RNA POLYMERASES FOR mRNA MANUFACTURING

Abstract

The present disclosure provides variant RNA polymerases with amino acid substitutions, and their use of which increases transcription yield, RNA integrity, capping efficiency, and reduces dsRNA formation during an in vitro transcription reaction.

Description

Attorney Docket Number: 14849-047-228 RNA POLYMERASES FOR mRNA MANUFACTURING CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 687,249 filed August 26, 2024, the disclosure of which is incorporated by reference herein in its entirety. SEQUENCE LISTING

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14849-047-228_SEQ_LISTING.xml”, was created on July 17, 2025, and is 9,618 bytes in size. BACKGROUND

[0003] Specific RNA sequences, for example for use in vaccines, therapeutics, diagnostics, R&D and agriculture are efficiently generated by in vitro transcription (IVT) from DNA templates using bacteriophage single-subunit RNA polymerases (RNApols). In principle, such reactions can be developed into large-scale manufacturing processes.

[0004] A robust solution to the problem of manufacturing large quantities of therapeutic grade RNA requires casting a wider net for RNApols with the desired catalytic activity. The present disclosure describes novel single-subunit RNApols that have desirable properties for RNA manufacturing in vitro.

[0005] Explorations of RNA as a molecule of clinical and biotechnological utility have increased dramatically in the past decade. For human therapeutic uses, RNA is being developed and used as a carrier of protein-coding information and gene regulatory activity to affect many aspects of human physiology (Rossbach 2010, Burnett 2011, Kole 2012, Kuhn 2012, Sahin 2014, Sergeeva 2016).

[0006] In agriculture, the development of spray technology for siRNAs as novel pesticides has led to an explosion of potential crop applications (Regalado 2015). NAI-5001873978v3

[0007] Messenger ribonucleic acid (mRNA) plays a key role as a carrier of genetic information flowing from DNA to protein. Since its discovery in 1961, mRNA has been the subject of consistent basic and applied research for various diseases (Sahin 2014). However, its short half- life and unfavorable immunogenicity has limited its use mainly as a research reagent. Only in the last decade, the breakthrough advances in the art of generating mRNA by in vitro transcription and the delivery in vivo by lipid nanoparticle technology have made mRNA as a potential new drug class possible (Pardi 2018, Hou 2021). mRNA can be administered in vivo to express therapeutic proteins and vaccine antigens and ex vivo for stem cell generation (Vallazza 2015, Weissman 2015, Pardi 2018, Chanda 2021). The power of mRNA technology was showcased by the successful launch of COVID-19 vaccines and will open door to address various global diseases ranging from cancers and infectious diseases to rare genetic diseases that can be treated by protein replacement (Sahin 2020; Xia, 2021). The heightened interest has created an unmet demand for inexpensive and efficient mRNA manufacturing process capable of generating large quantities of clinical-grade mRNA (Webb 2022, Whitley 2022) in the g to kg scale, while agricultural applications of RNA used in pesticides may require manufacturing scales of kg to ktons.

[0008] Clinical-grade mRNA requires mRNA of uniform and full length, high purity, high fidelity, with low levels of double stranded RNA (dsRNA) that causes adverse and harmful innate immune responses in patients and can lower mRNA efficacy (Kariko 2004, Kariko 2011, Ziemniak 2013, Shanmugasundaram 2022, Whitley 2022, Warminski 2023). Clinical-grade mRNA further requires a functional 5’ cap structure present on a high proportion (>80% or more) of mRNA molecules. Commercial development of RNA-based vaccines and therapeutics, as well as RNAs used in agriculture, has been slowed by the difficulty of manufacturing large quantities of commercially suitably material of uniform sequence, length and quality.

[0009] Because RNA is inherently unstable and immunogenic, RNA intended for human uses are chemically modified to stabilize the molecule, extend its shelf life and half-life in the human body and reduce immunogenicity (Majlessi 1998, Layzer 2004, Kraynack 2005, Jackson 2006, Wilson 2006, Ge 2010, Nelson 2020). Through variations in its structure and / or different delivery mechanisms, RNAs can be designed to affect both systemic and tissue-specific processes, further broadening its utility. The simplest way to modify RNA is to incorporate non- native nucleotides into RNA during synthesis, for example nucleotides blocked at their 2’ 2 NAI-5001873978position, or nucleotides like pseudouridine that contain modified bases (Nelson 2020). Tremendous progress has also been made in the development of various cap structures that mimic the structure of natural mRNA and enhance translation (Cougot 2004, Kariko 2008, Strenkowska 2016). Incorporation of cap analogs or modified nucleotides into RNA require development of better performing enzymes and processes to accommodate the newest cap and nucleotide structures and to streamline the manufacturing process.

[0010] Current mRNA manufacturing involves the use T7 RNA polymerase (T7 RNApol) to catalyze the polymerization of nucleotides using linearized plasmid DNA as a template in a bioreactor (Whitley, 2022). The products of the IVT reaction will go through DNase treatment to digest the DNA then one or more chromatography steps to remove the unincorporated nucleotides, enzymes, and other off-sized RNA. This process is far from being fast, cost- efficient, and suitable for all the DNA templates. T7 RNApol has several limitations that reduce its suitability for RNA manufacturing: 1) relatively low specific activity which requires long reaction times or high enzyme doses; 2) low efficiencies of incorporating modified nucleotides 3) low RNA quality due to a high percentage of aberrant transcripts (i.e. mutated, truncated, non- homogenous at the 3’ end), resulting in reduced efficiency of protein synthesis using the RNA as a template and potential off-target effects, and 4) High levels of double-stranded RNA, which is highly immunogenic (Lengyel 1987, Arnaud-Barbe 1998, Stark 1998, Majde 2000, Gantier 2007, Kariko 2011, Gholamalipour 2018, Mu 2018, Whitley, 2022).

[0011] Over the years, T7 RNApol has been modified by directed evolution or targeted mutagenesis based on structural modeling for higher incorporation of modified nucleotides (Padilla 2002, Chelliserrykattil 2004, Siegmund 2012, Boulain 2013, Ibach 2013, Meyer 2015). One particular study employed a directed evolution approach and identified a range of thermostable T7 mutants; they found that these thermostable mutants exhibited higher activity to incorporate modified nucleotides (Meyer 2015). Another study used a targeted mutagenesis approach, aiming to create variants with substitution in the C-helix and C-linker region of the N terminal region of the T7 RNApol. The resulting T7 RNApol variants exhibited less dsRNA and transcript with more 3’ homogeneity (Dousis 2023).

[0012] However, the improvements made to T7 RNApol in these studies were incremental and neglected to improve other important qualities of the enzyme, for example those related to 3 NAI-5001873978formation of double-stranded RNA and to efficient synthesis of primarily full-length RNA products from a double-stranded DNA template. Even the best available T7 RNApol variants (Padilla 2002, Chelliserrykattil 2004, Siegmund 2012, Ibach 2013, Meyer 2015) show deficiencies in all four of the above-listed performance indicators, and these enzymes fall far short of the requirements of a manufacturing process for clinical material. Current mRNA synthesis processes continue to use the wild type T7 RNApol enzyme. The manufacturing challenges associated with therapeutic mRNAs represent a significant hurdle for the clinical development and commercialization of a large number of potentially active RNA vaccines and therapeutics.

[0013] Rapid expansion of microbial and metagenome sequencing in the last two decades has led to the discovery and testing of single-subunit RNApols significantly distinct from T7 RNApol (Zhu 2013, Lu 2019, US 20230076421) expanding the enzymatic options available for RNA manufacturing.

[0014] The present disclosure provides variant compositions that were identified by mutagenesis to achieve desired performances. Random mutagenesis was applied as an enzyme diversification method. Random mutagenesis libraries were screened with ultra-high throughput screening methods. One of the screening methods creates a water-in-oil emulsion with a throughput in excess of 109enzyme variants per sample. When the enzyme variants were expressed and purified followed by activity validation, these variants were confirmed to have properties which allow for efficient mRNA production. Potentially beneficial amino acid substitutions were identified from 1) enzyme variants 2) next-generation sequencing (NGS) screening data, and 3) machine learning using zero-shot learning (Meier 2021, Mansoor 2023). Potentially beneficial substitutions were individually introduced into the wild-type RNApol background, and variants were validated to have properties that allow for efficient mRNA production. SUMMARY

[0015] Described herein are sequence variants of the RNApol from Stenotrophomonas phage IME15 (SEQ ID NO: 1) as well as amino acid positions and substitutions that are favorable for improved activity of this RNApol. The sequence variants include RNA polymerase mutants 4 NAI-5001873978containing single, double or multiple amino acid substitutions. Use of the variant RNA polymerases in in vitro transcription (IVT) reactions achieves higher yield of in vitro transcription with improved target mRNA yield and quality, and / or enhanced capping efficiency that ultimately leads to cost reduction of the mRNA manufacturing process.

[0016] In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 1; and (b) at least one amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 15, 22, 25, 40, 49, 53, 64, 65, 70, 74, 91, 94, 127, 128, 139, 149, 153, 163, 166, 177, 178, 198, 203, 205, 208, 217, 222, 232, 236, 251, 258, 260, 269, 279, 291, 320, 343, 346, 350, 351, 357, 363, 383, 388, 413, 421, 426, 440, 457, 459, 471, 473, 479, 480, 483, 487, 494, 498, 520, 522, 523, 534, 542, 555, 562, 565, 567, 576, 581, 583, 598, 601, 610, 625, 632, 633, 638, 641, 661, 665, 669, 693, 694, 707, 713, 736, 740, 775, 786, 795, 820, 839, 843, and 867 of SEQ ID NO: 1.

[0017] In one embodiment of the single-subunit RNA polymerase provided herein, the single-subunit RNA polymerase comprises at least one amino acid substitution corresponding to a substitution selected from the group consisting of: E15D, N22S, A25V, E40K, A49V, A49T, K53E, V64A, A65V, A70T, V74K, E91K, A94V, T127I, S128R, S139A, A149T, R153H, K163A, V166M, V177I, Y178C, G198N, S203N, H205Y, D208E, I217V, E222K, Q232R, V236I, A251T, A258V, A260V, Q269M, T279V, R291C, V320I, K343N, H346L, E350D, D351G, R357P, K363E, A383T, D388E, A413T, D421E, V426F, T440V, F457V, W459L, D471N, V473I, I479V, K480E, E483D, E487D, K494E, E498D, G520D, M522I, H523Y, L534P, G542V, G555V, L562V, E565D, V567P, K576R, I581L, Q583N, T598A, T601I, V610I, A625T, A625V, R632S, S633P, A638T, S641T, S661A, S661D, L665M, I669T, V693L, E694V, A707V, K713E, W736R, R740S, E775V, Q786L, V795I, D820N, C839N, A843V, and K867S of SEQ ID NO: 1.

[0018] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least two amino acid substitutions.

[0019] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 166, and 562 of SEQ ID NO: 1. 5 NAI-5001873978

[0020] In one embodiment, the single-subunit RNA polymerase provided herein comprises an N-terminal His-tag.

[0021] In one embodiment, the RNA yield in an in vitro transcription (IVT) reaction with the single-subunit RNA polymerase provided herein is increased by 2% to 10000% compared to the RNA yield in an IVT reaction with an RNA polymerase of SEQ ID NO: 1 or T7 RNA polymerase.

[0022] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 70, 166, 383, 562, and 567 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution corresponding to a substitution selected from the group consisting of: A25V, A70T, V166M, A383T, L562V, and V567P of SEQ ID NO: 1.

[0023] In one embodiment, the capping efficiency in an IVT reaction with the single-subunit RNA polymerase provided herein is increased by 2% to 10000% compared to the capping efficiency in an IVT reaction with an RNA polymerase of SEQ ID NO: 1 or T7 RNA polymerase.

[0024] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 74, 383, 567, 661, and 867 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution corresponding to a substitution selected from the group consisting of: A25V, V74K, A383T, V567P, S661D, and K867S of SEQ ID NO: 1.

[0025] In one embodiment, the quantity of double-stranded RNA (dsRNA) in an IVT reaction with the single-subunit RNA polymerase provided herein is decreased by 2% to 10000% compared to the quantity of double-stranded RNA in an IVT reaction with an RNA polymerase of SEQ ID NO: 1 or T7 RNA polymerase.

[0026] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one dsRNA-reducing amino acid substitution at a sequence position corresponding to a 6 NAI-5001873978sequence position selected from the group consisting of: 166, and 153 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one dsRNA-reducing amino acid substitution corresponding to a substitution selected from the group consisting of: V166M, and R153H of SEQ ID NO: 1.

[0027] In one embodiment, the RNA integrity in an IVT reaction with the single-subunit RNA polymerase provided herein is increased by 2% to 10000% compared to the RNA integrity in an IVT reaction with an RNA polymerase of SEQ ID NO: 1 or T7 RNA polymerase.

[0028] In one embodiment, the cap utilization efficiency in an IVT reaction with the single- subunit RNA polymerase provided herein is increased by 2% to 10000% compared to the cap utilization efficiency in an IVT reaction with an RNA polymerase of SEQ ID NO: 1 or T7 RNA polymerase.

[0029] In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and (b) at least one yield-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 70, 166, 383, 562, and 567 of SEQ ID NO: 1. In one embodiment, the at least one yield- enhancing amino acid substitution is at sequence position corresponding to sequence position 25 of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution corresponds to a substitution of A25V of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 70 of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution corresponds to a substitution of A70T of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 166 of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution corresponds to a substitution of V166M of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 383 of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution corresponds to a substitution of A383T of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 562 of SEQ ID NO: 1. In one embodiment, the at 7 NAI-5001873978least one yield-enhancing amino acid substitution corresponds to a substitution of L562V of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 567 of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution corresponds to a substitution of V567P of SEQ ID NO: 1.

[0030] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution corresponding to a substitution selected from the group consisting of: A25V, A70T, V166M, A383T, L562V, and V567P of SEQ ID NO: 1.

[0031] In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and (b) at least one capping-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 74, 383, 567, 661, and 867 of SEQ ID NO: 1. In one embodiment, the at least one capping- enhancing amino acid substitution is at sequence position corresponding to sequence position 25 of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution corresponds to a substitution of A25V of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution is at sequence position corresponding to sequence position 74 of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution corresponds to a substitution of V74K of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution is at sequence position corresponding to sequence position 383 of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution corresponds to a substitution of A383T of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution is at sequence position corresponding to sequence position 567 of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution corresponds to a substitution of V567P of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution is at sequence position corresponding to sequence position 661 of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution corresponds to a substitution of S661D of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution corresponds to a substitution of S661A of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution is at 8 NAI-5001873978sequence position corresponding to sequence position 867 of SEQ ID NO: 1. In one embodiment, the at least one capping-enhancing amino acid substitution corresponds to a substitution of K867S of SEQ ID NO: 1.

[0032] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution corresponding to a substitution selected from the group consisting of: A25V, V74K, A383T, V567P, S661D, S661A, and K867S of SEQ ID NO: 1.

[0033] In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and (b) at least one dsRNA-reducing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 166, 153 and 567 of SEQ ID NO: 1. In one embodiment, the at least one dsRNA-reducing amino acid substitution is at sequence position corresponding to sequence position 166 of SEQ ID NO: 1. In one embodiment, the at least one dsRNA-reducing amino acid substitution corresponds to a substitution of V166M of SEQ ID NO: 1. In one embodiment, the at least one dsRNA-reducing amino acid substitution is at sequence position corresponding to sequence position 153 of SEQ ID NO: 1. In one embodiment, the at least one dsRNA-reducing amino acid substitution corresponds to a substitution of R153H of SEQ ID NO: 1.

[0034] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one dsRNA-reducing amino acid substitution corresponding to a substitution selected from the group consisting of: V166M, R153H, and V567P of SEQ ID NO: 1.

[0035] In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; (b) at least one yield-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 70, 166, 383, 562, and 567 of SEQ ID NO: 1; (c) at least one capping-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 74, 383, 567, 661, and 867 of SEQ ID NO: 1; and (d) at least one dsRNA-reducing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 166, 153, and 567 of SEQ ID NO: 1. In one embodiment, the at least one 9 NAI-5001873978yield-enhancing amino acid substitution corresponds to a substitution of L562V of SEQ ID NO: 1, and the at least one dsRNA-reducing amino acid substitution corresponds to a substitution of V166M of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution corresponds to a substitution of L562V of SEQ ID NO: 1, the at least one capping- enhancing amino acid substitution corresponds to a substitution of S661D of SEQ ID NO: 1, and the at least one dsRNA-reducing amino acid substitution corresponds to a substitution of V166M of SEQ ID NO: 1.

[0036] In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises amino acid substitutions corresponding to the following positions of SEQ ID NO: 1: V166M, K343N, D351G, I479V, K494E, E498D, L562V, I581L, G618D, and S661A.

[0037] In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises amino acid substitutions corresponding to the following positions of SEQ ID NO: 1: V166M, K343N, E350D, D351G, I479V, E498D, L562V, I581L, G618D, and S661A.

[0038] In one aspect, provided herein is a nucleic acid composition encoding the single- subunit RNA polymerase provided herein. In one aspect, provided herein is a vector composition comprising the nucleic acid composition provided herein. In one aspect, provided herein is a host cell comprising the nucleic acid composition or the vector composition provided herein.

[0039] In one aspect, provided herein is a kit comprising the single-subunit RNA polymerase provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG.1: Single-subunit RNA Polymerase Structure Model. Shown are the structural domains of T7 RNA polymerase: an N-terminal domain, comprised of the C-helix, PDB and H subdomains and a catalytic domain, which is comprised of the thumb, palm and finger subdomains. The alignment positions that correspond to the domains are described in Table 4. 10 NAI-5001873978DETAILED DESCRIPTION

[0041] The following abbreviations and definitions are used for the interpretation of the specification and the claims.

[0042] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, are intended to cover a non- exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.

[0043] Unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or.” For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0044] Alignment Position: As used herein, “alignment position” is an integer that refers to the numerical position of an amino acid within a protein sequence, counted from the N-terminus of the protein towards the C-terminus, as defined by a multiple sequence alignment of several related proteins. Gaps introduced into the multiple sequence alignment mean that the alignment position of a specific amino acid differs from its numerical position as determined solely from the sequence of an individual protein and counted from the N-terminus of the protein towards the C-terminus.

[0045] Amino acid substitution: As used herein, an amino acid substitution is a change in the amino acid sequence of a peptide or protein from one amino acid to a different amino acid. This change in amino acid could be due to point mutation in the corresponding DNA sequence. An amino acid substitution can make a detectable change to the activity, stability, biochemical properties, manufacturability or other qualities of the protein, or it can leave the protein unaffected in terms of detectable changes to the same. An amino acid substitution can make a beneficial change to the useful qualities of a protein, or it can make a deleterious change. A protein or peptide variant can contain 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 11 NAI-500187397820, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 amino acid substitutions, or any number in between, when compared to the wild type or parental version of the same protein or peptide.

[0046] Cap, 5’ cap or mRNA cap: As used herein , the terms “cap”, “5’ cap” and “mRNA cap” refer to specialized nucleotides found at the 5’ ends of mRNAs, such as the 7- methylguanosine cap found in eukaryotic mRNAs or other capping structures found at the 5’ end of natural or synthetic RNAs. The term “cap analog” refers to a synthetic nucleic acid that is incorporated into an mRNA to serve the role of the 5’ cap. mRNAs synthesized in vitro can be capped at their 5’ ends by co- transcriptional incorporation of dinucleotide or trinucleotide, tetranucleotide or longer cap analogs by RNA polymerase.

[0047] Capping efficiency: As used herein, “capping efficiency” refers to the percentage of RNA molecules synthesized in vitro using an RNA polymerase that contain a 5’ cap.

[0048] Cap incorporation efficiency: As used herein, “cap incorporation efficiency” or “cap utilization efficiency” refer to the efficiency by which a single-subunit RNA polymerase incorporates a dinucleotide or trinucleotide or other cap analog into mRNA synthesized in vitro. For example, an RNA polymerase with high cap incorporation efficiency can achieve higher capping efficiency at lower concentrations of dinucleotide or trinucleotide cap analog in the reaction than an RNA polymerase with lower cap incorporation efficiency. For example, an RNA polymerase that achieves 90% capping at a concentration of cap analog in the IVT reaction of 10 mM has a high capping efficiency at that concentration of cap analog, but may only achieve 50% capping efficiency at a cap analog concentration of 4 mM. This RNA polymerase has the same capping efficiency at 10 mM cap analog but a lower cap incorporation efficiency at 4 mM cap analog than a second RNA polymerase that achieves 90% capping at 10 mM cap analog and 80% capping at 4 mM cap analog concentration in the IVT reaction.

[0049] Capped mRNA: As used herein, “capped mRNA” refers to an mRNA molecule containing a 5’ cap.

[0050] Capping-enhancing amino acid substitution: As used herein, “capping-enhancing amino acid substitution” refers to a change in the amino acid sequence of an RNA polymerase that results in higher capping efficiency or higher cap utilization efficiency of the variant RNA 12 NAI-5001873978polymerase containing the substitution, compared to the wild type or parental RNA polymerase. Higher capping efficiency or cap utilization efficiency can mean either an increase in the percentage of capped mRNA molecules produced in an IVT reaction, or an increase in the total amount of capped mRNA produced in an IVT reaction. Capping efficiency can be determined by, for example, mass spectrometry.

[0051] Complementary nucleotide sequence: As used herein, a complementary nucleotide sequence is a sequence in a polynucleotide chain in which all of the bases are able to form base pairs with a sequence of bases in another polynucleotide chain.

[0052] Control elements: The term “control elements” as used herein refers to nucleotide sequences located upstream (5’ control sequences), within, or downstream (3’ control sequences) of a coding sequence and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include but are not limited to promoters, terminators, translation leader sequences, 5’ untranslated sequences, 3’ untranslated sequences, Kozak sequences, ribosome binding sites, internal ribosome entry sites, other translation promoting sequences, enhancers, transcription factor binding sites, repressor binding sites, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.

[0053] Corresponding to: As used herein in the context of corresponding positions, the term “corresponding to” and grammatical variants thereof can refer to positions that lie across from one another when sequences are aligned (e.g., by the BLAST algorithm), or amino acid residues or nucleotides located in those corresponding positions of the aligned sequences, respectively.

[0054] As used herein, single amino acid residue substitution in a polypeptide is described using the nomenclature in the format such as “R150H” or “A159V”. In this nomenclature, the letter before the number is the one-letter code of the amino acid residue before the substitution, and the letter after the number is the one-letter code for the amino acid residue after the substitution. Depending on the context, the number can refer to (a) the sequence position in the polypeptide carrying such substitution, or alternatively, (b) the sequence position in a reference polypeptide corresponding to the position where such substitution is located in the polypeptide containing it. For clarity, as used herein, the expression such as “a substitution ‘corresponding 13 NAI-5001873978to’ R150H of a polypeptide,” or grammatical variant thereof, are used to indicate the latter case (b) described above.

[0055] Degenerate sequences: As used herein, the term “degenerate sequences” is defined as populations of sequences where specific sequence positions differ between different molecules or clones in the population. The sequence differences may be a single nucleotide or multiple nucleotides of any number, examples being 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides, or more, or any number in between. Sequence differences in a degenerate sequence may involve the presence of 2, 3 or 4 different nucleotides in that position within the population of sequences, molecules or clones. Examples of degenerate nucleotides in a specific position of a sequence are: A or C; A or G; A or T; C or G; C or T; G or T; A, C or G; A, C or T; A, G or T; C, G or T; A, C, G or T.

[0056] Double-stranded RNA-reducing amino acid substitution: As used herein, “double- stranded RNA-reducing amino acid substitution” or “dsRNA-reducing amino acid substitution” refers to a change in the amino acid sequence of an RNA polymerase that results in lower levels of double-stranded RNA (dsRNA) produced by the variant RNA polymerase containing the substitution, compared to the wild type or parental RNA polymerase. Lower dsRNA levels can be measured as absolute amounts of dsRNA produced in the reaction, or dsRNA level as a percentage of total RNA produced in the reaction. Double-stranded RNA can be measured, for example, using enzyme linked immunosorbent assay (ELISA) or dot blot assay.

[0057] Diversified sequence: As used herein, a “diversified sequence” refers to a nucleic acid sequence which has been derived from a parental nucleic acid sequence and altered to create one or more mutant or variant versions of the parental sequence. Alterations can be by mutagenesis (i.e. introduction of point mutations); introduction of insertions and deletions of varying lengths; sequence randomization (i.e. by replacement of one or more sequence stretches within the parental sequence with degenerate sequences or the same length or of different lengths); fusion with other sequences either at the 5’ or the 3’ end of the parental sequence; homologous sequence exchange with related or homologous sequences resulting in reassortment of polymorphisms; or combinations thereof; and any other means of creating sequence diversity. Diversified sequences are often created as populations of sequence variants, where a single nucleic acid sample contains nucleic acid molecules related to each other by sequence but differing in their specific sequence. 14 NAI-5001873978

[0058] Expression: The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid molecule or sequence, as well as the accumulation of polypeptide as a product of translation of mRNA.

[0059] Fidelity: As used herein, the term “fidelity” describes the accuracy of a nucleic acid polymerase, reflecting faithful copying of a template nucleic acid into a daughter nucleic acid strand. Fidelity also describes the accuracy by which a nucleic acid sample reflects the sequence of the template nucleic acid from which it was copied. For example, a high fidelity DNA or RNA polymerase makes few errors in copying a DNA strand and results in a DNA or RNA sample that is substantially free of mis-incorporated nucleotides that change the sequence from that of the template DNA when copied into an RNA or a DNA daughter strand. A high fidelity RNA sample is one that contains few mis-incorporated nucleotides that change the sequence from that of the template DNA from which the RNA sample was derived. Fidelity is often expressed as the error rate of a nucleic acid polymerase or fraction of a sequence length likely to contain a single error or misincorporation, for example an error rate of 1 / 100,000 implies the average of 1 error or misincorporation in 100,000 polymerized nucleotides.

[0060] Free nucleotide: As used herein, “free nucleotide” means a monomeric nucleotide, typically in solution.

[0061] Frequency rank: As used herein, “frequency rank” refers to data generated via next- generation sequencing. After the “maximum amino acid substitution frequency” is calculated for each position in an enzyme sequence, those positions are then sorted in descending order (i.e. ranked) by that frequency. The resulting ranks begin at 1 and end at a numerical value equal to the total number of positions in the enzyme sequence.

[0062] Full-length Open Reading Frame: As used herein, a “full-length open reading frame” refers to an open reading frame encoding a full-length protein which extends from its natural initiation codon to its natural final amino-acid coding codon, as expressed in a cell or organism. In cases where a particular open reading frame sequence gives rise to multiple distinct full-length proteins expressed within a cell or an organism, each open reading frame within this sequence, encoding one of the multiple distinct proteins, are considered full-length. A full-length open reading frame can be either continuous or interrupted by introns. 15 NAI-5001873978

[0063] Full-length RNA or full-length transcript: “Full-length RNA” or “full-length transcript” as used herein refers to an RNA synthesized from a nucleic acid template that covers the entire length of the nucleic acid template, from the transcription initiation site in a 3’ to 5’ direction along the template strand to the end of the nucleic acid template. An RNA molecule transcribed from a nucleic acid template may be considered full-length if it is substantially full- length, meaning that its length differs from the length of the nucleic acid template by a few or multiple nucleotides at either end, such that the migration of a full-length RNA molecule and the substantially full-length RNA molecule cannot be distinguished using commonly used methods of gel electrophoresis and capillary gel electrophoresis. Full-length RNA can also be alternatively referred to as “target RNA” because it represents the RNA species that is the primary goal of a manufacturing process. Full length RNA or target RNA may be a messenger RNA (mRNA), which contains an open reading frame that may be translated into protein.

[0064] Full-length Protein: As used herein, a “full-length protein” is a polypeptide which extends from its natural first amino acid to its natural final amino acid, as encoded in the genome of a cell or organism and expressed in the cell or organism.

[0065] Gene: As used herein, “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5’ control sequences), within and following (3’ control sequences) the coding sequence. “Native gene” or “natural gene” refers to a gene as found in nature in its natural host organism, complete with its natural control sequences including but not limited to a promoter, terminator, ribosome binding site or other translation promoting sequence, enhancer, and repressor binding sites. “Chimeric gene” refers to any gene that comprises regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Similarly, a “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes include native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure to result in a modified organism differing from the parental organism only in the introduced gene. 16 NAI-5001873978

[0066] In-Frame: The term “in-frame” as used herein , and particularly in the phrase “in-frame fusion polynucleotide,” refers to the reading frame of codons in an upstream or 5’ polynucleotide or ORF as being the same as the reading frame of codons in a polynucleotide or ORF placed downstream or 3’ of the upstream polynucleotide or ORF that is fused with the upstream or 5’ polynucleotide or ORF. Such in-frame fusion polynucleotides or fusion genes encode a fusion protein or fusion peptide encoded by both the 5’ polynucleotide and the 3’ polynucleotide. Collections of such in-frame fusion polynucleotides can vary in the percentage of fusion polynucleotides that contain upstream and downstream polynucleotides that are in-frame with respect to one another. The percentage in the total collection is at least 10% and can number 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or any number in between.

[0067] In vitro transcription (IVT) reaction: As used herein, “in vitro transcription reaction” or “IVT reaction” is a reaction designed to produce RNA by transcribing a DNA template in vitro. In vitro transcription reactions contain one or more DNA template molecules encoding the RNAs to be synthesized; one or more completely or partially purified RNA polymerases such as single-subunit RNA polymerases (RNApols); nucleoside triphosphates as substrates for the RNA polymerase(s) such as the four canonical ribonucleoside triphosphates ATP, CTP, GTP and TTP; buffers, divalent cations and salts as necessary for the RNApol to be active. IVT reactions can also contain additional enzymes such as a pyrophosphatase that degrades pyrophosphate released by the RNA polymerase during RNA synthesis and RNAse inhibitors to protect the synthesized RNA from the action of ribonucleases that may be contaminants in the reaction. The nucleic acid template contains a promoter sequence recognized by the RNApol and where the RNApol binds to initiate the transcript.

[0068] Integrity of a nucleic acid or RNA integrity: “Integrity of a nucleic acid” or “RNA integrity” as used herein, refers to the degree to which a collection of nucleic acid molecules have the expected length. For example, RNA molecules transcribed from a linear double-stranded DNA template that measures 2000 base pairs between the transcription start site and the end of the template (measured along the template strand and including the transcription start site) are expected to have a length of 2000 nucleotides. When measured by gel electrophoresis, capillary gel electrophoresis or equivalent nucleic acid size fractionation method, such RNA molecules transcribed from a 2000 base pair DNA template molecule may range in size from 250 17 NAI-5001873978nucleotides to 2000 nucleotides. If, as measured by gel electrophoresis, capillary gel electrophoresis or equivalent nucleic acid size fractionation method, half of the RNA molecules have the expected length of 2000 nucleotides and the other half are shorter, then the integrity of this RNA sample is 50%, or stated differently the sample has RNA integrity of 50%. The portion of the RNA molecules which, as measured by gel electrophoresis, capillary gel electrophoresis or equivalent nucleic acid size fractionation method, have a length of approximately 2000 base pairs corresponds to full-length and substantially full-length RNA molecules.

[0069] Integrity-enhancing amino acid substitution: As used herein, “integrity-enhancing amino acid substitution” refers to a change in the amino acid sequence of an RNA polymerase that results in higher integrity or purity of the mRNA produced by the variant RNA polymerase containing the substitution, compared to the wild type or parental RNA polymerase. Higher mRNA integrity or purity can mean mRNA integrity or purity as measured in any reaction condition of an IVT reaction, including at any temperature, pH, concentration of divalent cations, template DNA sequence, template DNA length, template DNA concentration, RNA polymerase concentration or any other aspect of an IVT reaction’s composition or reaction conditions that can be adjusted to alter or optimize the output of the IVT reaction.

[0070] In vitro translation reaction: as used herein, “in vitro translation reaction”, is a cell- free reaction designed to produce a protein by translating an RNA transcript in vitro. In vitro translation reactions contain one or more RNA transcripts to be translated, ribosomes, initiation and elongation factors, tRNAs charged with amino acids, ATP, and optionally accessory proteins to enhance protein folding.

[0071] Iterate / Iterative: As used herein, the terms “iterate” or “iterative” mean to apply a method or procedure repeatedly to a material or sample. Typically, the processed, altered or modified material or sample produced from each round of processing, alteration or modification is then used as the starting material for the next round of processing, alteration or modification. Iterative selection refers to a selection process that iterates or repeats the selection two or more times, using the survivors of or molecules remaining after one round of selection as starting material for the subsequent rounds.

[0072] Library: a “library” of genes or polynucleotide sequences as used herein is a collection of sequences that are different from each other and that are cloned into a vector for propagation of 18 NAI-5001873978the sequences. In different libraries, the sequences differ by sequence content, origin, source organism, length, structure, association with other sequences, and / or any other property of a polynucleotide sequence. For example, a library of amino acid repeat fusion genes is generated by cloning a starting ORF collection that contains multiple different ORFs encoded by the E. coli genome into a bacterial cloning and expression vector that contains a promoter, a sequence encoding an amino acid repeat oriented in a manner that this sequence will be joined directly and in-frame to the ORFs, a terminator, a plasmid backbone and an antibiotic resistance gene. The starting ORF collection can contain any number of ORFs that number 5 or greater, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000 or greater, or any number in between. In a specific aspect of the disclosure, the ORF collection used to generate the library contains a sufficient number of ORFs to give a high likelihood of encoding a specific desirable property of E. coli, for example 50% or more of the ORFs encoded by the E. coli genome, or 2074 or more ORFs when using the annotation of the E. coli strain MG1655 genome annotation prepared by the University of Wisconsin, Madison which lists a total of 4148 ORFs.

[0073] Linker sequence: As used herein, “linker sequence” refers to a polynucleotide sequence or polypeptide sequence separating two polynucleotides or polypeptides in a fusion polynucleotide or fusion polypeptide. For example, a fusion polynucleotide contains two or more ORFs that are separated by a linker sequence, which encodes a peptide which separates the two parts of the polypeptide that results from expression and translation of the fusion polynucleotide. A linker can also separate an epitope tag from a protein or enzyme. Linker sequences can have diverse length and / or sequence composition.

[0074] Maximum amino acid substitution frequency: As used herein, “maximum amino acid substitution frequency” refers to data generated via the analysis of next-generation DNA sequencing (NGS) results. For a particular sample, the NGS sequence reads are mapped to the reference gene and nucleic acid substitutions and their corresponding amino acid substitutions are determined. For all observed amino acid substitutions occurring at each amino acid position of the reference gene, the amino acid substitution frequency is determined as the number of reads containing the observed amino acid substitution divided by the total number of reads mapping to 19 NAI-5001873978that position. The maximum amino acid substitution frequency is then defined as the maximum frequency of any amino acid substitution for a given position.

[0075] Mutation: A “mutation” is an alteration in the nucleic acid sequence of a nucleic acid sequence, gene or the genome of an organism. Mutations include but are not limited to single nucleotide substitutions or point mutations; small deletions or small insertions, large insertions or deletions; sequence duplications; copy-number changes (amplifications or deletions) of repetitive sequences; translocations; chromosomal duplications or deletions; chromosomal rearrangements; genome duplications; or changes in ploidy. Mutations can be subdivided into deleterious mutations, which reduce the fitness or productivity of an organism, the function of a gene or the activity of the protein or enzyme encoded by a gene; beneficial mutations, which improve the fitness or productivity of an organism, the function of a gene or the activity and other desirable qualities of the protein or enzyme encoded by a gene; or neutral mutations, which do not measurably impact the qualities of an organism, gene or encoded protein or enzyme.

[0076] Non-homologous: The term “non-homologous” as used herein is defined as having sequence identity at the nucleotide level of less than 50%.

[0077] Nucleic acid: As used herein, “nucleic acid” refers to biopolymers, consisting of nucleotides joined to each other via phosphodiester linkages, phosphorothioate linkages or other linkages. “Nucleic acid”, “nucleic acid molecule” and “polynucleotide” can be used interchangeably. As used herein, “nucleic acid” can also refer to a single strand of nucleic acid. A nucleic acid can either consist of deoxyribonucleotide residues, in which case it is DNA, or ribonucleotide residues, in which case it is RNA, or it can contain both deoxyribonucleotide residues and ribonucleotide residues in which case it is a chimeric nucleic acid.

[0078] Nucleic acid polymerase: “Nucleic acid polymerase” as used herein is an enzyme that catalyzes the polymerization of a nucleic acid using nucleotide triphosphates and nucleic acids as substrates and sequentially adds single nucleotides to the 3’ end of the nucleic acid. Nucleic acid polymerases as described in the scientific literature typically fall into the classes of DNA polymerases and RNA polymerases, with DNA polymerases capable of polymerizing DNA and RNA polymerases capable of polymerizing RNA. However, specific enzymes may have the dual ability to catalyze the synthesis of both DNA and RNA. For example, a DNA polymerase may have the ability to add ribonucleotides to the 3’ end of a DNA or RNA molecule, and an RNA 20 NAI-5001873978polymerase may have the ability to add deoxyribonucleotides to the 3’ end of a DNA or RNA molecule. Nucleic acid polymerases may have the ability to synthesize a nucleic acid from different types of templates. For example, DNA polymerases can have DNA-dependent DNA polymerase activity or RNA-dependent DNA polymerase activity; RNA polymerases can have DNA-dependent RNA polymerase activity or RNA-dependent RNA polymerase activity. Nucleic acid polymerases may also have the ability to synthesize nucleic acids in the absence of a template, for example template-independent nucleic acid polymerases, template-independent DNA polymerases or template-independent RNA polymerases.

[0079] Nucleic acid synthesis: As used herein, “nucleic acid synthesis” is the process by which nucleic acids are produced in nature or by man, minimally requiring a nucleic acid polymerase, one or more nucleoside triphosphates as monomer building blocks, and a nucleic acid substrate. Nucleic acid synthesis can also occur in the absence of a nucleic acid substrate, in which case a template-independent nucleic acid polymerase synthesizes the nucleic acid in a template-independent manner.

[0080] De novo nucleic acid synthesis: As used herein, “de novo nucleic acid synthesis” refers to synthesis of man-made DNA, involving controlled addition of specific nucleotides to a nucleic acid substrate to create a specific sequence and structure of nucleic acid.

[0081] Nucleic acid template or template nucleic acid or template nucleic acid molecule: As used herein, “nucleic acid template”, “template nucleic acid” or “template nucleic acid molecule” is a nucleic acid molecule present in an in vitro or in vivo reaction in that serves as the template for synthesis of a homologous nucleic acid with a nucleic acid polymerase. For example, a double- stranded DNA template containing a specific promoter for a single-subunit RNA polymerase serves as the nucleic acid template for an RNA molecule homologous to the sense strand of the nucleic acid template. The nucleic acid template is often simply referred to as the “template.”

[0082] “Nucleotides” are the monomer building blocks of nucleic acids, made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleotides are deoxyribonucleotides, the building blocks of DNA and ribonucleotides, the building blocks of RNA. If the sugar is ribose, the nucleic acid is RNA; if the sugar is the ribose derivative deoxyribose, the nucleic acid is DNA. As used herein, a deoxyribonucleotide has the 21 NAI-5001873978group CH2 as the 2’ carbon in the ribose sugar. All other structures of the 2’ carbon are grouped under the term ribonucleotides. As used herein, a nucleotide can mean a nucleotide residue present within a nucleic acid, a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate or any derivative or modification thereof.

[0083] Nucleoside triphosphates: As used herein, “Nucleoside triphosphates” are defined as any of the ribonucleoside triphosphates ATP, CTP, GTP, ITP, UTP, Pseudo-UTP and XTP, etc. used in RNA synthesis, or any of the deoxyribonucleoside triphosphates dATP, dCTP, dGTP, dITP, dTTP and dXTP, etc. used in DNA synthesis, or any modified analogs, derivatives or variants thereof, including derivatives containing phosphorothioate linkages, modifications of the ribose sugar or modifications of the bases (e.g. N1methyl-pseudo UTP, 5-methyl CTP). Mixtures of the four canonical nucleoside triphosphates used in DNA synthesis (dATP, dCTP, dGTP, and dTTP) are denoted by the shorthand “dNTP” and mixtures of the four canonical nucleoside triphosphates used in RNA synthesis (ATP, CTP, GTP, and UTP) are denoted by the shorthand “NTP”.

[0084] Oligonucleotide: As used herein, “oligonucleotide” refers to a single stranded nucleic acid consisting of two or more nucleotides.

[0085] Open Reading Frame (ORF): An “ORF” is defined as any sequence of nucleotides in a nucleic acid that encodes a protein or peptide as a string of codons in a specific reading frame. Within this specific reading frame, an ORF can contain any codon specifying an amino acid, but does not contain a stop codon. The ORFs in the starting collection need not start or end with any particular amino acid. In different aspects of the disclosure, an ORF is either continuous or is interrupted by one or more introns.

[0086] Operably linked: The term “operably linked” as used herein refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. 22 NAI-5001873978

[0087] Parental: The term “parental”, as used herein, refers to an original or ancestral nucleic acid molecule, nucleic acid sequence, protein or polypeptide molecule or protein or polypeptide sequence that is diversified into variants of the same. The variants or mutants of the parental molecule or parental sequence that are derived from such molecule or sequence differ in nucleic acid or amino acid sequence from the parental molecule or sequence in one or more positions.

[0088] Peptide bond: A “peptide bond” is a covalent bond between a first amino acid and a second amino acid in which the alpha-amino group of the first amino acid is bonded to the alpha- carboxyl group of the second amino acid.

[0089] Percentage of sequence identity: The term “percent sequence identity” refers to the degree of identity between any given query sequence, e.g. SEQ ID NO: 1, and a subject sequence. A subject sequence typically has a length that is from about 80 percent to 200 percent of the length of the query sequence, e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, or 120, 130, 140, 150, 160, 170, 180, 190 or 200 percent of the length of the query sequence. A percent identity for any subject nucleic acid or polypeptide relative to a query nucleic acid or polypeptide is determined as follows. A query sequence (e.g. a nucleic acid or amino acid sequence) is aligned to one or more subject nucleic acid or amino acid sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment, Chenna 2003).

[0090] ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension 23 NAI-5001873978penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on. The ClustalW output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher website and at the European Bioinformatics Institute website on the World Wide Web (ebi.ac.uk / clustalw).

[0091] To determine a percent identity of a subject or nucleic acid or amino acid sequence to a query sequence, the sequences are aligned using ClustalW, the number of identical matches in the alignment is divided by the query length, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. Identical proteins have 100% sequence identity.

[0092] Plasmid and Vector: The terms “plasmid” and “vector” as used herein refer to genetic elements used for carrying genes which are not present in an unmodified or wild type cell or organism. Plasmids typically replicate extra-chromosomally as autonomous episomal genetic elements, while vectors can either integrate into the genome or can be maintained extra- chromosomally as linear or circular DNA fragments. Plasmids and vectors can be linear or circular, and can consist of single- and / or double-stranded DNA or RNA that is derived from any source. Plasmids and vectors can contain a number of nucleotide sequences from different sources which have been joined or recombined into a unique construction which is useful for introducing polynucleotide sequences into a cell or an organism and expressing genes within an organism. The sequences present on a plasmid or on a vector include but are not limited to: autonomously replicating sequences; centromere sequences; sequences homologous to a genome that facilitate integration; origins of replication; control sequences including but not limited to promoters, terminators, Kozak sequences, ribosome binding sites or internal ribosome entry sequences; open reading frames; selectable marker genes such as antibiotic resistance genes or auxotrophic marker genes; visible marker genes such as genes encoding fluorescent proteins; restriction endonuclease recognition sites; recombination sites; and / or sequences with no apparent or known function. The sequences within a plasmid or vector can be derived from any source or multiple sources. 24 NAI-5001873978

[0093] Polypeptide or protein: The terms “polypeptide” or “protein” as used herein denote a polymer composed of a plurality of amino acid monomers joined by peptide bonds. The polymer comprises 10 or more amino acid monomers, including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, or any length in between. A preferred polypeptide or protein of the disclosure is a single-subunit RNA polymerase.

[0094] Promoter: The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Promoters regulate the transcriptional activity of a gene, or the synthesis of RNA using the gene sequence as a template. In general, a coding sequence is located 3’ to or downstream of a promoter sequence. In different aspects, promoters are derived in their entirety from a native gene, or are composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0095] Protein coding sequence: The term protein coding sequence as used herein is used synonymously with “open reading frame” and refers to a nucleic acid sequence that encodes a polypeptide or protein.

[0096] Random / Randomized: As used herein, “random” or “randomized” as used herein means made or chosen without method or conscious decision.

[0097] Randomized sequence: As used herein, “randomized sequence” refers to a nucleic acid sequence in which one or more nucleotides have been replaced by degenerate nucleotides.

[0098] Reverse transcription-quantitative polymerase chain reaction, RT-qPCR, is used to quantitate the amount of an RNA sequence present in a nucleic acid sample. It can be used to analyze the transcriptional activity of an RNApol. 25 NAI-5001873978

[0099] RNA: As used herein, “RNA” is a nucleic acid that is a polymer of ribonucleotides. RNA occurs in single stranded or double stranded forms. As used herein, RNA contains nucleotide residues each of which has a 2’ carbon in a form other than CH2.

[0100] RNA polymerase: As used herein, “RNA polymerase” is an enzyme that synthesizes a single-stranded RNA molecule from a nucleic acid template, usually double-stranded DNA. RNA polymerase is sometimes abbreviated as RNApol or RNAP.

[0101] RNA quality: “RNA quality” as used herein refers to the intactness, purity or desired structure of RNA obtained in an in vitro transcription reaction. High RNA quality can mean high RNA integrity, high capping efficiency, low levels of double-stranded RNA, low levels of short, truncated RNAs, low levels of other undesirable side products other than the full-length RNA, high fidelity, high and / or uniform polyA tail length, high or desirable degree of substitution with a modified nucleotide, or any combinations thereof. Low RNA quality can mean low RNA integrity, low capping efficiency, high levels of double-stranded RNA, high levels of short, truncated RNAs, high levels of other undesirable side products, low RNA fidelity, low and / or non-uniform polyA tail length, low or undesirable degree of substitution with a modified nucleotide, or any combinations thereof. High RNA quality typically results in high rates of translation of the RNA into the functional or active protein encoded by the RNA.

[0102] Sequence: As used herein, “sequence,” when used in a biological context, can imply the sequence of nucleotides in a nucleic acid or the sequence of amino acids in a protein. As used herein, the term “sequence” has a meaning dependent on the context in which the term is used. For example, when used in the context suggesting nucleic acids such as genome sequences, gene sequences or ORFs, then sequence refers to a nucleotide sequence. In a context suggesting proteins or polypeptides, such as the proteome, proteins or enzymes, sequence refers to an amino acid sequence.

[0103] Sequence position: As used herein, “sequence position” or “amino acid position” refers to the numbered position of an amino acid residue within a protein sequence, with the N-terminal methionine residue in position 1 and counting from the methionine towards the protein’s C- terminus. All amino acid positions related to RNA polymerase RNApol225 that is the subject of the present disclosure uses SEQ ID NO: 1 as the reference sequence for defining sequence positions. 26 NAI-5001873978

[0104] Single-subunit RNA polymerase: A “single-subunit RNA polymerase”, as used herein, is an enzyme with DNA-dependent RNA polymerase activity capable of synthesizing RNA from a DNA template in vitro in a reaction in which the single-subunit RNA polymerase is present in a pure or substantially pure form, without the presence or addition of any other proteins or peptides into the reaction.

[0105] Template-independent Nucleic Acid Synthesis: As used herein, “template- independent nucleic acid synthesis” is a process by which a nucleic acid polymerase catalyzes the polymerization of a nucleic acid without use of a template strand that is base paired to the nucleic acid being synthesized and that serves as the template for the strand being synthesized.

[0106] Transcription start site (TSS) refers to the 2 first nucleotides in an RNA transcript synthesized by an RNA polymerase. Transcription start site also refers to the nucleotide positions in a template molecule that correspond to the 2 first nucleotides in an RNA transcript synthesized by an RNA polymerase. Transcription start sites for single-subunit RNA polymerases are typically found immediately 3’ to the RNA polymerase-specific promoter sequence.

[0107] Transcriptional 5’ end: the term “transcriptional 5’ end” refers to the first ribonucleotide in an RNA transcript. Transcripts generated by single-subunit RNA polymerases contain triphosphates at their transcriptional 5’ ends (Hornung 2006).

[0108] Transformed: As used herein, “transformed” means genetic modification by introduction of a polynucleotide sequence.

[0109] Transformation: As used herein, “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance of the nucleic acid fragment. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

[0110] Transformed Organism: As used herein, a “transformed organism” is an organism that has been genetically altered by introduction of a polynucleotide sequence into the organism’s genome.

[0111] Uncapped mRNA: As used herein, an “uncapped mRNA” is an mRNA molecule not containing a 5’ cap. 27 NAI-5001873978

[0112] Unfavorable Conditions: As used herein, “unfavorable conditions” implies any part of the growth condition, physical or chemical, or reaction conditions, physical or chemical, that results in slower growth than under normal growth conditions or lower protein activity than under normal reaction conditions, or that reduces the viability of cells compared to normal growth conditions.

[0113] Untranslated sequence: As used herein, “untranslated sequence” refers to untranslated regions (or UTRs) that occur on both sides (5’ and 3’) of a protein-coding sequence in a nucleic acid sequence. If it is found on the 5’ or leading side of the ORF or protein-coding sequence, it is called the 5’ UTR; if it is found on the 3’ side of the ORF or protein-coding sequence, it is called the 3’ UTR. The term “untranslated sequence” refers to sequences present within a protein encoding mRNA, which is transcribed from a corresponding DNA sequence, that are not translated into protein. Several regions of the mRNA, including 5’ UTRs, 3’ UTRs and polyA tails are untranslated sequences because they are usually not translated into protein.

[0114] Variant nucleic acids: As used herein, “variant nucleic acid” refers to mutated or altered versions of nucleic acid sequences. A variant nucleic acid may have point mutations, insertions, deletions, inversions, rearrangements or combinations thereof compared to the parental or reference sequence that it is derived from or related to. Sequences within a variant nucleic acid that contain mutations, insertions, deletions, inversions, rearrangements or combinations thereof compared to a reference or parental sequence that the variant nucleic acid is related to or derived from, may be of any length, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000 nucleotides or more, or any number in between. A variant nucleic acid may contain a single change (single mutation, insertion, etc.) compared to a reference or parental sequence, or multiple changes. A variant nucleic acid may have uniform sequences represented within a sample, where all molecules in a nucleic acid sample have the same sequence, or diverse sequences where a sample comprises nucleic acid molecules of different sequence. Variant nucleic acids comprising nucleic acid molecules of different sequence may differ from each other in sequence positions anywhere in the nucleic acid. Variant nucleic acids comprising nucleic acid molecules of different sequence may differ from each other in a particular region of the sequence, or have differences scattered over the entire length of the sequence, or combinations thereof. 28 NAI-5001873978Variant nucleic acids can contain degenerate or randomized positions, where a specific sequence or region has been replaced by a stretch of degenerate nucleotides. Randomized or degenerate positions within variant nucleic acids may involve adjacent nucleotides or non- adjacent nucleotides separated by nucleotides of a specific or fixed sequence. Variant nucleic acids are frequently employed in biotechnology to create variability within a sequence of interest (coding sequence or non-coding sequence) from which new nucleic acids with specific qualities of interest (for example higher efficiency of an encoded enzyme) can be isolated.

[0115] Variant proteins or variant enzymes: As used herein, a “variant protein” or “variant enzyme” refers to a protein or enzyme which is related to but distinct from a parental protein or enzyme by alteration of the parental amino acid sequence, resulting in one or more mutant or variant versions of the parental protein or enzyme. Alterations can be by mutagenesis (i.e. introduction of single amino acid changes); introduction of insertions and deletions of varying lengths; fusion with other sequences either at the N or the C-terminus of the parental protein; sequence exchange with related proteins resulting in hybrid or chimeric proteins containing blocks of sequence from two or more parental proteins; or combinations thereof; and any other means of creating sequence diversity. Variant proteins or enzymes are often created as populations of sequence variants, where a single protein sample contains protein molecules related to each other by sequence but differing in their specific amino acid sequence.

[0116] Sequence similarity or sequence identity of nucleic acid or amino acid sequences: As described herein “sequence similarity of nucleic acids or amino acid sequences” or “identity of nucleic acids or amino acid sequences” may be determined by methods known to those of skill in the art. In some aspects, amino acids are similar with regard to polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups or nonpolar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine. Thus, a similar amino acid may be an amino acid identified as suitable for a conservative amino acid substitution, e.g., as described in the literature and readily identified by methods known to those of skill in the art, for example, as shown in Table 1, listing conservative amino acid substitutions. In some aspects, a similar amino acid is an amino acid listed in Table 1, 29 NAI-5001873978second column (headed “I. Conservative Substitutions”) in the row corresponding to the original amino acid. In some aspects, a similar amino acid is an amino acid listed in Table 1 third column (headed “II. Alternative Substitutions”) in the row corresponding to the original amino acid.

[0117] In some aspects, a mutation results in the substitution of an amino acid with any other amino acid. In some aspects, the substitution is a non-conservative amino acid substitution. A non- conservative amino acid substitution can be readily selected by one of skill in the art. Table 1 provides examples of conservative amino acid substitutions (column I) and alternative conservative amino acid substitutions (II). In some aspects, a non-conservative substitution of an original amino acid (e.g., the amino acid in the wild-type protein) is a substitution with any amino acid not listed in (I) for the original amino acid. In some aspects, a non-conservative substitution of an original amino acid is any amino acid not listed in (II) for the original amino acid. In some aspects, a non- conservative amino acid substitution is any amino acid not listed in either (I) or (II) for the original amino acid.

[0118] Table 1: Similar Amino Acids Amino Acid I. Conservative II. Alternative Substitutions Substitutions30 NAI-5001873978Asn Asp, Gln, Gluany acidic amino acid or derivative thereof, or anyamide of any acidic amino acid or derivative thereof31 NAI-5001873978Glu Asn, Asp, Glnany acidic amino acid or derivative thereof, or anyamide of any acidic amino acid or derivative thereof32 NAI-5001873978Phe Trp, Tyrany aromatic amino acid or derivative thereof(Phe, Trp, Tyr)33 NAI-5001873978

[0119] Methods for diversifying a gene encoding a nucleic acid polymerase of interest, for the purpose of creating mutants or variants of this gene from which genes can be selected which encode improved variant nucleic acid polymerases, include but are not limited to: mutagenesis meaning introduction of point mutations; introduction of insertions and deletions of varying lengths within the enzyme coding sequence; fusion with other sequences either at the 5’ or the 3’ end of the coding sequence; homologous sequence exchange with related coding sequences resulting in reassortment of polymorphisms; and any other means of creating sequence diversity. Mutagenesis of a gene or coding sequence can be achieved by use of low-fidelity nucleic acid polymerases, for example during PCR amplification of the coding sequence; chemical mutagenesis of a cell harboring the sequence to be mutated; incorporation by a nucleic acid polymerase of modified nucleotides that are prone to causing mutations when replicated with normal nucleotides, for example during PCR amplification; propagation of the coding sequence to be mutated in a mutation-prone bacterial strain or other cell; or any other manner of introducing mutations into such sequence.

[0120] Different screening technologies and approaches have been described in the literature and can be adapted to screening for improved nucleic acid polymerase such as the ones described in this disclosure. Screening can be done in a low-throughput, high-throughput or ultra-high- throughput manner. Low throughput refers to processing and testing of individual samples or small numbers of samples; high throughput screening involves screening in 96- or 384-well plates which allows simultaneous testing of thousands of enzyme variants; ultra-high-throughput screening uses microbial cells or emulsion droplets to compartmentalize the screening of separate variants and allows millions of enzyme variants to be tested simultaneously. When a suitable screen can be developed that allows enrichment of improved variants of an enzyme, ultra-high- throughput screens allow exploration of a very large sequence space, or mutation space, for identifying improved variants of an enzyme. For example, an ultra-high-throughput screen allows exploration of all possible combinations of two point-mutations, or all possible combinations of three point-mutations, or all possible combinations of four point-mutations, etc., in a nucleic acid molecule.

[0121] In some aspects, the variant enzyme is a single-subunit RNA polymerase. Single- subunit RNA polymerases, such as bacteriophage-type RNA polymerases, comprise a catalytic domain with a fingers subdomain and palm subdomains, and an N-terminal domain. The fingers 34 NAI-5001873978subdomain is involved in the binding of NTPs with template, and the palm subdomains coordinate the NTPs with the template. The N-terminal domain is involved in promoter recognition and DNA strand separation. The domains of single-subunit RNA polymerases are shown in FIG.1 and Table 4.

[0122] In some aspects, high throughput or ultra-high-throughput screens are employed to identify RNA polymerase variants with desired attributes. The screens can be performed in vivo, that is in a living cell, or in vitro, that is outside of a living cell. Screens may be performed in one or more sequential rounds. In the present invention, two separate ultra-high-throughput screens were used to identify the RNA polymerase variants and the amino acid changes contained therein that are the subject of the present disclosure. Screen 1 uses a microbial system and has a per- sample throughput of approximately 1 million enzyme variants. Screen 2 is based on water-in oil emulsions, for example as described in International Publication No. WO2024 / 211850. Screen 2 has a per-sample throughput of approximately 1 billion enzyme variants. The present disclosure describes variant enzymes and amino acid substitutions identified in the RNA polymerases that are the subject of the disclosure. The variant enzymes and amino acid substitutions were identified by use of Screen 1 and Screen 2.

[0123] We describe variants of single-subunit RNA polymerases that are suitable for RNA manufacturing in vitro. These variant enzymes are substantially similar, by sequence, to the native single-subunit RNA polymerase RNApol225 (SEQ ID NOs: 1 and 2), from which they have been derived by mutagenesis. These single-subunit RNA polymerase variants are also related, by sequence and structure, to the T7 RNA polymerase (SEQ ID NOs: 3 and 4) that is widely used for RNA synthesis in vitro, in particular in the manufacture of RNA for use in pharmaceuticals, diagnostics, vaccines, medicaments, therapeutics, and cosmetics.

[0124] Different RNA polymerases vary in their ability to synthesize RNA. For example, the ability of a single-subunit RNA polymerase to synthesize a uniform population of RNA molecules in vitro decreases with the length of the DNA molecule used as a template for the RNA polymerase. Certain RNA polymerases have higher processivity than others, or an improved ability to synthesize full-length RNAs from longer templates, and are capable of synthesizing highly uniform RNAs >1 kb in length or >2 kb in length or >3 kb in length or >4 kb in length or >5 kb in length or >6 kb in length or>7 kb in length or >8 kb in length or >9 kb in 35 NAI-5001873978length or >10 kb in length or >11 kb in length or >12 kb in length or >13 kb in length or >14 kb in length or >15 kb in length or >16 kb in length or >17 kb in length or >18 kb in length or >19 kb in length or >20 kb in length or longer.

[0125] Certain RNA polymerases are capable of synthesizing RNAs of 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 21 kb, 22 kb, 23 kb, 24 kb, 25 kb, 26 kb, 27 kb, 28 kb, 29 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb in length or longer or shorter, or any length in between.

[0126] RNA synthesis by an RNA polymerase can also be influenced by the components or composition of the in vitro transcription reaction, which can include double-stranded DNA template molecules encoding the RNAs to be transcribed; single-subunit RNA polymerases; nucleoside triphosphates as monomers for RNA synthesis; buffers, divalent cations and salts as necessary for the RNApol to be active, other enzymes such as pyrophosphatase, other proteins such as RNase inhibitors, and other reaction additives of any kind. The reaction composition can be varied by varying the presence or concentration of each of the reaction components; by varying the types of reaction components such as buffers, salts or divalent cations; or by varying the pH.

[0127] Qualities of nucleic acid polymerases or RNA polymerases that are of interest to biotechnology and that can be improved for use in RNA production include, but are not limited to: transcription initiation from a specific transcription start site; recognition of and transcription initiation from a specific promoter; transcriptional activity from a specific promoter or template nucleic acid; the ability to synthesize a minimum number or specific number of RNA transcripts from a single nucleic acid template molecule within a specific time interval; yield of RNA synthesized in a reaction in which the RNA polymerase is used to transcribe a specific nucleic acid template; ability to incorporate modified nucleotides or nucleotides not typically incorporated by RNA polymerases such as nucleotides with modified sugars, bases, phosphodiester linkages, or deoxyribonucleotides, into a nucleic acid strand; ability to synthesize non-RNA nucleic acids such as DNA; integrity of RNA (that is, the percentage of full-length RNA transcribed from a nucleic acid template relative to total RNA) synthesized in a reaction in 36 NAI-5001873978which the RNA polymerase is used to transcribe a specific nucleic acid template; altered amounts of or the absence of undesirable side products, including but not limited to RNA transcripts shorter than the full-length transcript, antisense RNA molecules, or double-stranded RNA synthesized in a reaction in which the RNA polymerase is used to transcribe a specific nucleic acid template; incorporation of 1 or 2 phosphate groups at the transcriptional 5’ end; efficiency of incorporation of a 7-methyl-guanosine cap at the 5’ end of an RNA molecule, or of a different nucleotide cap, a di-nucleotide cap or cap analog, a tri-nucleotide cap or cap analog, or a longer cap used to initiate synthesis of RNA molecules synthesized in a reaction in which the RNA polymerase is used to transcribe a specific nucleic acid template; processivity of an RNA polymerase; ability of an RNA polymerase to synthesize long RNAs, for example RNAs in excess of 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 21 kb, 22 kb, 23 kb, 24 kb, 25 kb, 26 kb, 27 kb, 28 kb, 29 kb, 30 kb, 40 kb, 50 kb or longer or any size in between; RNA polymerase activity at a certain temperature; heat tolerance of an RNA polymerase; stability of an RNA polymerase; tolerance of an RNA polymerase to salts or other potentially inhibitory compounds present in an in vitro transcription reaction; or any other quality of the RNA polymerase that affects or alters its activity in the synthesis of RNA molecules or other nucleic acid molecules.

[0128] Single-subunit RNA polymerases and / or in vitro transcription reactions also differ in their ability to utilize non-natural nucleotides and incorporate these into the RNA molecule. Examples of such non-natural nucleotides are 2’-O-methyl NTPs, 2’-fluoro NTPs, pseudouridine-5’-triphosphate, N1-methylpseudouridine-5’-triphosphate, 1-ethylpseudouridine,2-thiouridine, 4 -thiouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl- pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine (mo5U) and 2 -O-methyl uridine. The 2’ hydroxyl of ribonucleotides hasfrequently been targeted for modification because this group is primarily responsible for the low stability of RNA under basic conditions. Various modifications at the 2’ position of nucleotides have been tested for increasing RNA stability. However, some single-subunit RNA polymerases incorporate such modified nucleotides inefficiently. Alternatively, RNA molecules containing such modified nucleotides may exhibit a high rate of sequence errors. Specific single-subunit 37 NAI-5001873978RNA polymerases among the ones described in this disclosure are able to incorporate modified nucleotides efficiently without compromising sequence fidelity.

[0129] Single-subunit RNA polymerases and / or in vitro transcription reactions differ in their RNA yield based on the nucleotides added to an in vitro transcription reaction. For example, a 1 ml in vitro transcription reaction containing 5 mM of each of the four nucleoside triphosphates ATP, CTP, GTP and TTP can yield up to about 6.43 mg of RNA (the ‘theoretical yield’) assuming equal representation of each of the nucleotides in the DNA template and complete incorporation of nucleoside triphosphates into RNA in the reaction. An RNA polymerase that synthesizes 2.5 mg of RNA in such a reaction has a yield of 38.9%. Higher-yielding RNA polymerases and / or in vitro transcription reactions are of value as they maximize the amount of RNA product made from a specific amount of nucleoside triphosphates added to a reaction of specific composition that is allowed to react in a specific set of conditions such as temperature and reaction time. For example, the single-subunit RNA polymerases disclosed herein produce a transcript yield that is greater than that generated by T7 RNA polymerase, and greater than that generated by the parental RNA polymerase from which such RNA polymerases are derived. In some cases, at temperatures less than 24°C or with templates longer than 5 kb, yield increases can be observed as much as a two-fold, three-fold, four-fold or higher increases. Similarly, when using modified nucleotides, the RNA yield of the single-subunit RNA polymerases disclosed herein can yield higher amounts of RNA, depending upon the modified nucleotide used as compared to T7 RNA polymerase.

[0130] Yield enhancement during in vitro transcription can mean increasing the absolute amount of RNA synthesized in the reaction with all reaction components being the same (approaching the theoretical yield) or increasing or maintaining the same RNA yield while reducing the reaction concentrations of the double-stranded DNA template or of the RNA polymerase. Such improved reactions can increase RNA yield on template or yield on RNA polymerase. Other proteins added to an in vitro transcription reaction can increase the RNA yield on template or increase the RNA yield on RNA polymerase or increase the RNA yield on any other reaction component that is expensive or otherwise limiting and for which it may benefit the producer of the RNA to lower the concentration of said component. 38 NAI-5001873978

[0131] RNA yield as described above can be expressed as total RNA yield, which includes all RNA molecules synthesized in the reaction, regardless of their length, or full-length RNA yield, which includes only the full-length and substantially full-length RNA molecules synthesized in the reaction. For example, an RNA polymerase or in vitro transcription reaction may produce a measurably higher RNA yield than full-length RNA yield. Use of specific RNA polymerases in an in vitro transcription reaction, addition of specific reaction components to an in vitro transcription reaction, or optimization of reaction composition and / or conditions of an in vitro transcription reaction may change either total RNA yield or full-length RNA yield.

[0132] Single-subunit RNA polymerases and / or in vitro transcription reactions differ in the amount of double-stranded RNA made in a reaction. Double-stranded RNA is a frequent and undesirable side product of in vitro transcription reactions (Arnaud-Barbe 1998, Mu 2018, Gholamalipour 2018), and its reduction or elimination reduces the cost of synthesizing pharmaceutical-grade RNA.

[0133] Single-subunit RNA polymerases and / or in vitro transcription reactions differ in the amount of short or truncated RNAs made in a reaction. Short or truncated RNAs can be any RNAs that are not full-length and are frequent and undesirable side products of in vitro transcription reactions. They represent aborted or incomplete transcription products of a template (Martin 1988); their reduction or elimination reduces the cost of synthesizing pharmaceutical- grade RNA.

[0134] Single-subunit RNA polymerases differ in their ability to synthesize polyA sequences encoded in DNA templates. RNAs synthesized with RNA polymerases that don’t efficiently synthesize polyA sequences may have truncated polyA sequences present in the transcribed RNA, or the polyA sequences present in the RNA may be of diverse length. For example, the ability to efficiently synthesize polyA sequences longer than 50 nucleotides, or to synthesize these in a uniform manner, with equal or near-equal length of the polyA sequence in each synthesized RNA molecule, is of great utility when synthesizing RNAs for use in biotechnology or medicine.

[0135] Single-subunit RNA polymerases and / or in vitro transcription reactions differ in their ability to incorporate a 5’-cap such as the 7-methylguanosine cap found in eukaryotic mRNAs or other capping structures into the 5’ end of RNAs. mRNAs used in biotechnology can be capped by incorporating a specialized dinucleotide or trinucleotide cap analog into the 5’ end of the 39 NAI-5001873978mRNA. Co-transcriptional incorporation of dinucleotide or trinucleotide cap analogs is catalyzed by the RNA polymerase during transcription initiation. Specific RNA polymerases as disclosed herein used in in vitro transcription reactions can increase the rate of cap incorporation and cap utilization.

[0136] A variant RNA polymerase can exhibit enhanced capping efficiency or cap utilization efficiency compared to a wild type or parental RNA polymerase, as exemplified by the following scenarios: 1) The variant RNA polymerase may have higher capping efficiency than the parental RNA polymerase under identical reaction conditions, as measured by the percentage of capped mRNA molecules produced in the reaction; 2) The variant RNA polymerase may have higher capping efficiency than the parental RNA polymerase under identical reaction conditions, as measured by the total amount of capped mRNA produced in the reaction; 3) The variant RNA polymerase may have higher capping utilization efficiency than the parental RNA polymerase under identical reaction conditions (typically using lower concentrations of cap analogs than in IVT reactions designed to maximize capping efficiency) as measured by the percentage of capped mRNA molecules produced in the reaction; 4) The variant RNA polymerase may have higher capping utilization efficiency than the parental RNA polymerase under identical reaction conditions (typically using lower concentrations of cap analogs than in IVT reactions designed to maximize capping efficiency) as measured by the total amount of capped mRNA produced in the reaction.

[0137] Single-subunit RNA polymerases and / or in vitro transcription reactions differ in their temperature specificity or reaction speed at varying temperatures, both of which are important parameters in RNA synthesis. Lower reaction temperatures such as between 10°C and 20°C can stabilize the RNA. However, certain RNA polymerases such as T7 RNA polymerase have very low activity at such temperatures. It is therefore of value to identify RNA polymerases active at low temperatures. Alternatively, mRNA synthesis may be more efficient at higher temperatures. Temperatures used for IVT reactions include 1°C, to 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C or any other temperature in between, or higher or lower temperatures. The reaction 40 NAI-5001873978temperature of an IVT reaction may also vary in the course of the reaction, from any of the temperatures listed above to any other temperature.

[0138] Single-subunit RNA polymerases and / or in vitro transcription reactions differ in their overall reaction speed, either at a specific temperature or irrespective of temperature. Faster enzymes are typically more desirable because shorter reaction times reduce RNA degradation.

[0139] Single-subunit RNA polymerases differ in their stability at different temperatures or in different reaction conditions. For example, certain RNA polymerases may show loss of activity at certain temperatures compared to other polymerases, indicating lower stability at these temperatures. For example, certain RNA polymerases may show loss of activity in certain in vitro transcription reaction conditions compared to other polymerases, indicating lower stability under these conditions. For certain applications, it may be useful to develop RNA polymerase with increased stability at temperatures at which T7 RNA polymerase or a parental RNA polymerase shows loss of activity, allowing longer reactions times that may increase the RNA yield in the reaction.

[0140] Single-subunit RNA polymerases and / or in vitro transcription reactions differ in their fidelity. High-fidelity RNA polymerases will produce RNAs that faithfully encode the sequence of the template DNA used to synthesize the RNA and faithfully encode a protein of interest. High-fidelity RNA polymerases therefore have higher utility when synthesizing RNAs for therapeutic or vaccine applications. Certain RNA polymerases may have different fidelity depending on reaction composition, reaction conditions such as temperature, template sequence, template length or incorporation of modified nucleotides into RNA.

[0141] Measurements of RNA polymerase activity, or quality metrics of RNA synthesized in in vitro transcription reactions, are generated using standardized methods and assays. RNA yield is measured by purification of the RNA after the in vitro transcription reaction, followed by spectroscopic or fluorescence measurement of RNA concentrations (Gandhi 2020, Hadi 2023). RNA yield and integrity are measured by gel electrophoresis (Henderson 2021, Tu 2024) and quantitation of the fluorescence intensity of RNA bands using ImageJ or related software (Schindelin 2012, Schneider 2012, Rueden 2017, Poveda 2019) or other methods for quantitating fluorescent band intensities. RNA yield and integrity are also determined with capillary electrophoresis-based methods (Poveda 2019, Warzak 2023) using commercially 41 NAI-5001873978available instruments such as the Fragment Analyzer manufactured by Agilent Corporation (Santa Clara, CA, USA). Capillary electrophoresis methods are also suitable for measuring other RNA qualities such as polyA tail length and uniformity (Di Grandi 2023, Tu 2024). RNA integrity can also be addressed using reverse transcription-qPCR (Poveda 2019, Di Grandi 2023). Double-stranded RNA present in RNA synthesized in in vitro transcription reactions is quantitated using dot blots or ELISA assays based on monoclonal antibodies that specifically bind double-stranded RNA (Aramburu 1991, Karikó 2011, Baiersdörfer 2019), such as the J2 IgG2a monoclonal antibody and the and the IgG2a K1 and IgM K2 monoclonal antibodies (Schönborn 1991) and the 9D5 monoclonal antibody (Son 2015). Double-stranded RNA levels can also be determined using reverse transcription-qPCR (Poveda 2019, Di Grandi 2023). Capping efficiency and cap incorporation efficiency can be measured with a variety of methods including gel electrophoresis, fluorescence spectroscopy (when using fluorescently labeled cap analogs), nanopore sequencing and liquid chromatography-mass spectrometry (Tu 2024). RNA polymerase and RNA fidelity are addressed by a variety of sequencing methods, including RNA sequencing following reverse transcription and nanopore sequencing (Gholamalipour 2018, Poveda 2019, Gunter 2023). RNA quality is also measured by in vitro translation followed by enzymatic assays (for RNAs encoding enzymes whose activity can be determined in vitro) and cell-based assays (Poveda 2019).

[0142] Single-subunit RNA polymerases differ in their RNA yield and capping efficiency in in vitro transcription reactions, depending on the transcription start site present downstream of the promoter sequence in a nucleic acid template.

[0143] Single-subunit RNA polymerases differ in their ability to incorporate a 5’-cap such as the 7-methylguanosine cap found in eukaryotic mRNAs or other capping structures found at the 5’ end of RNAs. mRNAs used in biotechnology can be capped by incorporating a specialized dinucleotide or trinucleotide cap analog into the 5’ end of the mRNA. Co- transcriptional incorporation of dinucleotide or trinucleotide cap analogs is catalyzed by the RNA polymerase during transcription initiation. The variant single-subunit RNA polymerases disclosed herein have higher rates of cap incorporation than T7 RNA polymerase.

[0144] The concentrations of cap analog in an IVT reaction can differ depending on the cap analog, the RNA polymerase and the percentage of capping to be achieved in the reaction. 42 NAI-5001873978Typical concentrations of cap analogs range from 1mM to 10mM, including 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM or 10mM, or any value in between. However, the concentrations of cap analog may also be higher than this range, for example 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM or higher or any value in between. Alternatively, the concentration of cap analog may also be lower, for example 0.01mM, 0.02mM, 0.03mM, 0.04mM, 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM or 1.0mM, or below 0.01mM, or any value in between.

[0145] In some aspects, the variant single-subunit RNA polymerase provided herein may have improved activity in an in vitro transcription reaction and / or increase RNA yields by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 5000%, 10000% or more, or any number in between, compared the corresponding wild type or parental single-subunit RNA polymerase or to T7 RNA polymerase. Increases in RNA yield can be reflected in total RNA yield of full-length RNA yield, or both. Increases in RNA yield can be of unmodified RNA, or RNA modified by incorporation of one or more modified nucleotides or nucleotide analogs.

[0146] Yield-enhancing amino acid substitution: As used herein, “yield-enhancing amino acid substitution” refers to a change in the amino acid sequence of an RNA polymerase that results in higher yields of the mRNA produced by the variant RNA polymerase containing the substitution during in vitro transcription, compared to the wild type or parental RNA polymerase. Higher mRNA yields can mean mRNA yield as measured in any reaction condition of an IVT reaction, including at any temperature, pH, concentration of divalent cations, template DNA sequence, template DNA length, template DNA concentration, RNA polymerase concentration or any other aspect of an IVT reaction’s composition or reaction conditions that can be adjusted to alter or optimize the output of the IVT reaction. Yield-enhancing amino acid substitution increases the absolute amount of RNA synthesized in the reaction with all reaction components being the same (approaching the theoretical yield) or increases or maintains the same RNA yield while reducing the reaction concentrations of the double-stranded DNA template or of the RNA polymerase itself. 43 NAI-5001873978

[0147] In some aspects, provided herein is a single-subunit RNA polymerase that comprises at least one yield-enhancing amino acid substitution. In some aspects, provided herein is a single- subunit RNA polymerase that comprises at least one yield-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 70, 166, 383, 562, and 567 of SEQ ID NO: 1 as listed in Table 3.

[0148] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution at a sequence position corresponding to sequence position 25 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution at a sequence position corresponding to sequence position 70 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution at a sequence position corresponding to sequence position 166 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution at a sequence position corresponding to sequence position 383 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution at a sequence position corresponding to sequence position 562 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution at a sequence position corresponding to sequence position 567 of SEQ ID NO: 1.

[0149] In some aspects, provided herein is a single-subunit RNA polymerase that comprises at least one yield-enhancing amino acid substitution corresponding to a substitution selected from the group consisting of: A25V, A70T, V166M, A383T, L562V, and V567P of SEQ ID NO: 1 as listed in Table 3.

[0150] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution corresponding to a substitution of A25V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution corresponding to a substitution of A70T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution corresponding to a 44 NAI-5001873978substitution of V166M of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution corresponding to a substitution of A383T of SEQ ID NO: 1. In one embodiment, the single- subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution corresponding to a substitution of L562V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution corresponding to a substitution of V567P of SEQ ID NO: 1.

[0151] In some aspects, use of the variant single-subunit RNA polymerase in an in vitro transcription reaction may increase RNA integrity by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%,, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 5000%, 10000% or higher, or any number in between, compared to use of the corresponding wild type single-subunit RNA polymerase or T7 RNA polymerase in an in vitro transcription reaction. Use of the variant RNA polymerase for in vitro RNA synthesis can result in integrity of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% or any number in between of RNA synthesized in in vitro transcription reactions.

[0152] In some aspects, the use of the variant single-subunit RNA polymerase in an in vitro transcription reaction may reduce the amount of double-stranded RNA (dsRNA) formed in the reaction, or reduce the amount of other undesirable side products such as short or truncated RNAs, by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or any number in between, compared to use of the corresponding wild type single-subunit RNA polymerase or T7 RNA polymerase in an in vitro transcription reaction. In some aspects, the use of the variant single-subunit RNA polymerase provided herein in an in vitro transcription reaction may decrease the quantity of double-stranded RNA by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 5000%, 10000% or more, or any number in between, compared to use of the 45 NAI-5001873978corresponding wild type single-subunit RNA polymerase or T7 RNA polymerase in an in vitro transcription reaction.

[0153] In some aspects, provided herein is a single-subunit RNA polymerase that comprises at least one dsRNA-reducing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 166, and 153 of SEQ ID NO: 1 as listed in Table 3.

[0154] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one dsRNA-reducing amino acid substitution at a sequence position corresponding to sequence position 166 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one dsRNA-reducing amino acid substitution at a sequence position corresponding to sequence position 153 of SEQ ID NO: 1.

[0155] In some aspects, provided herein is a single-subunit RNA polymerase that comprises at least one dsRNA-reducing amino acid substitution corresponding to a substitution selected from the group consisting of: V166M, and R153H of SEQ ID NO: 1 as listed in Table 3.

[0156] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one dsRNA-reducing amino acid substitution corresponding to a substitution of V166M of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one dsRNA-reducing amino acid substitution corresponding to a substitution of R153H of SEQ ID NO: 1.

[0157] In some aspects, the use of the variant single-subunit RNA polymerase provided herein in an in vitro transcription reaction may increase the capping efficiency by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 5000%, 10000% or more, or any number in between, compared to use of the corresponding wild type single-subunit RNA polymerase or T7 RNA polymerase in an in vitro transcription reaction. Use of the variant RNA polymerase for in vitro RNA synthesis can result in capping efficiency of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 46 NAI-500187397860%, 70%, 80%, 90%, 100% or any number in between of RNA synthesized in in vitro transcription reactions.

[0158] In some aspects, provided herein is a single-subunit RNA polymerase that comprises at least one capping-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 74, 383, 567, 661, and 867 of SEQ ID NO: 1 as listed in Table 3.

[0159] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution at a sequence position corresponding to sequence position 25 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution at a sequence position corresponding to sequence position 74 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution at a sequence position corresponding to sequence position 383 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution at a sequence position corresponding to sequence position 567 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution at a sequence position corresponding to sequence position 661 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution at a sequence position corresponding to sequence position 867 of SEQ ID NO: 1.

[0160] In some aspects, provided herein is a single-subunit RNA polymerase that comprises at least one capping-enhancing amino acid substitution corresponding to a substitution selected from the group consisting of: A25V, V74K, A383T, V567P, S661D, and K867S of SEQ ID NO: 1 as listed in Table 3.

[0161] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution corresponding to a substitution of A25V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution corresponding to a substitution of V74K of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one 47 NAI-5001873978capping-enhancing amino acid substitution corresponding to a substitution of A383T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution corresponding to a substitution of V567P of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution corresponding to a substitution of S661D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one capping-enhancing amino acid substitution corresponding to a substitution of K867S of SEQ ID NO: 1.

[0162] In some aspects, the use of the variant single-subunit RNA polymerase in an in vitro transcription reaction may increase the cap incorporation efficiency by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 5000%, 10000% or more, or any number in between, use of the corresponding wild type single-subunit RNA polymerase or T7 RNA polymerase in an in vitro transcription reaction. Use of the variant RNA polymerase for in vitro RNA synthesis can result in cap incorporation efficiency of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% or any number in between of RNA synthesized in in vitro transcription reactions.

[0163] We describe amino acid changes in RNA polymerase RNApol225 (SEQ ID NOs: 1 and 2) that can cause improvements in the enzyme’s activity and catalytic properties. Each amino acid polymorphism shown in the tables below can improve any of the qualities listed above of an RNA polymerase for RNA manufacturing. An amino acid change may improve the activity of an RNA polymerase either when present by itself (as the only amino acid change in the protein compared to the parental sequence) or when present in combination with other amino acid changes. When present with other amino acid changes there may be two or multiple amino acid changes in a single coding sequence and in the protein it encodes, including 2, 3, 4, 5, 6, 7, 8, 9.10.11.12.13.14.15.16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acid changes, or any number in between. 48 NAI-5001873978

[0164] Because the amino acid changes listed in the tables below were isolated in a screen involving a complex mixture of different mutations to an RNA polymerase coding sequence, a specific amino acid change can be beneficial for the activity of the RNA polymerase (the consequence of a beneficial amino acid substitution) or can be deleterious to the activity of the RNA polymerase (the consequence of a deleterious amino acid substitution), or can be neutral for the activity of the RNA polymerase (the consequence of a neutral amino acid substitution). The presence of other amino acid changes within an RNA polymerase may cause a beneficial amino acid change to become deleterious or may cause a beneficial amino acid change to become neutral or may cause a deleterious amino acid change to become beneficial or may cause a deleterious amino acid change to become neutral or may cause a neutral amino acid change to become beneficial or may cause a neutral amino acid change to become deleterious, or may not lead to any changes in the effects of a beneficial, deleterious or neutral amino acid substitution.

[0165] In some aspects, the amino acid substitutions described herein may be transferred to other, related single-subunit RNA polymerases and their variants at corresponding positions, with the same effect. As such, in certain aspects, this disclosure provides a non-naturally occurring variant of a naturally occurring, or wild type, single-subunit RNA polymerase, wherein the naturally occurring, or wild type, single-subunit RNA polymerase has an amino acid sequence that is 90%, 90.5%, 91%, 91.5%, 92%, 92.5%.93%, 93.5%, 94%, 94.5%, 95% 95.5%, 96%, 96.5%, 97%, 97.5% 98% 98.5%, 99% or 99.5% identical to RNApol225 (Table 2, SEQ ID NO: 1) or RNApol225 His6 tagged (Table 2, SEQ ID NO: 2) and comprises one or more amino acid substitutions relative the wild type single-subunit RNA polymerase, corresponding to one or more position as listed in Table 3 Amino acids correspond to each other when they occur at equivalent positions in aligned amino acid sequences and / or domains thereof, e.g. the catalytic domain or the N-terminal domain. Corresponding positions can be identified by alignment of protein or polypeptide sequences using a variety of methods as described in the art (e.g. BLASTP, ClustalW).

[0166] In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 1; and (b) at least one amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 15, 22, 25, 40, 49, 53, 64, 65, 70, 74, 91, 94, 127, 128, 139, 149, 153, 163, 166, 177, 178, 198, 203, 205, 208, 217, 49 NAI-5001873978222, 232, 236, 251, 258, 260, 269, 279, 291, 320, 343, 346, 350, 351, 357, 363, 383, 388, 413, 421, 426, 440, 457, 459, 471, 473, 479, 480, 483, 487, 494, 498, 520, 522, 523, 534, 542, 555, 562, 565, 567, 576, 581, 583, 598, 601, 610, 625, 632, 633, 638, 641, 661, 665, 669, 693, 694, 707, 713, 736, 740, 775, 786, 795, 820, 839, 843, and 867 of SEQ ID NO: 1.

[0167] The amino acid substitutions in RNA polymerase RNApol225 described in this disclosure may have an effect on any quality, or property of the RNA polymerase, or any aspects of its activity or performance, either individually or in combinations. The amino acid sequences of the parental RNA polymerase that the RNA polymerase variants described herein are derived from is RNApol225 as shown in Table 2. The RNA polymerase may encode an N-terminal poly- histidine tag, e.g. RNApol2256His tagged (Table 2).

[0168] Table 2: Single-subunit RNA Polymerase Amino Acid Sequences Described in this Disclosure Sequence SEQ ID Sequence name NO50 NAI-5001873978RNApol225 2 MHHHHHHGSTVIAIEKNDFSDVELAVIPFNTLADHYGEKLARE His6 tagged QLALEHEAYEMGEARFRKIFERQLKAGEVADNAAAKPLVATLL L51 NAI-5001873978T7 RNA 4 MHHHHHHGSNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQ Polymerase LALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLP Hi

[0169] Amino acid substitutions of interest are listed in Table 3. Activity improvements, or enhanced RNApol activity, observed in IVT reactions containing RNApol225 variants containing these substitutions include increased RNA yield, increased RNA integrity, reduced dsRNA formation, and / or enhanced capping efficiency. The listed substitutions were identified in clones isolated from high-throughput screening, populations of RNApol225 variants isolated in high throughput screens (detected by NGS) or were derived from machine learning (high zero- shot score).

[0170] Table 3: Amino acids substitutions found in screens or purified RNApol225 variants Position in RNA Position in RNA52 NAI-500187397825 A25V 473 V473I 40 E40K 479 I479VNAI-5001873978388 D388E 795 V795I 413 A413T 820 D820N [t least one amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 15, 22, 25, 40, 49, 53, 64, 65, 70, 74, 91, 94, 127, 128, 139, 149, 153, 163, 166, 177, 178, 198, 203, 205, 208, 217, 222, 232, 236, 251, 258, 260, 269, 279, 291, 320, 343, 346, 350, 351, 357, 363, 383, 388, 413, 421, 426, 440, 457, 459, 471, 473, 479, 480, 483, 487, 494, 498, 520, 522, 523, 534, 542, 555, 562, 565, 567, 576, 581, 583, 598, 601, 610, 625, 632, 633, 638, 641, 661, 665, 669, 693, 694, 707, 713, 736, 740, 775, 786, 795, 820, 839, 843, and 867 of SEQ ID NO: 1.

[0172] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 15 of SEQ ID NO: 1.

[0173] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 22 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 25 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 40 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 49 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 53 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 64 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 65 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid 54 NAI-5001873978substitution at a sequence position corresponding to sequence position 70 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 74 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 91 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 94 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 127 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 128 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 139 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 149 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 153 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 163 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 166 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 177 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 178 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 198 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 203 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 205 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid 55 NAI-5001873978substitution at a sequence position corresponding to sequence position 208 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 217 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 222 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 232 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 236 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 251 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 258 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 260 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 269 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 279 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 291 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 320 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 343 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 346 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 350 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 351 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid 56 NAI-5001873978substitution at a sequence position corresponding to sequence position 357 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 363 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 383 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 388 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 413 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 421 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 426 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 440 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 457 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 459 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 471 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 473 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 479 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 480 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 483 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 487 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid 57 NAI-5001873978substitution at a sequence position corresponding to sequence position 494 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 498 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 520 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 522 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 523 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 534 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 542 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 555 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 562 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 565 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 567 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 576 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 581 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 583 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 598 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 601 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid 58 NAI-5001873978substitution at a sequence position corresponding to sequence position 610 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 625 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 632 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 633 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 638 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 641 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 661 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 665 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 669 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 693 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 694 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 707 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 713 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 736 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 740 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 775 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid 59 NAI-5001873978substitution at a sequence position corresponding to sequence position 786 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 795 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 820 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 839 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 843 of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to sequence position 867 of SEQ ID NO: 1.

[0174] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 166, and 562 of SEQ ID NO: 1.

[0175] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution selected from the group consisting of: E15D, N22S, A25V, E40K, A49V, A49T, K53E, V64A, A65V, A70T, V74K, E91K, A94V, T127I, S128R, S139A, A149T, R153H, K163A, V166M, V177I, Y178C, G198N, S203N, H205Y, D208E, I217V, E222K, Q232R, V236I, A251T, A258V, A260V, Q269M, T279V, R291C, V320I, K343N, H346L, E350D, D351G, R357P, K363E, A383T, D388E, A413T, D421E, V426F, T440V, F457V, W459L, D471N, V473I, I479V, K480E, E483D, E487D, K494E, E498D, G520D, M522I, H523Y, L534P, G542V, G555V, L562V, E565D, V567P, K576R, I581L, Q583N, T598A, T601I, V610I, A625T, A625V, R632S, S633P, A638T, S641T, S661A, S661D, L665M, I669T, V693L, E694V, A707V, K713E, W736R, R740S, E775V, Q786L, V795I, D820N, C839N, A843V, and K867S of SEQ ID NO: 1.

[0176] In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E15D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of N22S of SEQ ID NO: 1. In one embodiment, the single- subunit RNA polymerase provided herein comprises at least one amino acid substitution 60 NAI-5001873978corresponding to a substitution of A25V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E40K of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A49V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A49T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of K53E of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V64A of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A65V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A70T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V74K of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E91K of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A94V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of T127I of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of S128R of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of S139A of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A149T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of R153H of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of K163A of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V166M of SEQ ID NO: 1. In one embodiment, the 61 NAI-5001873978single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V177I of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of Y178C of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of G198N of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of S203N of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of H205Y of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of D208E of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of I217V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E222K of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of Q232R of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V236I of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A251T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A258V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A260V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of Q269M of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of T279V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of R291C of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V320I of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid 62 NAI-5001873978substitution corresponding to a substitution of K343N of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of H346L of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E350D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of D351G of SEQ ID NO:1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of R357P of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of K363E of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A383T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of D388E of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A413T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of D421E of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V426F of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of T440V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of F457V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of W459L of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of D471N of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V473I of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of I479V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of K480E of SEQ ID NO: 1. In one 63 NAI-5001873978embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E483D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E487D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of K494E of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E498D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of G520D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of M522I of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of H523Y of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of L534P of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of G542V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of G555V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of L562V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E565D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V567P of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of K576R of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of I581L of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of Q583N of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of T598A of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one 64 NAI-5001873978amino acid substitution corresponding to a substitution of T601I of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V610I of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A625T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A625V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of R632S of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of S633P of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A638T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of S641T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of S661A of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of S661D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of L665M of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of I669T of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V693L of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E694V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A707V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of K713E of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of W736R of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of R740S of SEQ ID 65 NAI-5001873978NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of E775V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of Q786L of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of V795I of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of D820N of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of C839N of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of A843V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least one amino acid substitution corresponding to a substitution of K867S of SEQ ID NO: 1.

[0177] In one aspect, the single-subunit RNA polymerase provided herein comprises at least two amino acid substitutions. In one aspect, the single-subunit RNA polymerase provided herein comprises at least two amino acid substitutions listed in Table 3. In one embodiment, the single- subunit RNA polymerase provided herein comprises at least two amino acid substitutions corresponding to substitutions V166M and G618D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least two amino acid substitutions corresponding to substitutions V166M and L562V of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least two amino acid substitutions corresponding to substitutions L562V and G618D of SEQ ID NO: 1.

[0178] In one aspect, the single-subunit RNA polymerase provided herein comprises at least three amino acid substitutions. In one aspect, the single-subunit RNA polymerase provided herein comprises at least three amino acid substitutions listed in Table 3. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least three amino acid substitutions corresponding to substitutions V166M, L562V, and G618D of SEQ ID NO: 1.

[0179] In one aspect, the single-subunit RNA polymerase provided herein comprises at least four amino acid substitutions. In one aspect, the single-subunit RNA polymerase provided herein comprises at least four amino acid substitutions listed in Table 3. In one embodiment, the single- 66 NAI-5001873978subunit RNA polymerase provided herein comprises at least four amino acid substitutions corresponding to substitutions V166M, L562V, G618D, and S661D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least four amino acid substitutions corresponding to substitutions V166M, L562V, G618D, and G718K of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least four amino acid substitutions corresponding to substitutions V166M, L562V, G618D, and C839N of SEQ ID NO: 1.

[0180] In one aspect, the single-subunit RNA polymerase provided herein comprises at least five amino acid substitutions. In one aspect, the single-subunit RNA polymerase provided herein comprises at least five amino acid substitutions listed in Table 3. In one aspect, the single- subunit RNA polymerase provided herein comprises at least six amino acid substitutions. In one aspect, the single-subunit RNA polymerase provided herein comprises at least six amino acid substitutions listed in Table 3. In one aspect, the single-subunit RNA polymerase provided herein comprises at least seven amino acid substitutions. In one aspect, the single-subunit RNA polymerase provided herein comprises at least seven amino acid substitutions listed in Table 3. In one aspect, the single-subunit RNA polymerase provided herein comprises at least eight amino acid substitutions. In one aspect, the single-subunit RNA polymerase provided herein comprises at least eight amino acid substitutions listed in Table 3. In one aspect, the single- subunit RNA polymerase provided herein comprises at least nine amino acid substitutions. In one aspect, the single-subunit RNA polymerase provided herein comprises at least nine amino acid substitutions listed in Table 3.

[0181] In one aspect, the single-subunit RNA polymerase provided herein comprises at least ten amino acid substitutions. In one aspect, the single-subunit RNA polymerase provided herein comprises at least ten amino acid substitutions listed in Table 3. In one embodiment, the single- subunit RNA polymerase provided herein comprises at least ten amino acid substitutions corresponding to substitutions V166M, K343N, E350D, D351G, I479V, E498D, L562V, I581L, G618D, and S661A of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at least ten amino acid substitutions corresponding to substitutions V166M, E350D, D388E, E483D, E487D, L562V, E565D, K576R, Q583N, and G618D of SEQ ID NO: 1. In one embodiment, the single-subunit RNA polymerase provided herein comprises at 67 NAI-5001873978least ten amino acid substitutions corresponding to substitutions V166M, K343N, D351G, I479V, K494E, E498D, L562V, I581L, G618D, and S661A of SEQ ID NO: 1.

[0182] In one aspect, the single-subunit RNA polymerase provided herein comprises at least one yield-enhancing amino acid substitution and at least one capping-enhancing amino acid substitution. In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; (b) at least one yield-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 70, 166, 383, 562, and 567 of SEQ ID NO: 1; (c) at least one capping-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 74, 383, 567, 661, and 867 of SEQ ID NO: 1; and (d) at least one dsRNA-reducing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 166, 153, and 567 of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution corresponds to a substitution of L562V of SEQ ID NO: 1, and at least one dsRNA-reducing amino acid substitution corresponds to a substitution of V166M of SEQ ID NO: 1. In one embodiment, the at least one yield-enhancing amino acid substitution corresponds to a substitution of L562V of SEQ ID NO: 1, the at least one capping- enhancing amino acid substitution corresponds to a substitution of S661D of SEQ ID NO: 1, and the at least one dsRNA-reducing amino acid substitution corresponds to a substitution of V166M of SEQ ID NO: 1.

[0183] In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises amino acid substitutions corresponding to the following positions of SEQ ID NO: 1: V166M, K343N, D351G, I479V, K494E, E498D, L562V, I581L, G618D, and S661A.

[0184] In one aspect, provided herein is a single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises amino acid substitutions corresponding to the following positions of SEQ ID NO: 1: V166M, K343N, E350D, D351G, I479V, E498D, L562V, I581L, G618D, and S661A.

[0185] Alignment positions of the T7 RNA polymerase and RNApol225 amino acids are described in Table 4, showing the amino acid position of each amino acid residue in both 68 NAI-5001873978enzymes together with a common alignment position. This precise definition of alignment positions allows the identification of corresponding amino acids in the two RNA polymerase sequences, based on their corresponding sequence contexts.

[0186] Table 4: Alignment of single-subunit RNA Polymerases T7 RNAP RNApol225 SEQ ID (NP_041960) SEQ ID RNA Polymerase NO 3 NO: 1 l i ix ix ix ix ixNAI-500187397833 33 A 33 A N-term. domain, C-Helix 34 34 R 34 R N-term. domain, C-Helix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ix ixNAI-500187397875 75 T 75 A N-term. domain, PBD 76 76 T 76 T N-term. domain, PBDNAI-5001873978117 117 I 117 I N-term. domain, PBD 118 118 T 118 T N-term. domain, PBDNAI-5001873978159 159 A 159 A N-term. domain, H 160 160 K 160 K N-term. domain, HNAI-5001873978201 201 W 201 W N-term. domain, PBD 202 202 S 202 S N-term. domain, PBDNAI-5001873978243 243 T 243 T N-term. domain, PBD 244 244 I 244 L N-term. domain, PBDNAI-5001873978285 285 G 285 G N-term. domain 286 286 Y 286 Y N-term. domainNAI-5001873978327 327 A 327 A Cat. domain, Thumb 328 328 W 328 W Cat. domain, ThumbNAI-5001873978369 369 M 369 E Cat. domain, Thumb 370 370 N 370 N Cat. domain, ThumbNAI-5001873978411 411 H 411 H Cat. domain, Thumb 412 412 K 412 K Cat. domain, PalmNAI-5001873978453 453 G 453 G Cat. domain, Palm 454 454 K 454 K Cat. domain, PalmNAI-5001873978495 495 S 495 N Cat. domain, Palm 496 496 P 496 P Cat. domain, PalmNAI-5001873978537 537 D 537 D Cat. domain, Palm 538 538 G 538 G Cat. domain, PalmNAI-5001873978579 579 N 579 N Cat. domain, Fingers 580 580 E 580 E Cat. domain, FingersNAI-5001873978621 621 L 621 L Cat. domain, Fingers 622 622 A 622 A Cat. domain, FingersNAI-5001873978663 663 K 663 K Cat. domain, Fingers 664 664 G 664 G Cat. domain, FingersNAI-5001873978705 705 L 705 L Cat. domain, Fingers 706 706 L 706 L Cat. domain, FingersNAI-5001873978747 747 L 747 L Cat. domain, Fingers 748 748 N 748 N Cat. domain, FingersNAI-5001873978789 789 S 789 S Cat. domain, Palm 790 790 H 790 H Cat. domain, PalmNAI-5001873978831 831 T 831 T Cat. domain, Palm 832 832 M 832 M Cat. domain, PalmNAI-5001873978873 873 R 873 R Cat. domain, Palm 874 874 D 874 D Cat. domain, Palm90 NAI-5001873978EXAMPLES

[0187] Example 1: Generation, expression, purification, and activity testing of RNApol225 variants

[0188] Amino acid substitutions in rationally designed variants were generated via the Q5 site-directed mutagenesis kit from New England Biolabs (NEB catalog no. E0554S), substituting Phusion high-fidelity DNA polymerase (NEB) for Q5 DNA polymerase, and cloned as described above. Plasmid clones were sequenced by nanopore whole plasmid sequencing and capillary sequencing (Eurofins, San Diego) and the mutations were identified and mapped. Plasmids encoding specific RNApol variants were then transformed into E. coli BL21 (NEB catalog no. C2530) for expression. The transformants were grown at 30°C to reach an OD600 of 0.5-0.7, induced with 0.025% L-arabinose, and incubated at 28°C for 5 hours. The E. coli cells were pelleted by centrifugation and lysed with sonication and lysozyme. The variant proteins were purified with Ni- affinity purification followed by protein concentration with Amicon filters (catalog no. UFC805096).

[0189] The KPIs mRNA yield and integrity are measured on a capillary electrophoresis instrument, for example, a Fragment Analyzer (Agilent Corporation catalog no. M5311AA) or by photometric methods as described in Ziegenhals 2023. The target yield is calculated from the concentration of the expected mRNA full-sized transcript band. Integrity is the percentage of the target mRNA band divided by the total RNA. Capping efficiency is determined by mass spectrometry and calculated using the capped mRNA divided by the uncapped RNA. Double- stranded (ds) RNA is measured in an independent enzyme linked immunosorbent assay (ELISA) or dot blot assay using a J2 antibody (Nordic-MUbio SCICONS anti-dsRNA (J2) Part Number RNT-SCI-10010500) that specifically recognizes dsRNA.

[0190] RNApol225 variants were tested in IVT reactions on a DNA templates. Table 5 and Table 8 summarizes the performance of RNA polymerase variants using a 5 kb double-stranded DNA template encoding a beta-galactosidase (LacZ) - firefly luciferase (luc) fusion protein with an AG TSS, showing total RNA yield, and RNA integrity compared to the parental RNApol225 enzyme. The yield data corresponds to total mRNA yield in a 0.02 ml in vitro transcription reaction (IVT). IVTs were incubated at 30°C for 2 hours and treated with DNase I. Yield and integrity of the IVT reactions were assessed with a Fragment Analyzer. RNA polymerase 91 NAI-5001873978variants show improved yield and integrity compared to the wild-type parental RNApol225 enzyme.

[0191] Table 5: Performance of single amino acid substitution variants on a 5 kb template Position Substitution Variant name RNA yield [μg / μl IVT]Integrity[%][amino acid substitutions using a 2 kb double-stranded DNA template encoding firefly luciferase (luc) protein with an AG TSS, showing total RNA yield, RNA integrity, dsRNA, and capping efficiency compared to the parental RNApol225 enzyme and T7 RNApol (Table 6). The yield data corresponds to total mRNA yield in a 0.2 ml in vitro transcription reaction (IVT). IVTs were incubated at 37 C for 2 hours in the presence of 1.5 mM CleanCap AG 3’OMe analogue (TriLink), treated with DNase I. Yield of RNA in purified samples was determined by photometric methods as described in Ziegenhals 2023. Integrity of the IVT reactions was assessed with a Fragment Analyzer, dsRNA with dot blots using J2 antibody, and capping % using mass spectrometry. RNA polymerase variants show improved yield, integrity, capping, or reduced dsRNA compared to the wild-type parental RNApol225 enzyme and T7 RNApol. 92 NAI-5001873978

[0193] Table 6: Performance of rationally designed variants in a 0.2 ml IVT reaction with a 2 kb firefly luciferase template and 1.5 mM cap analog present. Positions Substitutio Variant name RNA Integrit dsRNA Cappin ns yield y [%] [pg / μg g [%] RNA]

[0194] Activities were determined for RNApol225 variants with single and multiple-amino acid substitutions using a 2 kb double-stranded DNA template encoding firefly luciferase (luc) 93 NAI-5001873978protein with an AG TSS, showing total RNA yield, RNA integrity, dsRNA, and capping efficiency compared to the parental RNApol225 enzyme and T7 RNApol (Table 7). The yield data corresponds to total mRNA yield in a 0.2 ml in vitro transcription reaction (IVT). IVTs were incubated at 37°C for 2 hours in the presence of 3 mM CleanCap AG 3’OMe analogue (TriLink), treated with DNase I. Yield or the quantity of RNA obtained per unit volume of the IVT reaction, in purified samples was determined by photometric methods and integrity of the IVT reactions was assessed with a Fragment Analyzer, dsRNA with dot blots using J2 antibody, and capping % using mass spectrometry. RNA polymerase variants show improved yield, integrity, capping, or reduced dsRNA compared to the wild-type parental RNApol225 enzyme and T7 RNApol.

[0195] Table 7: Performance of rationally designed variants in a 0.2 ml IVT reaction with a 2 kb firefly luciferase template and 3 mM cap analog present. Positions Substitutions Variant name RNA Integrit dsRNA Cappin yield y [%] [pg / μg g [%]NAI-5001873978565, 576, 583, E483D, E487D, 618 L562V, E565D,

[0196] RNApol225 multiple substitution and single substitution variants were tested in IVT reactions on the 5 kb DNA template. Table 8 summarizes the performance of RNA polymerase variants using a 5 kb double-stranded DNA template encoding a beta-galactosidase (LacZ) - firefly luciferase (luc) fusion protein with an AG TSS, showing target RNA yield, and RNA integrity compared to the parental RNApol225 enzyme. The yield data corresponds to target mRNA yield in a 0.02 ml in vitro transcription reaction (IVT). IVTs were incubated at 30°C for 2 hours and treated with DNase I. Yield and integrity of the IVT reactions were assessed with a Fragment Analyzer. RNA polymerase variants show improved yield and integrity compared to the wild-type parental RNApol225 enzyme.

[0197] Table 8: Performance of rationally designed variants in a 0.02 ml IVT reaction with a 5 kb template and 1.5 mM cap analog present. RNA yield Ex rim nt P iti n S b tit ti n V ri nt n m [ / l Integrity95 NAI-5001873978166, 343, V166M, K343N, 350, 351, E350D, D351G,96 NAI-5001873978REFERENCES

[0198] Andries O, Mc Cafferty S, De Smedt SC, Weiss R, Sanders NN, Kitada T (2015). N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control Release 217:337-344.

[0199] Aramburu J, Navas-Castillo J, Moreno P, Cambra M (1991). Detection of double- stranded RNA by ELISA and dot immunobinding assay using an antiserum to synthetic polynucleotides. J Virol Methods 33(1-2):1-11.

[0200] Arnaud-Barbe N, Cheynet-Sauvion V, Oriol G, Mandrand B, Mallet F (1998). Transcription of RNA templates by T7 RNA polymerase. Nucleic Acids Res.26(15):3550-3554.

[0201] Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T (2001). Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Engineering 14 (8): 529-532.

[0202] Bagdasarian M, Lurz R, Rückert B, Franklin FC, Bagdasarian MM, Frey J, Timmis KN (1981). Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16(1- 3):237-247.

[0203] Baiersdörfer M, Boros G, Muramatsu H, Mahiny A, Vlatkovic I, Sahin U, Karikó K (2019). A Facile Method for the Removal of dsRNA Contaminant from In vitro-Transcribed mRNA. Mol Ther Nucleic Acids 15:26-35.

[0204] Boulain JC, Dassa J, Mesta L, Savatier A, Costa N, Muller BH, L’hostis G, Stura EA, Troesch A, Ducancel F (2013). Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase. Protein Eng Des Sel.6(11):725-34.

[0205] Burnett JC, Rossi JJ, Tiemann K (2011). Current progress of siRNA / shRNA therapeutics in clinical trials. Biotechnol J.6(9):1130-1146.

[0206] Butler ET, Chamberlin MJ (1982). Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme. J Biol Chem.257(10):5772-5778. 97 NAI-5001873978

[0207] Chang AC, Cohen SN (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 134(3):1141-1156.

[0208] Chelliserrykattil J, Ellington AD (2004). Evolution of a T7 RNA polymerase variant that transcribes 2’-O-methyl RNA. Nat Biotechnol.22(9):1155-1160.

[0209] Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31(13):3497-3500.

[0210] Cougot N, van Dijk E, Babajko S, Séraphin B (2004). ‘Cap-tabolism’. Trends Biochem Sci.29(8):436-444.

[0211] Darzynkiewicz E, Stepinski J, Ekiel I, Jin Y, Haber D, Sijuwade T, Tahara SM (1988). Beta-globin mRNAs capped with m7G, m2.7(2)G or m2.2.7(3)G differ in intrinsic translation efficiency. Nucleic Acids Res.16(18):8953-62.

[0212] Ge Q, Dallas A, Ilves H, Shorenstein J, Behlke MA, Johnston BH (2010). Effects of chemical modification on the potency, serum stability, and immunostimulatory properties of short shRNAs. RNA 16(1):118-130.

[0213] Di Grandi D, Dayeh DM, Kaur K, Chen Y, Henderson S, Moon Y, Bhowmick A, Ihnat PM, Fu Y, Muthusamy K, Palackal N, Pyles EA (2023). A single-nucleotide resolution capillary gel electrophoresis workflow for poly(A) tail characterization in the development of mRNA therapeutics and vaccines. J Pharm Biomed Anal.236:115692.

[0214] Dousis A, Ravichandran K, Hobert EM, Moore MJ, Rabideau AE (2023). An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts. Nat Biotechnol.41,560-568.

[0215] Gandhi V, O’Brien MH, Yadav S (2020). High-quality and high-yield RNA extraction method from whole human saliva. Biomark Insights.15:1177271920929705.

[0216] Gantier MP, Williams BR (2007). The response of mammalian cells to double- stranded RNA. Cytokine Growth Factor Rev.18(5-6):363-71. 98 NAI-5001873978

[0217] Ge Z, Mehta P, Richards J, Karzai AW (2010). Non-stop mRNA decay initiates at the ribosome. Mol Microbiol.78(5):1159-70.

[0218] Gholamalipour Y, Karunanayake MA, Martin CT (2018).3’ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character - RNA-Seq analyses. Nucleic Acids Res.;46(18):9253-9263.

[0219] Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods.6(5):343- 345.

[0220] Gibson DG, Smith HO, Hutchison CA 3rd, Venter JC, Merryman C. (2010). Chemical synthesis of the mouse mitochondrial genome. Nat Methods.7(11):901-903.

[0221] Gunter HM, Idrisoglu S, Singh S, Han DJ, Ariens E, Peters JR, Wong T, Cheetham SW, Xu J, Rai SK, Feldman R, Herbert A, Marcellin E, Tropee R, Munro T, Mercer TR (2023). mRNA vaccine quality analysis using RNA sequencing. Nat Commun.14(1):5663.

[0222] Hadi M, Stacy EA (2023). An optimized RNA extraction method for diverse leaves of Hawaiian Metrosideros, a hypervariable tree species complex. Appl Plant Sci.11(3):e11518.

[0223] Henderson JM, Ujita A, Hill E, Yousif-Rosales S, Smith C, Ko N, McReynolds T, Cabral CR, Escamilla-Powers JR, Houston ME (2021). Cap 1 messenger RNA synthesis with co- transcriptional CleanCap® analog by In vitro Transcription. Curr Protoc.1(2):e39.

[0224] Hornung V, Ellegast J, Kim S, Brzózka K, Jung A, Kato H, Poeck H, Akira S, Conzelmann KK, Schlee M, Endres S, Hartmann G (2006).5’-Triphosphate RNA is the ligand for RIG-I. Science 314(5801):994-997.

[0225] Ibach J, Dietrich L, Koopmans KR, Nöbel N, Skoupi M, Brakmann S (2013). Identification of a T7 RNA polymerase variant that permits the enzymatic synthesis of fully 2’- O-methyl-modified RNA. J Biotechnol.167(3):287-295.

[0226] Imburgio D, Rong M, Ma K, McAllister WT (2000). Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. Biochemistry 39(34):10419-10430. 99 NAI-5001873978

[0227] International Publication No. WO2024 / 211850.

[0228] Irwin CR, Farmer A, Willer DO, Evans DH (2012). In-fusion® cloning with vaccinia virus DNA polymerase. Methods Mol Biol.890:23-35.

[0229] Jackson AL, Burchard J, Leake D, Reynolds A, Schelter J, Guo J, Johnson JM, Lim L, Karpilow J, Nichols K, Marshall W, Khvorova A, Linsley PS (2006). Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12(7):1197- 1205.

[0230] Jeong J, Cho N, Jung D, Bang D (2013). Genome-scale genetic engineering in Escherichia coli. Biotechnol Adv.31(6):804-810.

[0231] Karikó K, Ni H, Capodici J, Lamphier M, Weissman D (2004). mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem.279(13):12542-12550.

[0232] Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D (2008). Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther.16(11):1833-1840

[0233] Karikó K, Muramatsu H, Ludwig J, Weissman D (2011). Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucl. Acids Res.39(21), e142.

[0234] Kole R, Krainer AR, Altman S (2012). RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat Rev Drug Discov.11(2):125-140.

[0235] Konarska MM, Padgett RA, Sharp PA (1984). Recognition of cap structure in splicing in vitro of mRNA precursors. Cell 38(3):731-736.

[0236] Kraynack BA, Baker BF (2005). Small interfering RNAs containing full 2’-O- methylribonucleotide-modified sense strands display Argonaute2 / eIF2C2-dependent activity. RNA 12(1):163-176. 100 NAI-5001873978

[0237] Kuhn AN, Bei ert T, Simon P, Vallazza B, Buck J, Davies BP, Tureci O, Sahin U(2012). mRNA as a versatile tool for exogenous protein expression. Curr Gene Ther.12(5):347- 361.

[0238] Layzer JM, McCaffrey AP, Tanner AK, Huang Z, Kay MA, Sullenger BA (2004). In vivo activity of nuclease-resistant siRNAs. RNA 10(5):766-771.

[0239] Lathe R, Kieny MP, Skory S, Lecocq JP (1984). Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini. DNA 3(2): 173-182

[0240] Lengyel P (1987). Double-stranded RNA and interferon action. J Interferon Res. 7(5):511-519.

[0241] Leprince A, van Passel MW, dos Santos VA (2012). Streamlining genomes: toward the generation of simplified and stabilized microbial systems. Curr Opin Biotechnol.23(5):651- 658.

[0242] Li MZ, Elledge SJ. (2007). Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods.4(3): 251-256.

[0243] Li C, Wen A, Shen B, Lu J, Huang Y, Chang Y. (2011). FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnol.11:92.

[0244] Li MZ, Elledge SJ. (2012). SLIC: a method for sequence- and ligation-independent cloning. Methods Mol Biol.852:51-59.

[0245] Lobban PE, Kaiser AD (1973). Enzymatic end-to end joining of DNA molecules. J Mol Biol.78(3): 453-471.

[0246] Lu X, Wu H, Xia H, Huang F, Yan Y, Yu B, Cheng R, Drulis-Kawa Z, Zhu B (2019). Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis. Front Microbiol. 10:2487. 101 NAI-5001873978

[0247] Madyagol M, Al-Alami H, Levarski Z, Drahovská H, Tur a J, Stuchlík S (2011).Gene replacement techniques for Escherichia coli genome modification. Folia Microbiol (Praha) 56(3):253-263.

[0248] Majde JA (2000). Viral double-stranded RNA, cytokines, and the flu. J Interferon Cytokine Res.20(3):259-272.

[0249] Majlessi M, Nelson NC, Becker MM (1998). Advantages of 2’-O-methyl oligoribonucleotide probes for detecting RNA targets. Nucleic Acids Res.26(9):2224-2229

[0250] Mansoor S, Baek M, Juergens D, Watson JL, Baker D (2023). Zero-shot mutation effect prediction on protein stability and function using RoseTTAFold. Protein Sci.32(11):e4780.

[0251] McGraw NJ, Bailey JN, Cleaves GR, Dembinski DR, Gocke CR, Joliffe LK, MacWright RS, McAllister WT (1985). Sequence and analysis of the gene for bacteriophage T3 RNA polymerase. Nucleic Acids Res.13(18):6753-6766.

[0252] Meier J, Rao R, Verkuil R, Liu J, Sercu T, Rives A (2021). Language models enable zero-shot prediction of the effects of mutations on protein function. bioRxiv. doi: https: / / doi.org / 10.1101 / 2021.07.09.450648

[0253] Meyer R, Figurski D, Helinski DR (1975). Molecular vehicle properties of the broad host range plasmid RK2. Science 190(4220):1226-1228.

[0254] Meyer AJ, Garry DJ, Hall B, Byrom MM, McDonald HG, Yang X, Yin YW, Ellington AD (2015). Transcription yield of fully 2’-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants. Nucleic Acids Res. 43(15):7480- 7488.

[0255] Monsion B, Incarbone M, Hleibieh K, Poignavent V, Ghannam A, Dunoyer P, Daeffler L, Tilsner J, Ritzenthaler C (2018). Efficient detection of long dsRNA in vitro and in vivo using the dsRNA binding domain from FHV B2 Protein. Front Plant Sci.9: 70.

[0256] Mu X, Greenwald E, Ahmad S, Hur S (2018). An origin of the immunogenicity of in vitro transcribed RNA. Nucleic Acids Res.46(10): 5239–5249. 102 NAI-5001873978

[0257] Nelson J, Sorensen EW, Mintri S, Rabideau AE, Zheng W, Besin G, Khatwani N, Su SV, Miracco EJ, Issa WJ, Hoge S, Stanton MG, Joyal JL (2020). Impact of mRNA chemistry and manufacturing process on innate immune activation. Sci Adv.2020 Jun 24;6(26):eaaz6893. doi: 10.1126 / sciadv.aaz6893. eCollection 2020 Jun.

[0258] Padilla R, Sousa R (2002). A Y639F / H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs. Nucleic Acids Res. 30(24):e138.

[0259] Pardi N, Weissman D (2017). Nucleoside Modified mRNA Vaccines for Infectious Diseases. Methods Mol Biol.1499:109-121.

[0260] Pasquinelli AE, Dahlberg JE, Lund E (1995). Reverse 5’ caps in RNAs made in vitro by phage RNA polymerases. RNA 1(9):957-967.

[0261] Potapov V, Fu X, Dai N, Corrêa IR Jr, Tanner NA, Ong JL (2018). Base modifications affecting RNA polymerase and reverse transcriptase fidelity. Nucleic Acids Res. 46(11):5753-5763.

[0262] Poveda C, Biter AB, Bottazzi ME, Strych U (2019). Establishing preferred product characterization for the evaluation of RNA vaccine antigens. Vaccines 7(4), 131.

[0263] Quan J, Tian J (2009). Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One.4(7): e6441.

[0264] Quan J, Tian J (2011). Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat Protoc.6(2):242-251.

[0265] Regalado, A (2015). The Next Great GMO Debate. MIT Technology Review Aug 11, 2015. Available on the technologyreview website.

[0266] Richter K, Gescher J (2012); The molecular toolbox for chromosomal heterologous multiprotein expression in Escherichia coli. Biochem Soc Trans.40(6):1222-1226.

[0267] Rose RE (1988). The nucleotide sequence of pACYC184. Nucleic Acids Res. 16(1):355. 103 NAI-5001873978

[0268] Rossbach M (2010). Small non-coding RNAs as novel therapeutics. Curr Mol Med. 10(4):361-368.

[0269] Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, Eliceiri KW (2017). ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics, 18(1). doi:10.1186 / s12859-017-1934-z

[0270] Sahin U, Karikó K, Türeci Ö (2014). mRNA-based therapeutics--developing a new class of drugs. Nat Rev Drug Discov.13(10):759-780.

[0271] Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, Plainview, New York.

[0272] Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Cardona A (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682.

[0273] Schmidhauser TJ, Filutowicz M, Helinski DR (1983). Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria. Plasmid 9(3):325-330.

[0274] Schmidhauser TJ, Helinski DR (1985). Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bacteriol.164(1):446-455.

[0275] Schneider CA, Rasband WS, Eliceiri, KW (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671–675.

[0276] Schönborn J, Oberstrass J, Breyel E, Tittgen J, Schumacher J, Lukacs N (1991). Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res.19(11):2993-3000.

[0277] Schweizer H (2008). Bacterial genetics: past achievements, present state of the field, and future challenges. Biotechniques 44(5):633-641.

[0278] Sergeeva OV, Koteliansky VE, Zatsepin TS (2016). mRNA-Based Therapeutics - Advances and Perspectives. Biochemistry (Mosc) 81(7):709-22. 104 NAI-5001873978

[0279] Shanmugasundaram M, Senthilvelan A, Anilkumar RK (2022). Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines. Chem Rec.22(8): e202200005.

[0280] Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M (1992). Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci U S A.89(18):8794-8797.

[0281] Siegmund V, Santner T, Micura R, Marx A (2012). Screening mutant libraries of T7 RNA polymerase for candidates with increased acceptance of 2’-modified nucleotides. Chem Commun (Camb).48(79):9870-9872.

[0282] Son KN, Liang Z, Lipton HL (2015). Double-stranded RNA Is detected by immunofluorescence analysis in RNA and DNA virus infections, including those by negative- stranded RNA viruses. J Virol.89(18):9383-9392.

[0283] Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD (1998). How cells respond to interferons. Annu Rev Biochem.67:227-264.

[0284] Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads RE (2001). Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(3’-O- methyl)GpppG and 7-methyl (3’-deoxy)GpppG. RNA 7(10):1486-1495.

[0285] Strenkowska M, Grzela R, Majewski M, Wnek K, Kowalska J, Lukaszewicz M, Zuberek J, Darzynkiewicz E, Kuhn AN, Sahin U, Jemielity J (2016). Cap analogs modified with 1,2-dithiodiphosphate moiety protect mRNA from decapping and enhance its translational potential. Nucleic Acids Res.44(20):9578-9590.

[0286] Studier FW, Moffatt BA (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol.189(1):113-130.

[0287] Thieme F, Engler C, Kandzia R, Marillonnet S (2011). Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non- specific mixes. PLoS One 6(6): e20556 105 NAI-5001873978

[0288] Tsygankov YD, Chistoserdov AY (1985). Specific-purpose broad-host-range vectors. Plasmid 14(2):118-125.

[0289] Tu Y, Das A, Redwood-Sawyerr C, Polizzi KM (2024). Capped or uncapped? Techniques to assess the quality of mRNA molecules. Curr. Opin. Systems Biol.37: 100503.

[0290] Vroom JA, Wang CL (2008). Modular construction of plasmids through ligation-free assembly of vector components with oligonucleotide linkers. Biotechniques 44(7): 924-926.

[0291] Wang R, Xue Y, Wu X, Song X, Peng J (2010). Enhancement of engineered trifunctional enzyme by optimizing linker peptides for degradation of agricultural by-products. Enzyme and Microb. Technol.47 (5): 194-199.

[0292] Warminski M, Mamot A, Depaix A, Kowalska J, Jemielity J (2023). Chemical Modifications of mRNA Ends for Therapeutic Applications. Acc. Chem. Res.56(20): 2814– 2826.

[0293] Warzak DA, Pike WA, Luttgeharm KD (2023). Capillary electrophoresis methods for determining the IVT mRNA critical quality attributes of size and purity. SLAS Technol. 28(5):369-374.

[0294] Webb C, Ip S, Bathula NV, Popova P, Soriano SKV, Ly HH, Eryilmaz B, Nguyen Huu VA, Broadhead R, Rabel M, Villamagna I, Abraham S, Raeesi V, Thomas A, Clarke S, Ramsay EC, Perrie Y, Blakney AK (2022). Current status and future perspectives on mRNA drug manufacturing. Mol Pharm.19(4):1047-1058.

[0295] Weber F, Wagner V, Rasmussen SB, Hartmann R, Paludan SR (2006). Double- stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol.80(10): 5059–5064.

[0296] Whitley J, Zwolinski C, Denis C, Maughan M, Hayles L, Clarke D, Snare M, Liao H, Chiou S, Marmura T, Zoeller H, Hudson B, Peart J, Johnson M, Karlsson A, Wang Y, Nagle C, Harris C, Tonkin D, Fraser S, Capiz L, Zeno CL, Meli Y, Martik D, Ozaki DA, Caparoni A, Dickens JE, Weissman D, Saunders KO, Haynes BF, Sempowski GD, Denny TN, Johnson MR (2022). Development of mRNA manufacturing for vaccines and therapeutics: mRNA platform 106 NAI-5001873978requirements and development of a scalable production process to support early phase clinical trials. Transl Res.242:38-55.

[0297] Wilson C, Keefe AD (2006). Building oligonucleotide therapeutics using non-natural chemistries. Curr Opin Chem Biol.10(6):607-614.

[0298] Yanisch-Perron C, Vieira J, Messing J (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33(1):103- 119.

[0299] Zangger H, Ronet C, Desponds C, Kuhlmann FM, Robinson J, Hartley MA, Prevel F, Castiglioni P, Pratlong F, Bastien P, Müller N, Parmentier L, Saravia NG, Beverley SM, Fasel N (2013). Detection of Leishmania RNA virus in Leishmania parasites. PLoS Negl Trop Dis. 7(1):e2006.

[0300] Zhu B, Cai G, Hall EO, Freeman GJ (2007). In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. BioTechniques 43:354-359.

[0301] Zhu B, Tabor S, Raytcheva DA, Hernandez A, King JA, Richardson CC (2013). The RNA polymerase of marine cyanophage Syn5. J Biol Chem.288(5):3545-3552.

[0302] Zhu B, Tabor S, Richardson CC (2014). Syn5 RNA polymerase synthesizes precise run-off RNA products. Nucleic Acids Res.42(5):e33.

[0303] Ziegenhals T, Frieling R, Wolf P, Göbel K, Koch S, Lohmann M, Baiersdörfer M, Fesser S, Sahin U, Kuhn AN. (2023) Formation of dsRNA by-products during in vitro transcription can be reduced by using low steady-state levels of UTP.10:1291045.

[0304] Ziemniak M, Strenkowska M, Kowalska J, Jemielity J (2013). Potential therapeutic applications of RNA cap analogs. Future Med Chem.5(10):1141-1172. 107 NAI-5001873978

Claims

CLAIMSWhat is claimed is:

1. A single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 1; and (b) at least one amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 15, 22, 25, 40, 49, 53, 64, 65, 70, 74, 91, 94, 127, 128, 139, 149, 153, 163, 166, 177, 178, 198, 203, 205, 208, 217, 222, 232, 236, 251, 258, 260, 269, 279, 291, 320, 343, 346, 350, 351, 357, 363, 383, 388, 413, 421, 426, 440, 457, 459, 471, 473, 479, 480, 483, 487, 494, 498, 520, 522, 523, 534, 542, 555, 562, 565, 567, 576, 581, 583, 598, 601, 610, 625, 632, 633, 638, 641, 661, 665, 669, 693, 694, 707, 713, 736, 740, 775, 786, 795, 820, 839, 843, and 867 of SEQ ID NO:

1.

2. The single-subunit RNA polymerase of claim 1, wherein the single-subunit RNA polymerase comprises at least one amino acid substitution corresponding to a substitution selected from the group consisting of: E15D, N22S, A25V, E40K, A49V, A49T, K53E, V64A, A65V, A70T, V74K, E91K, A94V, T127I, S128R, S139A, A149T, R153H, K163A, V166M, V177I, Y178C, G198N, S203N, H205Y, D208E, I217V, E222K, Q232R, V236I, A251T, A258V, A260V, Q269M, T279V, R291C, V320I, K343N, H346L, E350D, D351G, R357P, K363E, A383T, D388E, A413T, D421E, V426F, T440V, F457V, W459L, D471N, V473I, I479V, K480E, E483D, E487D, K494E, E498D, G520D, M522I, H523Y, L534P, G542V, G555V, L562V, E565D, V567P, K576R, I581L, Q583N, T598A, T601I, V610I, A625T, A625V, R632S, S633P, A638T, S641T, S661A, S661D, L665M, I669T, V693L, E694V, A707V, K713E, W736R, R740S, E775V, Q786L, V795I, D820N, C839N, A843V, and K867S of SEQ ID NO:

1.

3. The single-subunit RNA polymerase of claim 1 or claim 2, wherein the single-subunit RNA polymerase comprises at least two amino acid substitutions.

4. The single-subunit RNA polymerase of any one of claims 1-3, wherein the single-subunit 108 NAI-5001873978RNA polymerase comprises at least one amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 166, and 562 of SEQ ID NO:

1.

5. The single-subunit RNA polymerase of any one of claims 1-4, wherein the single-subunit RNA polymerase comprises an N-terminal His-tag.

6. The single-subunit RNA polymerase of any one of claims 1-5, wherein the RNA yield in an in vitro transcription (IVT) reaction with the single-subunit RNA polymerase is increased by 2% to 10000% compared to the RNA yield in an IVT reaction with an RNA polymerase of SEQ ID NO: 1 or T7 RNA polymerase.

7. The single-subunit RNA polymerase of claim 6, wherein the single-subunit RNA polymerase comprises at least one yield-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 70, 166, 383, 562, and 567 of SEQ ID NO:

1.

8. The single-subunit RNA polymerase of claim 7, wherein the single-subunit RNA polymerase comprises at least one yield-enhancing amino acid substitution corresponding to a substitution selected from the group consisting of: A25V, A70T, V166M, A383T, L562V, and V567P of SEQ ID NO:

1.

9. The single-subunit RNA polymerase of any one of claims 1-5, wherein the capping efficiency in an IVT reaction with the single-subunit RNA polymerase is increased by 2% to 10000% compared to the capping efficiency in an IVT reaction with an RNA polymerase of SEQ ID NO: 1 or T7 RNA polymerase.

10. The single-subunit RNA polymerase of claim 9, wherein the single-subunit RNA polymerase comprises at least one capping-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 74, 383, 567, 661, and 867 of SEQ ID NO:

1.

11. The single-subunit RNA polymerase of claim 10, wherein the single-subunit RNA polymerase comprises at least one capping-enhancing amino acid substitution corresponding to a substitution selected from the group consisting of: A25V, V74K, 109 NAI-5001873978A383T, V567P, S661D, and K867S of SEQ ID NO:

1.

12. The single-subunit RNA polymerase of any one of claims 1-5, wherein the quantity of double-stranded RNA (dsRNA) in an IVT reaction with the single-subunit RNA polymerase is decreased by 2% to 10000% compared to the quantity of double-stranded RNA in an IVT reaction with an RNA polymerase of SEQ ID NO: 1 or T7 RNA polymerase.

13. The single-subunit RNA polymerase of claim 12, wherein the single-subunit RNA polymerase comprises at least one dsRNA-reducing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 166, and 153 of SEQ ID NO:

1.

14. The single-subunit RNA polymerase of claim 13, wherein the single-subunit RNA polymerase comprises at least one dsRNA-reducing amino acid substitution corresponding to a substitution selected from the group consisting of: V166M, and R153H of SEQ ID NO:

1.

15. The single-subunit RNA polymerase of any one of claims 1-5, wherein the RNA integrity in an IVT reaction with the single-subunit RNA polymerase is increased by 2% to 10000% compared to the RNA integrity in an IVT reaction with an RNA polymerase of SEQ ID NO: 1 or T7 RNA polymerase.

16. The single-subunit RNA polymerase of any one of claims 1-5, wherein the cap utilization efficiency in an IVT reaction with the single-subunit RNA polymerase is increased by 2% to 10000% compared to the cap utilization efficiency in an IVT reaction with an RNA polymerase of SEQ ID NO: 1 or T7 RNA polymerase.

17. A single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and (b) at least one yield-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 70, 166, 383, 562, and 567 of SEQ ID NO:

1. NAI-500187397818. The single-subunit RNA polymerase of claim 17, wherein the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 25 of SEQ ID NO:

1.

19. The single-subunit RNA polymerase of claim 18, wherein the at least one yield-enhancing amino acid substitution corresponds to a substitution of A25V of SEQ ID NO:

1.

20. The single-subunit RNA polymerase of claim 17, wherein the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 70 of SEQ ID NO:

1.

21. The single-subunit RNA polymerase of claim 20, wherein the at least one yield-enhancing amino acid substitution corresponds to a substitution of A70T of SEQ ID NO:

1.

22. The single-subunit RNA polymerase of claim 17, wherein the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 166 of SEQ ID NO:

1.

23. The single-subunit RNA polymerase of claim 22, wherein the at least one yield-enhancing amino acid substitution corresponds to a substitution of V166M of SEQ ID NO:

1.

24. The single-subunit RNA polymerase of claim 17, wherein the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 383 of SEQ ID NO:

1.

25. The single-subunit RNA polymerase of claim 24, wherein the at least one yield-enhancing amino acid substitution corresponds to a substitution of A383T of SEQ ID NO:

1.

26. The single-subunit RNA polymerase of claim 17, wherein the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 562 of SEQ ID NO:

1.

27. The single-subunit RNA polymerase of claim 26, wherein the at least one yield-enhancing amino acid substitution corresponds to a substitution of L562V of SEQ ID NO:

1. 111 NAI-500187397828. The single-subunit RNA polymerase of claim 17, wherein the at least one yield-enhancing amino acid substitution is at sequence position corresponding to sequence position 567 of SEQ ID NO:

1.

29. The single-subunit RNA polymerase of claim 28, wherein the at least one yield-enhancing amino acid substitution corresponds to a substitution of V567P of SEQ ID NO:

1.

30. The single-subunit RNA polymerase of claim 17, wherein the single-subunit RNA polymerase comprises at least one yield-enhancing amino acid substitution corresponding to a substitution selected from the group consisting of: A25V, A70T, V166M, A383T, L562V, and V567P of SEQ ID NO:

1.

31. A single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and (b) at least one capping-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 74, 383, 567, 661, and 867 of SEQ ID NO:

1.

32. The single-subunit RNA polymerase of claim 31, wherein the at least one capping- enhancing amino acid substitution is at sequence position corresponding to sequence position 25 of SEQ ID NO:

1.

33. The single-subunit RNA polymerase of claim 32, wherein the at least one capping- enhancing amino acid substitution corresponds to a substitution of A25V of SEQ ID NO:

1.

34. The single-subunit RNA polymerase of claim 31, wherein the at least one capping- enhancing amino acid substitution is at sequence position corresponding to sequence position 74 of SEQ ID NO:

1.

35. The single-subunit RNA polymerase of claim 34, wherein the at least one capping- enhancing amino acid substitution corresponds to a substitution of V74K of SEQ ID NO:

1. 112 NAI-500187397836. The single-subunit RNA polymerase of claim 31, wherein the at least one capping- enhancing amino acid substitution is at sequence position corresponding to sequence position 383 of SEQ ID NO:

1.

37. The single-subunit RNA polymerase of claim 36, wherein the at least one capping- enhancing amino acid substitution corresponds to a substitution of A383T of SEQ ID NO:

1.

38. The single-subunit RNA polymerase of claim 31, wherein the at least one capping- enhancing amino acid substitution is at sequence position corresponding to sequence position 567 of SEQ ID NO:

1.

39. The single-subunit RNA polymerase of claim 38, wherein the at least one capping- enhancing amino acid substitution corresponds to a substitution of V567P of SEQ ID NO:

1.

40. The single-subunit RNA polymerase of claim 31, wherein the at least one capping- enhancing amino acid substitution is at sequence position corresponding to sequence position 661 of SEQ ID NO:

1.

41. The single-subunit RNA polymerase of claim 40, wherein the at least one capping- enhancing amino acid substitution corresponds to a substitution of S661D of SEQ ID NO:

1.

42. The single-subunit RNA polymerase of claim 40, wherein the at least one capping- enhancing amino acid substitution corresponds to a substitution of S661A of SEQ ID NO:

1.

43. The single-subunit RNA polymerase of claim 31, wherein the at least one capping- enhancing amino acid substitution is at sequence position corresponding to sequence position 867 of SEQ ID NO:

1.

44. The single-subunit RNA polymerase of claim 43, wherein the at least one capping- enhancing amino acid substitution corresponds to a substitution of K867S of SEQ ID NO:

1. NAI-45. The single-subunit RNA polymerase of claim 31, wherein the single-subunit RNA polymerase comprises at least one capping-enhancing amino acid substitution corresponding to a substitution selected from the group consisting of: A25V, V74K, A383T, V567P, S661D, S661A, and K867S of SEQ ID NO:

1.

46. A single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: (a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and (b) at least one dsRNA-reducing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 166, 153, and 567 of SEQ ID NO:

1.

47. The single-subunit RNA polymerase of claim 46, wherein the at least one dsRNA-reducing amino acid substitution is at sequence position corresponding to sequence position 166 of SEQ ID NO:

1.

48. The single-subunit RNA polymerase of claim 47, wherein the at least one dsRNA-reducing amino acid substitution corresponds to a substitution of V166M of SEQ ID NO:

1.

49. The single-subunit RNA polymerase of claim 46, wherein the at least one dsRNA-reducing amino acid substitution is at sequence position corresponding to sequence position 153 of SEQ ID NO:

1.

50. The single-subunit RNA polymerase of claim 49, wherein the at least one dsRNA-reducing amino acid substitution corresponds to a substitution of R153H of SEQ ID NO:

1.

51. The single-subunit RNA polymerase of claim 46, wherein the single-subunit RNA polymerase comprises at least one dsRNA-reducing amino acid substitution corresponding to a substitution selected from the group consisting of: V166M, R153H, and V567P of SEQ ID NO:

1.

52. A single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises: 114 NAI-5001873978(a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; (b) at least one yield-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 70, 166, 383, 562, and 567 of SEQ ID NO: 1; (c) at least one capping-enhancing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 25, 74, 383, 567, 661, and 867 of SEQ ID NO: 1; and (d) at least one dsRNA-reducing amino acid substitution at a sequence position corresponding to a sequence position selected from the group consisting of: 166, 153, and 567 of SEQ ID NO:

1.

53. The single-subunit RNA polymerase of claim 52, wherein the at least one yield-enhancing amino acid substitution corresponds to a substitution of L562V of SEQ ID NO: 1, and at least one dsRNA-reducing amino acid substitution corresponds to a substitution of V166M of SEQ ID NO:

1.

54. The single-subunit RNA polymerase of claim 52, wherein the at least one yield-enhancing amino acid substitution corresponds to a substitution of L562V of SEQ ID NO: 1, the at least one capping-enhancing amino acid substitution corresponds to a substitution of S661D of SEQ ID NO: 1, and the at least one dsRNA-reducing amino acid substitution corresponds to a substitution of V166M of SEQ ID NO:

1.

55. A single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises amino acid substitutions corresponding to the following positions of SEQ ID NO: 1: V166M, K343N, D351G, I479V, K494E, E498D, L562V, I581L, G618D, and S661A.

56. A single-subunit RNA polymerase, wherein the single-subunit RNA polymerase comprises amino acid substitutions corresponding to the following positions of SEQ ID NO: 1: V166M, K343N, E350D, D351G, I479V, E498D, L562V, I581L, G618D, and S661A.

57. A nucleic acid composition encoding the single-subunit RNA polymerase of any one of claims 1-56. 115 NAI-500187397858. A vector composition comprising the nucleic acid composition of claim 57.

59. A host cell comprising the nucleic acid composition of claim 57 or the vector composition of claim 58.

60. A kit comprising the single-subunit RNA polymerase of any one of claims 1-56. 116 NAI-5001873978

Images