcription fidelity Polymerase fidelity was measured based on direct sequencing of multiple RT-PCR clones derived from mRNA transcribed from the 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 allowed for measurement of the contribution, if any, of this cap analog to the error rates of wild-type and variant polymerases under process-relevant conditions. A 1.7 kb luciferase template DNA (SEQ ID NO: 8) served as the DNA template for transcription using wild-type and variant T7 RNAP to generate full-length mRNA transcripts. RNA was isolated using the Zymo RNA Clean and Concentrator-25 Kit (Zymo Research), and residual DNA was removed from the RNA samples by two sequential treatments with a DNA-free DNAase I Kit (Ambion / Thermo Fisher). The sample was purified by oligo-(dT) 25 Primer (SEQ ID NO: 40) was used to reverse transcribe the luciferase template using Accuprime Reverse Transcriptase (Agilent) and anneal to the poly(A) tail on the luciferase template. The RT reaction was then reverse transcribed using HF buffer (New England Biolabs) to synthesize the PHUSION RT-PCR product. (登録商標) A 1675-bp amplicon was generated using gene-specific primers (SEQ ID NO: 12, SEQ ID NO: 13) that anneal to the luciferase coding sequence using a high-fidelity DNA polymerase. The amplified fragment was digested with BglI (New England Biolabs), ligated into a cloning vector, and transformed to generate a single clone in E. coli.

[0152] Individual clones were picked and sequenced on the Ion Torrent PGM platform (Thermo Fisher) using a multiplexed barcoding strategy. Barcode reads were deconvoluted, and then the sequence of each clone was assembled against its predicted 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 matched. Most observed mutations were substitutions, although insertions and deletions were also observed. The total number of mutations per base for sequenced mRNA-derived clones was calculated and is reported in Tables 7.1 and 7.2. Based on the literature, the predicted overall rate of mutations per base was 1.0 for T7RNAP (1 x 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 a total of 1.72 × 10 -4 Most of the variant polymerases exhibited overall error rates close to the reported literature value for T7 RNAP, even in the presence of 0.5 mM m7G(5')ppp(5')m7G.

[0153] A one-sided (right-sided) binomial test was used to calculate the probability of sampling the observed number of errors (or more) given the number of bases sequenced, if the actual error rate in an experiment was equal to the observed overall error rate of T7RNAP-WT in separate experiments shown in Tables 7.1 and 7.2. The fidelity of a given variant was considered indistinguishable from WT T7RNAP in this assay for a p-value greater than 0.05. In Table 7.1, in the observed error rate results column, a "+" indicates a p-value of 1.7*10 -4 is less than 1.7*10 -4In Table 7.2, in the Observed Error Rate Results column, a "+" indicates p>0.05 and a "-" indicates 0<0.05, while in the Binomial Test Results column, a "+" indicates 1.5 x 10 -4 is less than 1.5 x 10 -4 while in the binomial test result column, "+" means p>0.05 and "-" means p<0.05. [Table 7.1] [Table 7.2]

[0154] While the invention has been described with reference to specific embodiments thereof, various modifications may be made and equivalents may be substituted to adapt to a particular situation, material, composition of matter, process, process step or steps, so that the benefits of the invention are achieved without departing from the scope of what is claimed.

[0155] For all purposes in the United States, each and every publication and patent document cited in this application is incorporated herein by reference 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 intended as an indication that any such document is pertinent prior art, and does not constitute an admission as to the contents or date thereof. The present invention also provides the following items. (Item 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 of SEQ ID NO: 4 and / or 15, or a functional fragment thereof, wherein the engineered RNA polymerase comprises at least one substitution or set of substitutions in the polypeptide sequence, wherein the amino acid substitutions in the polypeptide sequence are numbered with reference to SEQ ID NO: 4 or 15. (Item 2) 2. The engineered RNA polymerase of item 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. (Item 3) At least one substitution or set of substitutions is 397, 397 / 513, 397 / 513 / 635, 397 / 513 / 635, 397 / 513 / 635 / 656, 397 / 513 / 635 / 656 / 660, 397 / 513 / 635 / 656 / 660 / 664, 397 / 513 / 635 / 656 / 664, 397 / 513 / 635 / 660, 397 / 513 / 635 / 660 / 664, 397 / 513 / 635 / 664, 397 / 513 / 656 / 660, 397 / 513 / 660 , 397 / 513 / 660 / 664, 397 / 513 / 664, 397 / 513, 397 / 635, 397 / 635 / 656 / 660 / 664, 397 / 635 / 656 / 664, 397 / 635 / 660, 397 / 635 / 664, 397 / 635 / 664 / 850, 397 / 660, 397 / 664, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:4. (Item 4) At least one substitution or set of substitutions is 113 / 137 / 513, 136 / 357 / 404 / 514, 136 / 357 / 514, 136 / 394 / 404 / 446, 136 / 401, 136 / 401 / 404, 136 / 404 / 446, 136 / 404 / 514, 136 / 446, 136 / 514, 137, 137 / 401, 137 / 401 / 513, 137 / 401 / 513, 137 / 513, 137 / 513 / 621, 137 / 635, 137 / 656, 357 / 394 / 446 2. The engineered RNA polymerase of item 1, wherein the amino acid positions are selected from the following: 01 / 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. (Item 5) 2. The engineered RNA polymerase of 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 shown in Table 5.3, Table 5.4, Table 5.5, and / or Table 5.6. (Item 6) 2. The engineered RNA polymerase of item 1, wherein 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. (Item 7) 2. The engineered RNA polymerase of 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 set forth in SEQ ID NOs: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and / or 39. (Item 8) 2. The engineered RNA polymerase of item 1, wherein the engineered RNA polymerase comprises 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. (Item 9) 9. The engineered RNA polymerase of any of items 1 to 8, wherein the engineered polymerase exhibits at least one improved property compared to wild-type T7 RNA polymerase. (Item 10) 10. The engineered RNA polymerase of item 9, wherein the at least one improved property is selected from improved selectivity for the cap analog over GTP during transcription initiation, improved protein expression, improved stability in a storage buffer, and improved stability under reaction conditions. (Item 11) 11. The engineered RNA polymerase of any of paragraphs 1 to 10, wherein the polymerase maintains RNA yield, transcription fidelity, thermostability, protein expression, stability at -20°C, or stability under reaction conditions equivalent to wild-type T7 RNA polymerase. (Item 12) 12. The engineered RNA polymerase according to any one of items 1 to 11, wherein the engineered polymerase is purified. (Item 13) 13. A polynucleotide sequence encoding at least one engineered RNA polymerase according to any of items 1 to 12. (Item 14) A polynucleotide sequence encoding at least one engineered RNA polymerase, or a functional fragment thereof, that comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence of SEQ ID NO: 4 and / or 15, wherein the engineered RNA polymerase comprises at least one substitution at one or more amino acid positions. (Item 15) A polynucleotide sequence encoding at least one engineered RNA polymerase, or a functional fragment thereof, comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 4, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and / or 39. (Item 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 NOs: 3, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and / or 38. (Item 17) 17. The polynucleotide sequence according to any one of items 13 to 16, wherein the polynucleotide sequence is operably linked to a regulatory sequence. (Item 18) 17. The polynucleotide sequence according to any one of items 13 to 16, wherein the polynucleotide sequence is codon-optimized. (Item 19) 19. An expression vector comprising at least one polynucleotide sequence according to any one of items 13 to 18. (Item 20) 20. A host cell comprising at least one expression vector according to item 19. (Item 21) 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 to produce at least one engineered RNA polymerase. (Item 22) 22. The method of claim 21, further comprising recovering the at least one engineered RNA polymerase from the culture and / or host cell. (Item 23) 23. The method of items 21 and / or 22, further comprising purifying the at least one engineered RNA polymerase. (Item 24) 13. A composition comprising at least one engineered RNA polymerase according to any of items 1 to 12. (Item 25) 13. A method for producing capped RNA transcripts, the method comprising: i) providing a composition comprising at least one engineered RNA polymerase according to any one of items 1 to 12, a dinucleotide cap analog, and ii) a DNA template; and exposing the DNA template to the composition under conditions such that the engineered RNA polymerase produces capped RNA transcripts. (Item 26) The dinucleotide cap analog is α,γ-bis(N7-methylguanosine) triphosphate (m7G(5')ppp(5')m7G) or the anti-reverse cap analog 3'-O-Me-m 7 Item 26. The method according to item 25, wherein the nucleotide sequence is G(5')ppp(5')G. (Item 27) 27. The method of claim 25, wherein the dinucleotide cap analog is α,γ-bis(N7-methylguanosine) triphosphate. (Item 28) 28. The method according to any one of items 25 to 27, further comprising adding inorganic pyrophosphatase.

Claims

[Claim 1] The product and method described in the present specification.

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