Docket No.11555-013WO1 METHODS OF PREPARING A CATALYST FOR RNA PRODUCTION CROSS REFERENCE TO RELATED APPLICATIONS [1] This application claims the benefit of priority to U.S. Provisional Application No. 63/593,687, filed October 27, 2023, which is incorporated by reference herein in its entirety. GOVERNMENT RIGHTS IN INVENTION [2] This invention was made with government support under 1R01GM134042 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING [3] The sequence listing submitted on October 25, 2024, as an .XML file entitled “11555-013WO1_ST26.xml” created on October 24, 2024, and having a file size of 76,138 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). TECHNICAL FIELD [4] The present application relates to methods of preparing a catalyst configured to produce RNA from a functional template DNA without the need for amplification of the functional template DNA, and more particularly to methods of preparing such catalysts from plasmid DNA and from enzymatically synthesized DNA. BACKGROUND ART [5] The synthesis / manufacturing of RNA is mostly carried out with two primary components (an RNA polymerase and an RNA-encoding DNA) free in solution. Some implementations immobilize the RNA-encoding DNA to a solid support, but retain the RNA polymerase enzyme free in solution. Both approaches suffer from the problem that accumulating free RNA begins to compete with promoter DNA to bind the free RNA polymerase. This re-binding can lead to the synthesis of double stranded RNA impurities. [6] Functionally co-tethering RNA polymerase to RNA-encoding DNA (this co- complex optionally bound to a solid support) favors promoter re-binding and allows initiation of transcription at salt concentrations that reduce RNA re-binding and that reduce the
Docket No.11555-013WO1 generation of dsRNA contaminants. Such an approach, as applied to RNA-encoding DNA produced by PCR and using chemically modified synthetic oligonucleotide PCR primers, is described in U.S. Patent No. 11,578,348, which is hereby incorporated by reference in its entirety. [7] In the manufacturing of RNA, linearized plasmid DNA is widely used as the RNA-encoding DNA and is generally preferred over RNA-encoding DNA produced by amplification, e.g., PCR. Plasmid DNA generated in cultured cells is generally less expensive and has higher fidelity than DNA prepared through many rounds of PCR amplification. PCR amplification also becomes increasingly challenging with increasing length of the RNA encoding DNA and so is less commonly used for manufacture of very long RNAs. Segmented polyA tails are now routinely accommodated in plasmid-based DNA templates. [8] More recently, enzymatically synthesized DNA, produced by enzymatic methods that do not involve amplification, have also become an attractive alternative to plasmid DNA. SUMMARY [9] In accordance with one embodiment, there is provided a method of preparing a catalyst for RNA production configured to produce RNA from an RNA-encoding sequence of a functional template DNA, the method comprising: providing the functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and the RNA-encoding sequence, the RNA-encoding sequence being downstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus, wherein the 5’ terminus of the nontemplate strand extends past the 3’ terminus of the template strand, thereby forming a first 5’ overhang; enzymatically adding nucleotides to the 3’ terminus of the template strand by filling in the first 5’ overhang, wherein a nucleotide added to the 3’ terminus of the template strand comprises a first handle; and functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA.
Docket No.11555-013WO1 [10] In accordance with another embodiment, there is provided a method of preparing a catalyst for RNA production configured to produce RNA from an RNA-encoding sequence of a functional template DNA, the method comprising: providing the functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and the RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus, wherein the 3’ terminus of the template strand extends past the 5’ terminus of the nontemplate strand, thereby forming a first 3’ overhang; enzymatically adding nucleotides to the 3’ terminus of the template strand using a terminal transferase, wherein a nucleotide added to the 3’ terminus of the template strand comprises a first handle; and functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA. [11] In accordance with one embodiment, there is provided a method of preparing a catalyst for RNA production configured to produce RNA from an RNA-encoding sequence of a functional template DNA, the method comprising: providing the functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and the RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus; and ligating a first adapter, comprising a first handle, to the 3’ terminus of the template strand and the 5’ terminus of the nontemplate strand, wherein an RNA polymerase is functionally coupled to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA.
Docket No.11555-013WO1 [12] In accordance with another embodiment, there is provided a method of preparing a catalyst for RNA production configured to produce RNA from an RNA-encoding sequence of a functional template DNA, the method comprising: providing the functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a promoter sequence and the RNA-encoding sequence, the RNA- encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand, enzymatically nicking a site of the nontemplate strand using a nicking endonuclease, thereby generating a gap in the nontemplate strand, the gap being a single stranded portion of the template strand without a corresponding complementary portion of the nontemplate strand, wherein the site is within a region of the nontemplate strand selected from the group consisting of: (a) a region of the nontemplate strand complementary to, and base paired with, the promoter sequence, (b) 1–100 nucleotides upstream of a sequence of the nontemplate strand complementary to, and base paired with, the promoter sequence, and (c) combinations thereof; and hybridizing an invading nucleic acid molecule to the single stranded portion of the template strand, the invading nucleic acid molecule being complementary to the single stranded portion of the template strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA. [13] In accordance with one embodiment, there is provided a method of preparing a catalyst for RNA production configured to produce RNA from an RNA-encoding sequence of a functional template DNA, the method comprising: providing the functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a promoter sequence and the RNA-encoding sequence, the RNA- encoding sequence being upstream of, and operably linked to, the promoter sequence; and
Docket No.11555-013WO1 (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand, enzymatically nicking a site of the template strand using a nicking endonuclease, thereby generating a gap in the template strand, the gap being a single stranded portion of the nontemplate strand without a corresponding complementary portion of the template strand, wherein the site is within a region of the template strand selected from the group consisting of: (a) a region of the promoter sequence, (b) 1–100 nucleotides upstream of the promoter sequence, and (c) combinations thereof; and hybridizing an invading nucleic acid molecule to the single stranded portion of the nontemplate strand, the invading nucleic acid molecule being complementary to the single stranded portion of the nontemplate strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA. [14] In some embodiments, the nicking endonuclease is a Cas9 nickase. [15] In some embodiments, the functional template DNA is a plasmid DNA or an enzymatically produced DNA synthesized in vitro. In some embodiments, the enzymatically produced DNA synthesized in vitro is produced by enzymatic methods that do not involve amplification. The functional template DNA may be a digestion product. [16] The template strand may comprise a 5’ terminus and the nontemplate strand may comprise a 3’ terminus. In some embodiments, the 5’ terminus of the template strand extends past the 3’ terminus of the nontemplate strand thereby forming a second 5’ overhang, the method further comprising enzymatically adding nucleotides to the 3’ terminus of the nontemplate strand thereby filling in the second 5’ overhang, wherein a nucleotide added to the 3’ terminus of the nontemplate strand comprises a second handle. In some embodiments, a 3’ terminal nucleotide added to the 3’ terminus of the nontemplate strand is a dideoxynucleotide. [17] In some embodiments, the method further comprises enzymatically adding nucleotides to the 3’ terminus of the nontemplate strand using terminal transferase, wherein a nucleotide added to the 3’ terminus of the nontemplate strand comprises a second handle. [18] In other embodiments, the method further comprises ligating a second adapter to the 3’ terminus of the nontemplate strand and the 5’ terminus of the template strand, the second adapter comprising a second handle.
Docket No.11555-013WO1 [19] The invading nucleic acid molecule may comprise a second handle. In some embodiments, the invading nucleic acid molecule comprises a modified nucleotide. The modified nucleotide may be a locked nucleic acid nucleotide or a peptide nucleic acid nucleotide. In some embodiments, the method further comprises—after hybridizing the invading nucleic acid molecule—ligating a terminus of the invading nucleic acid molecule to an adjacent terminus of a nicked strand of the functional template DNA. [20] In some embodiments, the first handle of the catalyst is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides upstream of a start of the RNA encoding sequence. The first handle of the catalyst may be 21 nucleotides upstream of the start of the RNA encoding sequence. [21] In some embodiments, the RNA polymerase is a T-phage RNA polymerase. The T-phage RNA polymerase may be a T7 RNA polymerase, a T3 RNA polymerase, a K11 RNA polymerase, a SP6 RNA polymerase, a Syn5 RNA polymerase, or a variant of any of the foregoing. [22] The first handle may comprise a moiety selected from the group consisting of an alkyl halide, O6-benzylguanine, O2-benzylcytosine, organoarsenic, trimethoprim ligand, and biotin. In some embodiments, the first handle is bifunctional. [23] The RNA polymerase may comprise a tag domain, configured to bind the first handle, selected from the group consisting of a HaloTag domain, an AviTag domain, a SNAP- Tag domain, FlAsH-Tag domain, ReAsH-Tag domain, a TMP-Tag domain, and a CLIP-Tag domain. BRIEF DESCRIPTION OF THE DRAWINGS [24] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [25] The foregoing features of embodiments will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which: [26] Fig. 1A is an illustration of a double-stranded DNA having a template strand and a nontemplate strand, the 5’ terminus of the nontemplate strand functionally coupled to a RNA polymerase, forming a catalyst for RNA production. The catalyst for RNA production may optionally be coupled to a solid support (such as the surface of a material, for example, the surface of a bead, a hydrogel, or other material known to those of skill in the art, now and
Docket No.11555-013WO1 in the future, for use in a flow reactor), either at the 5’ terminus of the nontemplate strand, or at the 5’ terminus of the template strand, in accordance with embodiments of the invention. Fig.1B is an illustration of a double-stranded DNA having a template strand and a nontemplate strand, the 3’ terminus of the nontemplate strand functionally coupled to a RNA polymerase, forming a catalyst for RNA production. The catalyst for RNA production may optionally be coupled to a solid support, either at the 3’ terminus of the nontemplate strand, or at the 3’ terminus of the template strand, in accordance with embodiments of the invention. [27] Fig.2A is an illustration of a functional template DNA having a template strand and a nontemplate strand, a 3’ terminus of the template strand functionally coupled to the N- terminal domain of a RNA polymerase forming a catalyst for RNA production, a 3’ terminus of the nontemplate strand being coupled to a solid support, in accordance with embodiments of the invention. The RNA polymerase comprises a HaloTag domain and is coupled to a first handle (e.g., comprising a ligand such as an alkyl halide configured to bind the HaloTag domain) at the 3’ terminus of the template strand. A second handle (e.g., comprising biotin) at the 3’ terminus of the non-template strand couples the catalyst for RNA production to a streptavidin of a solid support. Fig. 2B is an illustration of a functional template DNA having a template strand and a nontemplate strand, a 3’ terminus of the template strand functionally coupled (here, indirectly) to the N-terminal domain of a RNA polymerase forming a catalyst for RNA production, the 3’ terminus of the template strand being coupled to a solid support, in accordance with embodiments of the invention. The RNA polymerase comprises an AviTag and is coupled to a streptavidin of a solid support, the streptavidin being coupled to a first handle (e.g., comprising biotin) at the 3’ terminus of the template strand. Fig. 2C is an illustration of a functional template DNA having a template strand and a nontemplate strand, a 3’ terminus of the template strand functionally coupled to the N-terminal domain of a RNA polymerase forming a catalyst for RNA production, the 3’ terminus of the template strand also being coupled to a solid support, in accordance with embodiments of the invention. The RNA polymerase comprises a HaloTag domain and is coupled to a bifunctional first handle (e.g., comprising a ligand such as an alkyl halide configured to bind the HaloTag domain) at the 3’ terminus of the template strand. The bifunctional first handle further comprises biotin and couples the catalyst for RNA production to a streptavidin of a solid support. [28] Figs. 3A-3F show a schematic showing an exemplary method of creating a catalyst for RNA production from a plasmid functional template DNA, in accordance with embodiments of the invention. The plasmid DNA (Fig. 3A) is first digested with restriction enzyme BspQ1 (Figs. 3B-D), generating a digestion product having 5’ overhangs (Fig. 3E).
Docket No.11555-013WO1 The digestion product comprises a template strand and a nontemplate strand. The 5’ overhangs are then filled-in using Klenow polymerase, incorporating a modified nucleotide comprising a first handle (aminoallyl dUTP or alkyl-Cl dUTP) into the template strand of the digestion product, and a modified nucleotide comprising a second handle (biotin-16-dCTP) into the nontemplate strand of the digestion product (Fig.3F). The alkyl-Cl first handle of the template strand may then be coupled to a T7 RNA polymerase comprising a HaloTag domain. Alternatively, the aminoallyl first handle of the template strand may be derivatized with an alkyl halide and coupled to the T7 RNA polymerase comprising a HaloTag domain. The biotin second handle of the nontemplate strand may be coupled to streptavidin of a solid support. Sequences in Figs.3A-3F are provided in Table 1. Table 1. Sequences in Figs.3A-3F.
Docket No.11555-013WO1 [29] Fig 4A is a PAGE gel demonstrating production of a 70 nucleotide (“nt”) RNA using a catalyst for RNA production prepared by the method exemplified in Figs.3A-3F, and using an alkyl-Cl first handle, in accordance with embodiments of the invention. The biotin second handle was coupled to streptavidin-coated magnetic beads prior to RNA production. Fig.4B is a PAGE gel demonstrating the reusability and stability of the same catalyst for RNA production after 10 repeat rounds of production of the 70 nucleotide RNA, in accordance with embodiments of the invention. Immobilization of the catalyst to the streptavidin-coated magnetic beads allows product solution to be removed and fresh reagents to be re-supplied for additional (repeat) rounds of RNA synthesis from the same bead-immobilized catalyst for RNA production). [30] Fig 5A is an agarose gel demonstrating production of an 851 nucleotide mRNA encoding nanoluciferase using a catalyst for RNA production prepared by the method exemplified in Figs. 3A-3F, and using an alkyl-Cl first handle, in accordance with embodiments of the invention. Fig. 5B are agarose gels demonstrating the reusability and stability of the same catalyst for RNA production after 1, 2, 3, and 4 repeat rounds of production of the 851 nucleotide mRNA encoding nanoluciferase, in accordance with embodiments of the invention. [31] Fig. 6A is a schematic illustrating, including RNA synthesis conditions, a catalyst for RNA production prepared from a plasmid functional template DNA used to produce a 2.4 kb mRNA encoding a GFP-Fluc fusion protein, in accordance with embodiments of the invention. Fig 6B is an agarose gel demonstrating production of the 2.4 kb (2,393 nucleotides) mRNA encoding a GFP-Fluc fusion protein using the catalyst for RNA production, which was prepared by the method exemplified in Figs.3A-3F, and using an alkyl- Cl first handle, in accordance with embodiments of the invention. Fig. 6C is an agarose gel demonstrating the reusability and stability of the same catalyst for RNA production after 1, 2, 3, 4, 5, 6, 7, 8, and 9 repeat rounds of production of the 2.4 kb mRNA encoding the GFP-Fluc fusion protein, in accordance with embodiments of the invention. Fig. 6D is a PAGE gel showing successful translation of the 2.4 kb mRNA encoding the GFP-Fluc fusion protein in a wheat germ extract, in accordance with embodiments of the invention. The 80 kD GFP-Fluc fusion protein can be seen in the last lane of the gel (controls include 72 kD and 60 kD markers, the latter expressed in the same wheat germ extract system). [32] Fig.7A is a crystal structure of a promoter-bound T7 RNA polymerase initiation complex (PDB 1QLN) with upstream DNA modeled as a B-form duplex. The location of a
Docket No.11555-013WO1 HaloTag domain is estimated by overlaying the HaloTag C-terminus near the polymerase N- terminus, as constructed genetically. Distances are indicated to the polymerase N-terminus, but of course it is the distances (likely shorter) to the Halo-Tag active site that matters. Fig. 7B is a gel showing transcription products produced using a catalyst for RNA production created from a plasmid functional template DNA and having a T7 RNA polymerase coupled (tethered) to a first handle that has been incorporated into a template strand at different positions upstream of the transcription start site, as indicated at the top of the gel, in accordance with embodiments of the invention. [33] Fig. 8 shows derivatization of an aminoallyl handle with an alkyl halide to produce an alkylated amino allyl handle, which may be coupled to a HaloTag domain of an RNA polymerase, in accordance with embodiments of the invention. [34] Figs. 9A-9F show a schematic showing an exemplary method of creating a catalyst for RNA production from a plasmid functional template DNA, in accordance with embodiments of the invention. The plasmid (Fig.9A) is first digested with restriction enzymes Bmt I and NotI (Fig. 9B), generating a digestion product having a 3’ overhang upstream of a promoter and an RNA-encoding sequence downstream of the promoter. In addition, a 5’ overhang is generated downstream of the RNA-encoding sequence. The digestion product comprises a template strand and a nontemplate strand. The 5’ overhang is then filled-in using Klenow polymerase, incorporating a modified nucleotide comprising a second handle (biotin- dCTP) and ddGTP (3’ deoxy GTP) into the nontemplate strand of the product (Fig.9C and Fig. 9E). Next, a modified nucleotide comprising a first handle (NH
2-dUTP) is added to the terminus of the 3’ overhang using terminal transferase (Fig.9D). The amine at the 3’ terminus may then be derivatized with an alkyl halide and coupled to an RNA polymerase comprising a HaloTag domain. Alternatively, a modified nucleotide comprising an alkyl-Cl dUTP first handle may be added to the terminus of the 3’ overhang using terminal transferase and directly coupled to an RNA polymerase having a HaloTag domain (not shown). The biotin second handle of the nontemplate strand may be coupled to streptavidin of a solid support. Sequences in Figs.9A-9F are provided in Table 2. Table 2. Sequences in Figs.9A-9F.
Docket No.11555-013WO1
[35] Fig 10 is an agarose gel demonstrating production of a 2.4 kb (2,393 nucleotides) mRNA encoding a GFP-Fluc fusion protein using a catalyst for RNA production prepared by the method exemplified in Figs.9A-9F. [36] Figs. 11A-11F show a schematic showing an exemplary method of creating a catalyst for RNA production from a plasmid functional template DNA by ligation of a universal adapter, in accordance with embodiments of the invention. Here, a first DNA adapter comprising a first handle is ligated to restriction-enzyme-cut plasmid DNA upstream of a promoter of an RNA-encoding sequence. (Fig.11A) Starting plasmid DNA may be cut with a restriction enzyme at a unique restriction site upstream of the promoter, and at a site downstream of the RNA-encoding sequence (using a different restriction enzyme). (Fig.11B) Cleavage leaves “sticky ends.” (Fig. 11C) A DNA adapter containing a first handle R is then ligated to the sticky end upstream of the promoter. (Fig.11D) As before, e.g., Figs.3A-3F, the first handle may be coupled to an RNA polymerase or derivatized before the coupling to the RNA polymerase. A second adapter comprising a second handle may similarly be ligated to the sticky end downstream of the RNA-encoding sequence. The second handle may, for example, comprise biotin, which can be coupled to a streptavidin of a solid support. Such adapters are “universal” in that they can be used with functional template DNA encoding any RNA. Such an adapter may be referred to as a universal adapter. (Fig.11E) An agarose gel is shown demonstrating production of a 2.4 kb (2,393 nucleotides) mRNA encoding a GFP-Fluc fusion protein using a catalyst for RNA production from a plasmid functional template DNA prepared by the method exemplified in Figs. 11A-11D using T7 RNA polymerase (either uncoupled to the first handle, or coupled to the first handle using a HaloTag domain). The
Docket No.11555-013WO1 relatively lower expression from the latter likely reflects that the first handle is at position -18, which may not be ideal. (Fig. 11F) A universal adapter can be either a single oligonucleotide containing a hairpin or two complementary nucleic acid strands. Sequences in Figs. 11A-11F are provided in Table 3. Table 3. Sequences in Figs.11A-11F.
Docket No.11555-013WO1 [37] Figs. 12A-12C show a schematic showing an exemplary method of creating a catalyst for RNA production from a functional template DNA using an invading DNA approach for incorporating a first handle and a second handle. Here, a custom sequence (Fig. 12A), containing recognition sites for multiple “nicking” endonucleases (Nb.BtsI and Nb.BssSI) which, after nicking, generates a product having a large gap in one strand, is engineered immediately upstream of a promoter sequence. (Fig. 12B) DNA complementary to the gap (referred to herein as a “universal reagent”) is derivatized with a first handle (e.g., an alkyl halide for coupling to HaloTag T7 RNA polymerase) and a second handle (e.g., biotin for coupling to a streptavidin of a solid support), the first handle coupled to an RNA polymerase comprising a HaloTag domain and the second handle coupled to a solid support (such as the surface of a material, for example, the surface of a bead, a hydrogel, or other material known to those of skill in the art, now and in the future, for use in a flow reactor) to produce a catalyst for RNA production. Use of such a universal reagent requires only a specific sequence upstream of the promoter and is universal to any downstream RNA-encoding sequence. (Fig. 12C) An agarose gel demonstrates successful batch synthesis of a 24 nucleotide RNA from the catalyst for RNA production. Sequences in Figs.12A-12C are provided in Table 4. Table 4. Sequences in Figs.12A-12C.
[38] Fig. 13A is an illustration of a scheme wherein a universal reagent hybridizes to a gapped DNA sequence immediately upstream of a promoter, in accordance with embodiments of the invention. Fig. 13B is an illustration of a scheme wherein a universal reagent hybridizes to a gapped DNA sequence overlapping a promoter, in accordance with embodiments of the invention.
Docket No.11555-013WO1 DETAILED DESCRIPTION [39] Disclosed herein are methods of preparing a catalyst for RNA production by incorporating “handles” into a functional template DNA, e.g., plasmid DNA, thereby facilitating coupling of an RNA polymerase to the functional template DNA (and optionally immobilization of the catalyst for RNA production to a solid support), obviating the need for PCR amplification and the need for chemically modified synthetic oligonucleotide PCR primers in generating templates for RNA production at scale. The methods and systems described herein allow for repeated synthesis of RNA from a functional template DNA template in vitro and at scale, while avoiding problems related to double stranded RNA impurities, and other problems related PCR-derived templates, which have limits on their length and are prone to the introduction of sequence errors during amplification. DEFINITIONS [40] As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires: [41] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. [42] A “template strand” of a double-stranded nucleic acid molecule is a DNA strand that enters an RNA polymerase active site and serves as a template for polymerization of RNA. [43] A “nontemplate strand” of a double-stranded nucleic acid molecule is a DNA strand complementary to the template strand. [44] A “promoter” or “promoter sequence” means a sequence of DNA to which one or more proteins bind, for example, a DNA-dependent RNA polymerase, to initiate transcription of a RNA transcript from an RNA-encoding sequence of DNA operably linked to the promoter. A DNA sequence of a template strand that is transcribed into a RNA transcript is a “RNA-encoding sequence.” [45] The term “polynucleotide” is interchangeable with nucleic acid and includes any compound and/or substance that comprise a polymer of nucleotides. Exemplary polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic
Docket No.11555-013WO1 acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ^-D-ribo configuration, ^- LNA having an ^-L-ribo configuration (a diastereomer of LNA), 2^-amino-LNA having a 2^- amino functionalization, and 2^-amino-^-LNA having a 2^-amino functionalization) or hybrids thereof. Polynucleotides may include naturally occurring nucleotides as well as modified nucleotides such as pseudouridine 1-N-pseudouridine, dye-labeled nucleotides, biotin-labeled nucleotide, and others. [46] A “functional template” refers to a double-stranded polynucleotide including a RNA-encoding sequence operably linked to a promoter sequence. A “functional template DNA” is a double stranded DNA molecule (e.g., a plasmid DNA or other double stranded DNA molecule, including enzymatically produced double-stranded DNA synthesized in vitro) having a RNA-encoding sequence operably linked to a promoter sequence. [47] “Operably linked” refers to a functional connection between two or more polynucleotide sequences. For example, an RNA-encoding sequence operably linked to a promoter allows binding of an RNA polymerase to the promoter and subsequent transcription of RNA from the RNA-encoding sequence by the RNA polymerase. [48] Any number of DNA-dependent RNA polymerases or variants may be used in the methods described herein. The polymerase may be selected from, but is not limited to, a T- phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, an SP6 RNA polymerase, a K11 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids, polymerases showing reduced abortive cycling, and polymerases with increased thermostability. As used herein, the terms a T7 RNA polymerase, a T3 RNA polymerase, a K11RNA polymerase, and an SP6 RNA polymerase include both wild type, mutant and truncated polymerases, so long as RNA polymerase activity is maintained. [49] RNA polymerases may be modified by inserting or deleting amino acids of the RNA polymerase sequence. As a non-limiting example, the RNA polymerase may be modified to exhibit an increased ability to incorporate a 2^-modified nucleotide triphosphate compared to an unmodified RNA polymerase (see International Publication WO2008078180 and U.S. Pat. No. 8,101,385; herein incorporated by reference in their entireties). Variants may be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and/or nucleic acid sequence and/or by using other methods known in the art. [50] “Downstream” means in a direction of transcription, the direction of transcription being from a promoter sequence to a RNA-encoding sequence. For a template
Docket No.11555-013WO1 strand of a double-stranded DNA molecule, the direction of transcription is 3’ to 5’. For a nontemplate strand of the double-stranded DNA molecule, the direction of transcription is 5’ to 3’. “Upstream” means in a direction opposite the direction of transcription. “Upstream” and “downstream” may be used in reference to either strand of a double-stranded DNA molecule even when relative to a sequence on one strand of a double-stranded DNA molecule. [51] “Filling in,” or performing a “fill in” reaction, and the like, means enzymatically adding nucleotides to a 3’ terminus of a first nucleic acid molecule, the added nucleotides being complementary to a single-stranded 5’ overhang of a second nucleic acid molecule. A 5’ overhang is a single-stranded region at the 5’ terminus of a one strand of a double-stranded nucleic acid molecule that extends beyond its complementary strand, here, the first strand. Filling in a 5’ overhang is accomplished using a DNA polymerase and dNTPs, under conditions suitable for DNA polymerization, as known in the art. The polymerase preferably lacks or substantially lacks 5’ to 3’ exonuclease activity, e.g., Klenow fragment. The dNTPs comprise at least one nucleoside triphosphate selected from the group consisting of dATP, dTTP, dCTP, dGTP, dUTP, and derivatives thereof. The dNTPs may comprise a nucleoside triphosphate that has been modified to include a chemical moiety, such as a handle. [52] A “handle” means a chemical moiety of a nucleic acid molecule that is configured to be coupled to a molecule other than the nucleic acid molecule. The chemical moiety may be a modification, such as a functional group, that has been introduced into a nucleotide of the nucleic acid. A “first handle” is a handle comprising a moiety configured to functionally couple an RNA polymerase to a functional template DNA. In some embodiments, the catalyst comprises a “second handle,” the second handle being a handle comprising a moiety configured to couple catalyst for RNA production to a solid support, e.g., a material surface, for example, the surface of a bead, a hydrogel, or other material known to those of skill in the art, now and in the future, for use in a repeat batch or flow reactor. In some embodiments, the first handle may be bifunctional, being further configured to couple a catalyst for RNA production to a solid support. [53] In some embodiments, a first handle comprises an alkyl halide moiety, e.g., alkyl-Cl, configured to bind the HaloTag domain of an RNA polymerase comprising a HaloTag domain. In other embodiments, a first handle comprises an O
6-benzylguanine moiety configured to bind the SNAP-Tag domain of an RNA polymerase comprising a SNAP-Tag domain. In some embodiments, a first handle comprises an O
2-benzylcytosine moiety configured to bind the CLIP-Tag domain of an RNA polymerase comprising a CLIP-Tag domain. In other embodiments, a first handle comprises an organoarsenic moiety configured
Docket No.11555-013WO1 to bind the FlAsH-Tag domain of an RNA polymerase comprising a FlAsH-Tag domain. In some embodiments, a first handle comprises a trimethoprim ligand moiety configured to bind the TMP-Tag domain of an RNA polymerase comprising a TMP-Tag domain. In other embodiments, a first handle comprises a biotin moiety configured to bind an intermediate streptavidin, the intermediate streptavidin bound to the AviTag domain of an RNA polymerase comprising an AviTag domain. Moieties suitable for first handles, along with their associated protein tags, are described in Wang et al. RSC Advances Issue 14 (2014) doi: 10.1039/C3RA46991C, which is hereby incorporated by reference in its entirety. Other chemical moieties suitable for a first handle, along with their corresponding protein tag domains, known to those of skill in the art now and in the future, are also contemplated as part of the present disclosure. [54] In some embodiments, a second handle comprises a biotin moiety configured to bind a streptavidin of a streptavidin-coated solid support. In other embodiments, a second handle comprises a moiety having a primary amine configured to react with an aldehyde of a solid support, thereby coupling the second handle to the solid support. Other chemical moieties suitable for a second handle, along with their corresponding solid support couplings, known to those of skill in the art now and in the future, including but not limited to click chemistries, are also contemplated as part of the present disclosure. [55] A “solid support” means a material surface for use in a flow reactor, as is known to those of skill in the art, now and in the future. Not limiting examples of a solid support include a bead (magnetic or otherwise) surface and a hydrogel surface. [56] “Functionally coupling,” and the like, means that, after coupling, a DNA retains its ability to be transcribed by an RNA polymerase. For example, with respect to a first handle, functionally coupling means coupling an RNA polymerase to a first handle of a nucleic acid molecule, e.g., a functional template DNA, such that the RNA polymerase retains its activity and can transcribe RNA from an RNA-encoding sequence of the functional template DNA, under conditions suitable for transcription, as known in the art. [57] “Terminal deoxynucleotidyl transferase,” or “terminal transferase,” is a DNA polymerase that does not require a template, and which catalyzes the addition of nucleotides to the 3’ terminus of a DNA strand. The preferred substrate of terminal transferase is a 3'- overhang, although it can also add nucleotides to blunt or recessed 3’ ends. Nucleotides may be added to a 3’ terminus of a DNA strand using a terminal transferase and dNTPs, under conditions suitable for terminal transferase activity, as known in the art. The dNTPs comprise at least one nucleoside triphosphate selected from the group consisting of dATP, dTTP, dCTP,
Docket No.11555-013WO1 dGTP, dUTP, and derivatives thereof. The dNTPs may comprise a nucleoside triphosphate that has been modified to include a handle. [58] As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant of an RNA polymerase comprising one or more changes in amino acid residues as compared to a wild type RNA polymerase amino acid sequence. The term “variant” encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence. [59] An “adapter” is a double-stranded nucleic acid molecule, 8–50 nucleotides in length, which is configured to be enzymatically ligated, e.g., using DNA ligase under conditions suitable for ligation, as known in the art, to a target double-stranded nucleic acid molecule, such as a functional template DNA. An adapter may have a blunt end, a 5’ overhang, or a 3’ overhang. A blunt end of the adapter may be ligated to a blunt end of the target double- stranded nucleic acid molecule. A 5’ overhang of the adapter may be ligated to a 3’ overhang of the target double-stranded nucleic acid molecule. A 3’ overhang of the adapter may be ligated to a 5’ overhang of the target double-stranded nucleic acid molecule. An adapter may comprise a handle. [60] “Enzymatically digesting” means digesting a nucleic acid molecule with a restriction enzyme. A nucleic acid produced by digesting the nucleic acid molecule is a “digestion product.” [61] An “invading nucleic acid molecule” is a single-stranded nucleic acid molecule, 8–100 nucleotides in length, comprising a first handle. In some embodiments, the first handle is functionally coupled to an RNA polymerase. In some embodiments, the invading nucleic acid molecule comprises a second handle. The invading nucleic acid molecule is complementary to, and configured to hybridize to, a sequence upstream of (or overlapping) the promoter of one strand of a functional template DNA. [62] A key advantage of using an invading nucleic acid molecule, as disclosed herein, is that a “universal” reagent comprised of the invading DNA functionally coupled to an RNA polymerase, and optionally coupled to a solid or other flow support, can be used to generate RNA of any sequence. The only sequence restriction on a functional template DNA is that it have a cleavable target sequence upstream of (or overlapping) the promoter (outside
Docket No.11555-013WO1 of an RNA-encoding region) that is suitable for preparation for hybridization with an invading nucleic acid molecule. [63] A universal reagent may be prepared at high volume (and high purity), ready to be used to produce RNA of any sequence. Minimal digestion of a functional template DNA is required to prepare it for invasion, i.e., hybridization to a complementary single-stranded sequence upstream of (or overlapping) the promoter of one strand of the functional template DNA. [64] Use of a universal reagent avoids the need for fill-in or terminal transferase reactions and subsequent purification of the reaction product. Preparation of functional template DNA would involve only simple digestion of the functional template DNA with appropriate enzymes. METHODS [65] Fig. 1A is an illustration of a double-stranded DNA having a template strand and a nontemplate strand, the 5’ terminus of the nontemplate strand functionally coupled to a RNA polymerase, forming a catalyst for RNA production. The catalyst for RNA production may optionally be coupled to a solid support, either at the 5’ terminus of the nontemplate strand, or at the 5’ terminus of the template strand, in accordance with embodiments of the invention. Fig.1B is an illustration of a double-stranded DNA having a template strand and a nontemplate strand, the 3’ terminus of the nontemplate strand functionally coupled to a RNA polymerase, forming a catalyst for RNA production. The catalyst for RNA production may optionally be coupled to a solid support, either at the 3’ terminus of the nontemplate strand, or at the 3’ terminus of the template strand, in accordance with embodiments of the invention. [66] The double-stranded DNA is comprised of a nontemplate strand and a complementary template strand. The template strand enters the RNA polymerase active site and serves as a template for polymerization of RNA (RNA polymerase transcribes in a “downstream” direction). In Fig. 1A, incorporation of a first handle at the 5’ terminus of the nontemplate strand functionally couples the RNA polymerase to the double-stranded DNA. In Fig.1B, incorporation of a first handle at the 3’ terminus of the nontemplate strand functionally couples the RNA polymerase to the double-stranded DNA. RNA polymerase is coupled to the double-stranded DNA at the upstream terminus of the double-stranded DNA in order to localize the RNA polymerase near a promoter. Immobilization of the double-stranded DNA to a solid support may be achieved at either terminus (either directly from a second handle of a terminal nucleotide or from a handle of a nucleotide near, e.g., within 20 nucleotides of, the terminal
Docket No.11555-013WO1 nucleotide), as illustrated (though it is also possible to immobilize the polymerase to a support, not shown). [67] In accordance with one embodiment, there is provided a method of preparing a catalyst for RNA production configured to produce RNA from an RNA-encoding sequence of a functional template DNA, the method comprising: providing the functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and the RNA-encoding sequence, the RNA-encoding sequence being downstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus, wherein the 5’ terminus of the nontemplate strand extends past the 3’ terminus of the template strand, thereby forming a first 5’ overhang; enzymatically adding nucleotides to the 3’ terminus of the template strand by filling in the first 5’ overhang, wherein a nucleotide added to the 3’ terminus of the template strand comprises a first handle; and functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA. [68] In accordance with another embodiment, there is provided a method of preparing a catalyst for RNA production configured to produce RNA from an RNA-encoding sequence of a functional template DNA, the method comprising: providing the functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and the RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus, wherein the 3’ terminus of the template strand extends past the 5’ terminus of the nontemplate strand, thereby forming a first 3’ overhang;
Docket No.11555-013WO1 enzymatically adding nucleotides to the 3’ terminus of the template strand using a terminal transferase, wherein a nucleotide added to the 3’ terminus of the template strand comprises a first handle; and functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA. [69] In accordance with one embodiment, there is provided a method of preparing a catalyst for RNA production configured to produce RNA from an RNA-encoding sequence of a functional template DNA, the method comprising: providing the functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and the RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus; and ligating a first adapter, comprising a first handle, to the 3’ terminus of the template strand and the 5’ terminus of the nontemplate strand, wherein an RNA polymerase is functionally coupled to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA. [70] In accordance with another embodiment, there is provided a method of preparing a catalyst for RNA production configured to produce RNA from an RNA-encoding sequence of a functional template DNA, the method comprising: providing the functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a promoter sequence and the RNA-encoding sequence, the RNA- encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand, enzymatically nicking a site of the nontemplate strand using a nicking endonuclease, thereby generating a gap in the nontemplate strand, the gap being a single stranded portion of the template strand without a corresponding complementary portion of the nontemplate strand,
Docket No.11555-013WO1 wherein the site is within a region of the nontemplate strand selected from the group consisting of: (a) a region of the nontemplate strand complementary to, and base paired with, the promoter sequence, (b) 1–100 nucleotides upstream of a sequence of the nontemplate strand complementary to, and base paired with, the promoter sequence, and (c) combinations thereof; and hybridizing an invading nucleic acid molecule to the single stranded portion of the template strand, the invading nucleic acid molecule being complementary to the single stranded portion of the template strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA. [71] In accordance with one embodiment, there is provided a method of preparing a catalyst for RNA production configured to produce RNA from an RNA-encoding sequence of a functional template DNA, the method comprising: providing the functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a promoter sequence and the RNA-encoding sequence, the RNA- encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand, enzymatically nicking a site of the template strand using a nicking endonuclease, thereby generating a gap in the template strand, the gap being a single stranded portion of the nontemplate strand without a corresponding complementary portion of the template strand, wherein the site is within a region of the template strand selected from the group consisting of: (a) a region of the promoter sequence, (b) 1–100 nucleotides upstream of the promoter sequence, and (c) combinations thereof; and hybridizing an invading nucleic acid molecule to the single stranded portion of the nontemplate strand, the invading nucleic acid molecule being complementary to the single stranded portion of the nontemplate strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA.
Docket No.11555-013WO1 [72] In some embodiments, the site is within a region of the template strand that is at least 1 nucleotide (e.g., at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides) upstream of the promoter sequence. In some embodiments, the site is within a region of the template strand that is up to 100 nucleotides (e.g., up to 95 nucleotides, up to 90 nucleotides, up to 85 nucleotides, up to 80 nucleotides, up to 75 nucleotides, up to 70 nucleotides, up to 65 nucleotides, up to 60 nucleotides, up to 55 nucleotides, up to 50 nucleotides, up to 45 nucleotides, up to 40 nucleotides, up to 35 nucleotides, up to 30 nucleotides, up to 25 nucleotides, up to 20 nucleotides, up to 15 nucleotides, up to 10 nucleotides, up to 9 nucleotides, up to 8 nucleotides, up to 7 nucleotides, up to 6 nucleotides, up to 5 nucleotides, up to 4 nucleotides, up to 3 nucleotides, up to 2 nucleotides, up to 1 nucleotide) upstream of the promoter sequence. [73] It is considered that the site can be within a region of the template strand that is a number of nucleotides upstream of the promoter sequence ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the site is within a region of the template strand that is 1 to 100 nucleotides (e.g., 2 to 95 nucleotides, 3 to 90 nucleotides, 4 to 85 nucleotides, 5 to 80 nucleotides, 6 to 75 nucleotides, 7 to 70 nucleotides, 8 to 65 nucleotides, 9 to 60 nucleotides, 10 to 55 nucleotides, 15 to 50 nucleotides, 20 to 45 nucleotides, 25 to 40 nucleotides, 30 to 35 nucleotides, 1 to 35 nucleotides, 2 to 30 nucleotides, 3 to 25 nucleotides, 4 to 20 nucleotides, 5 to 15 nucleotides, 6 to 10 nucleotides, 7 to 9 nucleotides, 30 to 100 nucleotides, 35 to 95 nucleotides, 40 to 90 nucleotides, 45 to 85 nucleotides, 50 to 80 nucleotides, 55 to 75 nucleotides, 60 to 70 nucleotides) upstream of the promoter sequence. [74] In some embodiments, the nicking endonuclease is a Cas9 nickase. [75] In some embodiments, the functional template DNA is a plasmid DNA or an enzymatically produced DNA synthesized in vitro. In some embodiments, the enzymatically produced DNA synthesized in vitro is produced by enzymatic methods that do not utilize DNA amplification. The functional template DNA may be a digestion product.
Docket No.11555-013WO1 [76] The template strand may comprise a 5’ terminus and the nontemplate strand may comprise a 3’ terminus. In some embodiments, the 5’ terminus of the template strand extends past the 3’ terminus of the nontemplate strand thereby forming a second 5’ overhang, the method further comprising enzymatically adding nucleotides to the 3’ terminus of the nontemplate strand thereby filling in the second 5’ overhang, wherein a nucleotide added to the 3’ terminus of the nontemplate strand comprises a second handle. In some embodiments, a 3’ terminal nucleotide added to the 3’ terminus of the nontemplate strand is a nucleotide analog, for example, a dideoxynucleotide or a canonical nucleotide comprising any of the disclosed handles. [77] In some embodiments, the method further comprises enzymatically adding nucleotides to the 3’ terminus of the nontemplate strand using terminal transferase, wherein a nucleotide added to the 3’ terminus of the nontemplate strand comprises a second handle. [78] In other embodiments, the method further comprises ligating a second adapter to the 3’ terminus of the nontemplate strand and the 5’ terminus of the template strand, the second adapter comprising a second handle. [79] The invading nucleic acid molecule may comprise a second handle. In some embodiments, the invading nucleic acid molecule comprises a modified nucleotide. The modified nucleotide may be a locked nucleic acid nucleotide or a peptide nucleic acid nucleotide. In some embodiments, the method further comprises—after hybridizing the invading nucleic acid molecule—ligating a terminus of the invading nucleic acid molecule to an adjacent terminus of a nicked strand of the functional template DNA. [80] In some embodiments, the first handle of the catalyst is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides upstream of a start of the RNA encoding sequence. The first handle of the catalyst may be 21 nucleotides upstream of the start of the RNA encoding sequence. [81] In some embodiments, the RNA polymerase is a T-phage RNA polymerase or related single subunit. The T-phage RNA polymerase may be a T7 RNA polymerase, a T3 RNA polymerase, a K11 RNA polymerase, a SP6 RNA polymerase, a Syn5 RNA polymerase, or a variant of any of the foregoing. [82] The first handle may comprise a moiety selected from the group consisting of an alkyl halide, O6-benzylguanine, O2-benzylcytosine, organoarsenic, trimethoprim ligand, and biotin. In some embodiments, the first handle is bifunctional. [83] The RNA polymerase may comprise a tag domain, configured to bind the first handle, selected from the group consisting of a HaloTag domain, an AviTag domain, a SNAP-
Docket No.11555-013WO1 Tag domain, FlAsH-Tag domain, ReAsH-Tag domain, a TMP-Tag domain, and a CLIP-Tag domain. [84] In some embodiments, RNA-encoding sequence is at least 50 nucleotides (e.g., at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1,000 nucleotides, at least 1,500 nucleotides, at least 2,000 nucleotides, at least 2,500 nucleotides, at least 3,000 nucleotides, at least 3,500 nucleotides, at least 4,000 nucleotides, at least 4,500 nucleotides, at least 5,000 nucleotides, at least 5,500 nucleotides, at least 6,000 nucleotides, at least 6,500 nucleotides, at least 7,000 nucleotides, at least 7,500 nucleotides, at least 8,000 nucleotides, at least 8,500 nucleotides, at least 9,000 nucleotides, at least 9,500 nucleotides, at least 10,000 nucleotides) in length. In some embodiments, the RNA-encoding sequence is up to 10,000 nucleotides (e.g., up to 9,500 nucleotides, up to 9,000 nucleotides, up to 8,500 nucleotides, up to 8,000 nucleotides, up to 7,500 nucleotides, up to 7,000 nucleotides, up to 6,500 nucleotides up to 6,000 nucleotides, up to 5,500 nucleotides, up to 5,000 nucleotides, up to 4,500 nucleotides, up to 4,000 nucleotides, up to 3,500 nucleotides, up to 3,000 nucleotides, up to 2,500 nucleotides, up to 2,000 nucleotides, up to 1,500 nucleotides, up to 1,000 nucleotides, up to 900 nucleotides, up to 800 nucleotides, up to 700 nucleotides, up to 600 nucleotides, up to 500 nucleotides, up to 450 nucleotides, up to 400 nucleotides, up to 350 nucleotides, up to 300 nucleotides, up to 250 nucleotides, up to 200 nucleotides, up to 150 nucleotides, up to 100 nucleotides, up to 50 nucleotides) in length. [85] It is considered that the RNA-encoding sequence can be a number of nucleotides in length ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the RNA-encoding sequence is from 50 to 10,000 nucleotides (e.g., from 100 to 9,500 nucleotides, from 150 to 9,000 nucleotides, from 200 to 8,500 nucleotides, from 250 to 8,000 nucleotides, from 300 to 7,500 nucleotides, from 350 to 7,000 nucleotides, from 400 to 6,500 nucleotides, from 450 to 6,000 nucleotides, from 500 to 5,500 nucleotides, from 600 to 5,000 nucleotides, from 700 to 4,500 nucleotides, from 800 to 4,000 nucleotides, from 900 to 3,500 nucleotides, from 1,000 to 3,000 nucleotides, from 1,500 to 2,500 nucleotides, from 50 to 2,500 nucleotides, from 100 to 2,000 nucleotides, from 150 to 1,500 nucleotides, from 200 to 1,000 nucleotides, from 250 to 900 nucleotides, from 300 to 800 nucleotides, from 350 to 700 nucleotides, from 400 to 600 nucleotides, from 450 to 500 nucleotides, from 2,000 to 10,000 nucleotides, from 2,500 to
Docket No.11555-013WO1 9,500 nucleotides, from 3,000 to 9,000 nucleotides, from 3,500 to 8,500 nucleotides, from 4,000 to 8,000 nucleotides, from 4,500 to 7,500 nucleotides, from 5,000 to 7,000 nucleotides, from 5,500 to 6,500 nucleotides) in length. [86] The methods disclosed herein for preparing a catalyst for RNA production from a functional template DNA are merely exemplary. It is contemplated as part of the present disclosure that the methods disclosed herein for introducing a first handle and a second handle into plasmid DNA may be used in various combinations suitable for preparing a catalyst for RNA production. Example 1: Fill-in Reaction for Generation of a Catalyst for Production of RNA from Plasmid DNA [87] Here, a study was conducted which used BspQI restriction digestion of plasmids, each having a promoter driving expression of RNA of various lengths from an RNA- encoding sequence, followed by fill-in reactions in order to add a first handle and a second handle to the digested plasmid DNA, the first handle being functionally coupled to an RNA polymerase thereby forming a catalyst for RNA production. The second handle may be coupled to a solid support, for example a bead. Although the present example functionally couples T7 RNA polymerase comprising a HaloTag domain to a first handle comprising an alkyl halide (Fig.2A), other RNA polymerases known in the art, now and in the future, may be functionally coupled to the first handle. Moreover, other methods known in the art may be used to functionally couple the RNA polymerase to the first handle. For example, a polymerase having an AviTag may be coupled to the first handle by an intermediate streptavidin (Fig.2B). In some embodiments, the first handle may be bifunctional (Fig.2C). [88] The study designed plasmids having RNA-encoding sequences of various lengths with a first BspQI recognition sequence upstream of a promoter and a second BspQI recognition sequence downstream of the corresponding RNA-encoding sequence, as shown in Fig.3A. [89] Digestion of plasmid DNA with BspQI creates a product having a 5’ overhang at each cleaved BspQI recognition sequence (Fig. 3B–D). The sequences within these overhanging regions were chosen so that a DNA polymerase one pot “fill in” reaction would uniquely incorporate a dU base comprising a first handle in the upstream overhang, and a dC base comprising a second handle in the downstream overhang (Fig. 3E–F). Other restriction enzyme sequences known in the art, now and in the future, may also be used as would be appreciated by one of ordinary skill in the art.
Docket No.11555-013WO1 [90] In some embodiments, a fill-in reaction may be used to uniquely incorporate a dC base comprising a first handle in the upstream overhang, and a dU base comprising a second handle in the downstream overhang. Although any base comprising a first handle or second handle may be used, the study used dU and dC because variants of these bases containing appropriate handles are commercially available or can readily be generated from commercially available precursors. [91] Unless otherwise stated, the experiments disclosed in these examples functionally couple T7 RNA polymerase comprising a HaloTag domain to a first handle comprising alkyl chloride (of incorporated alkyl-Cl-dUTP). In some embodiments, an aminoallyl handle may be derivatized with an alkyl halide to produce an alkylated amino allyl handle, which may then be coupled to a HaloTag domain of an RNA polymerase. Other RNA polymerase coupling moieties known to those of skill in the art, now and in the future, are also contemplated as part of the present disclosure for use as first handles. [92] To prepare alkyl-Cl dUTP, the study reacted Halo succinimidyl ester (O4) with amino-allyl-dUTP (both commercially available), as shown in Fig.8. An identical reaction may be carried out with an amino allyl-labeled DNA. [93] In addition, a second handle comprising biotin (of an incorporated biotin-16- dCTP) was coupled to streptavidin coated beads. Other solid support surface coupling moieties known to those of skill in the art, now and in the future, are also contemplated as part of the present disclosure for use as second handles. [94] Klenow fragment was used for all fill-in reactions. In some embodiments, other suitable DNA polymerases known in the art, now and in the future, e.g., Taq DNA polymerase, phi29 DNA polymerase, and DNA polymerase I, may be used to perform fill-in reactions. [95] Production of 70mer RNA from synthetic DNA [96] Fig.4A shows a gel demonstrating successful batch synthesis of a 70-nucleotide RNA (70mer) using a catalyst for RNA production prepared (as exemplified in Fig. 3A–F) from a plasmid functional template DNA having a RNA-encoding sequence encoding the 70mer. Plasmid DNA was used directly, without PCR amplification, to create the catalyst for RNA production. A reduction in higher molecular weight impurities was observed with increasing RNA synthesis salt concentrations. Fig. 4B is a gel demonstrating efficient repeat- batch synthesis (10 repeat rounds) of the 70mer, with a high level of purity, indicating that the catalyst for RNA production is stable and may be reused for repeated RNA syntheses.
Docket No.11555-013WO1 [97] Production of 851 nucleotide RNA from plasmid DNA [98] Fig. 5A shows a gel demonstrating successful batch synthesis of an 851- nucleotide mRNA encoding nanoluciferase using a catalyst for RNA production prepared (as exemplified in Fig.3A–F) from a plasmid functional template DNA having an 851-nucleotide RNA-encoding sequence encoding the nanoluciferase mRNA. Plasmid DNA was used directly, without PCR amplification, to create the catalyst for RNA production. Fig. 5B is a gel demonstrating efficient repeat-batch synthesis (1, 2, 3, and 4 repeat rounds) of the nanoluciferase mRNA from the catalyst for RNA production, indicating that the catalyst for RNA production is stable and reusable. [99] Production of 2.4 kb RNA from plasmid DNA [100] Fig. 6A is a schematic, including RNA synthesis conditions, illustrating a catalyst for RNA production prepared from a plasmid functional template DNA used to produce a 2.4 kb mRNA encoding a GFP-firefly luciferase (Fluc) fusion protein. Fig.6B shows a gel demonstrating successful batch synthesis of a 2.4 kb (2,393 nucleotides) mRNA encoding a GFP-Fluc fusion protein using the catalyst for RNA production created (as exemplified in Fig. 3A–F) from the plasmid functional template DNA having a 2.4 kb RNA-encoding sequence encoding the GFP-Fluc mRNA. Plasmid DNA was used directly, without PCR amplification, to create the catalyst for RNA production. The GFP-Fluc mRNA was transcribed with CleanCAP AG and comprises a poly(A) tail. Fig. 6C is a gel demonstrating efficient repeat-batch synthesis (1, 2, 3, 4, 5, 6, 7, 8, and 9 repeat rounds) of the GFP-Fluc mRNA from the catalyst for RNA production, indicating that the catalyst for RNA production is stable and reusable. Since the GFP-Fluc mRNA was transcribed with CleanCAP AG and comprises a poly(A) tail, translatability of the GFP-Fluc mRNA was demonstrated by in vitro translation of the GFP-Fluc mRNA in a wheat germ extract, followed by denaturing gel electrophoresis (Fig.6D). The 80 kD protein band provides evidence that the GFP-Fluc mRNA was translated into GFP-Fluc fusion protein. Translatability of the GFP-Fluc mRNA was also confirmed in transfected induced Bone Marrow Derived Macrophages cells using a luciferase activity assay (data not shown). [101] Production of 8 kb RNA from plasmid DNA [102] The study also successfully produced an 8 kb mRNA encoding an ELYS- emerin-EGFP fusion protein from a catalyst for RNA production prepared by the method disclosed above for production of GFP-Fluc mRNA.
Docket No.11555-013WO1 Example 2: Optimization of First Handle Position [103] For the RNA polymerases used in Example 1, above, a HaloTag domain is genetically fused to T7 RNA polymerase via the HaloTag C-terminus joined to T7 RNA polymerase. Although there is no crystal structure for this HaloTag-T7 RNA polymerase, a study was conducted which approximated a structural model of the HaloTag-T7 RNA polymerase functionally coupled to a first handle of a catalyst for RNA production. The model, shown in Fig.7A, brings the two termini in close overlap, with the HaloTag domain rotated to allow proximity to the DNA upstream of a promoter (the HaloTag domain structure shows a fluorescent probe anchored at the coupling site). The placement of a first handle (functionally coupled to the HaloTag-T7 RNA polymerase) at (or very near) the upstream terminus of the DNA likely provides flexibility in connectivity, as the ends of the DNA will “breathe” dynamically. In contrast, incorporating a first handle relatively farther from the upstream terminus likely places more constraints on the coupling of the first handle to the HaloTag-T7 RNA polymerase. [104] In Fig.7A, DNA upstream of position -17 is modeled as B-form DNA. One can see that the major groove from position -18 to -22 generally points in the direction in which the Halo-Tag domain is expected to reside. Distances between major groove elements (chemistries used currently derivatize base functionalities in the major groove) and the N- terminus of T7 RNA polymerase are shown. Note that the distances shown in grey pass sterically through the bases. In other words, the major groove is pointing away from the N- terminus at some positions. Also note that the relevant distance is from the DNA attachment site to the HaloTag domain active site. This modeling suggests a shorter distance for this. [105] The study then determined, empirically, the optimal location for incorporating a first handle to functionally couple to a HaloTag T7 RNA polymerase. The study prepared several DNA constructs (having a promoter driving expression of an RNA-encoding sequence) with the PEG-alkly-chloride at the positions shown in Fig. 7B, each construct directing the transcription of the same 34 base RNA encoded by the RNA-encoding sequence. Transcription from the coupled constructs was then carried out at either 0 M or 0.4 M added NaCl. The former allows for strong (native) promoter binding, while 0.4 M added NaCl weakens binding. [106] Fig. 7B is a gel showing transcription products produced using a catalyst for RNA production created from a plasmid functional template DNA and having a T7 RNA polymerase coupled (tethered) to a first handle that has been incorporated into a template strand at different positions upstream of the transcription start site, as indicated at the top of the gel. These results suggest that coupling of T7 RNA polymerase to a first handle at position at -21
Docket No.11555-013WO1 of a template strand of the catalyst for RNA production results in optimal production of RNA from the catalyst. Although other first handle positions show significant transcription at a low salt concentration, they are more inhibited at a higher salt concentration, suggesting that a strain in the coupling effectively weakens binding of the T7 RNA polymerase to the DNA (as evidenced by higher sensitivity to salt). In the experiments that follow, the study introduced a first handle at position -21, i.e., 21 bases upstream of a given transcription start site, of the template strand of a plasmid functional template DNA. Example 3: Terminal Transferase for Generation of a Catalyst for Production of RNA from Plasmid DNA [107] An alternative approach to introduce a first and/or second handle into plasmid functional template DNA is to add a similarly modified nucleotide, e.g., alkyl-Cl-dU or biotin- dC, to a 3’ end of a linearized plasmid DNA using a terminal transferase enzyme. This enzyme adds a substrate dNTP to blunt or recessed DNA, but has a preference for 3’ overhangs. [108] As shown in Figure 9A-B, a study was conducted which prepared a plasmid functional template DNA (having a promoter configured to drive expression of a downstream sequence encoding a GFP-Fluc fusion mRNA) by digesting the plasmid with restriction enzymes BmtI and NotI. The former generates a 3’ overhang, while the latter generates a 5’ overhang. The 5’ overhang was then filled-in using Klenow polymerase, incorporating a modified nucleotide comprising a second handle (biotin-dCTP) and ddGTP (3’ deoxy GTP) into a nontemplate strand of the prepared plasmid DNA (Figs. 9C and 9E). ddGTP was incorporated into the nontemplate strand to prevent terminal transferase from acting on the 3’ terminus of the nontemplate strand in the following step. A modified nucleotide comprising a first handle (NH2-dUTP) was added to the terminus of the 3’ overhang using terminal transferase (Figs.9D and 9F). The amine at the 3’ terminus was then derivatized with an alkyl halide and functionally coupled to a T7 RNA polymerase comprising a HaloTag domain. The resulting catalyst for RNA production was coupled to streptavidin coated beads and batch RNA synthesis was performed. [109] Fig.10 shows a gel demonstrating successful batch synthesis of a 2.4 kb (2,393 nucleotides) mRNA encoding a GFP-Fluc fusion protein using the catalyst for RNA production created by the method illustrated in Figs. 9A–F from a plasmid functional template DNA having a 2.4 kb RNA-encoding sequence encoding the GFP-Fluc mRNA by the method shown in Figs.9A–F.
Docket No.11555-013WO1 Example 4: Adapter Ligation for Generation of a Catalyst for Production of RNA from Plasmid DNA [110] Another study was conducted which developed another method of preparing a catalyst for RNA production prepared from a plasmid functional template DNA that includes ligation of an adapter to digested plasmid DNA, the adapter comprising a first handle. In some embodiments, a second adapter comprising a second handle is also ligated to the digested plasmid DNA. [111] Here, a first DNA adapter comprising two paired nucleic acid strands and having a first handle was ligated to restriction-enzyme-cut plasmid DNA upstream of a promoter of an RNA-encoding sequence (encoding a GFP-Fluc fusion mRNA). In some embodiments, a single oligonucleotide comprising a handle (first or second handle) may be used as an adapter, e.g., as shown in Fig. 11F for a second adapter having a second handle. Starting plasmid DNA was cut with a restriction enzyme at a unique restriction site upstream of the promoter, and at a site downstream of the RNA-encoding sequence (using a different restriction enzyme) (Fig. 11A), creating a digestion product having “sticky ends” (Fig. 11B). A DNA adapter containing a first handle R (alkyl-Cl) was then ligated to the sticky end upstream of the promoter (Fig.11C). As in Examples 1 and 3, the first handle was functionally coupled to a T7 RNA polymerase comprising a HaloTag domain, thereby forming a catalyst for RNA production (Fig. 11D). A second DNA adapter comprising two paired nucleic acid strands and having a second handle (biotin) was similarly ligated to the sticky end downstream of the RNA-encoding sequence, the ligated construct was coupled to a streptavidin coated bead (Fig.11F, right), and batch RNA synthesis was performed. [112] Importantly, the adapters comprising a first or second handle are “universal” in that they can be used with a plasmid functional template DNA encoding any RNA. Such an adapter may be referred to herein as a universal adapter. Any functional template DNA comprising a T7 RNA polymerase promoter sequence, an RNA-encoding sequence downstream of the T7 promoter, and a restriction site upstream of the T7 promoter that provides a complementary overlap with an adapter after digestion, may then be added to the universal adapter and ligated to provide a catalyst for RNA production. In some embodiments, a second universal adapter coupled to a solid support may similarly be utilized. [113] Fig.11E shows a gel demonstrating successful batch synthesis of a 2.4 kb (2,393 nucleotides) mRNA encoding a GFP-Fluc fusion protein using the catalyst for RNA production prepared from a plasmid functional template DNA by the method illustrated in Figs. 11A–D
Docket No.11555-013WO1 and Fig.11F. Here, a control synthesis reaction was also run using a T7 RNA polymerase that did not comprise a HaloTag domain, and thus was not coupled to plasmid DNA. [114] The restriction sequences and adapter sequences shown in Figs.11A–D and 11F are exemplary and any suitable sequences as known to one of ordinary skill in the art may similarly be used. Example 5: Invading DNA for Generation of a Catalyst for Production of RNA from Plasmid DNA [115] Yet another approach towards introducing handles into plasmid (or other long) DNA is shown in Figs. 12A-12C. In this approach, a reagent (referred to as an “invading strand” or “invading DNA”) may be prepared that will function with any plasmid DNA, requiring only minor plasmid editing upstream of a promoter of an RNA-encoding sequence of a plasmid. Such an invading strand is referred to as a “universal reagent.” [116] A custom sequence, containing recognition sites in a nontemplate strand for multiple “nicking” endonucleases (Nb.BtsI and Nb.BssSI) upstream of a promoter for a 24 nucleotide RNA-encoding sequence of a plasmid, is engineered into a functional template DNA. The functional template DNA is incubated with Nb.BtsI and Nb.BssSI. After nicking, the relatively short fragments denature from their corresponding complementary sequences of the template strand, generating a large gap in the nontemplate strand of the functional template DNA, immediately upstream of the promoter (Fig. 12A). DNA complementary to the gap (referred to herein as a “universal reagent”) is (i) derivatized with an alkyl-Cl first handle and coupled to a HaloTag T7 RNA polymerase and (ii) derivatized with a biotin second handle and coupled to streptavidin coated beads (Fig.12A). The gapped functional template DNA is then admixed with the streptavidin coated beads, hybridizing with the universal reagent (Fig.12B), thereby forming a catalyst for RNA production. [117] Fig. 12C shows a gel demonstrating successful batch synthesis of an encoded 24 nucleotide RNA using the catalyst for RNA production. [118] Although, here, a gap is generated in a nontemplate strand and the universal reagent is complementary to a sequence of a template strand, generation of a gap in a template strand, upstream of a promoter, and use of a universal reagent complementary to a corresponding sequence of a nontemplate strand is also contemplated as part of the present disclosure. In addition, a gap for hybridizing with a universal reagent may be generated upstream of a promoter (Fig.13A), as in the present Example, or may overlap with the promoter (Fig.13B).
Docket No.11555-013WO1 [119] The embodiments of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims. [120] The publications (including patent publications), web sites, company names, books, manuals, treatise, and scientific literature referred to herein establish the knowledge that is available to those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter.
Docket No.11555-013WO1 SEQUENCES SEQ ID NO: 1 5’-GCTCTTCNNNNTAAT-3’ (NNNN = 4-8 base pair restriction site) SEQ ID NO: 2 3’-CGAGAAGNNNNATTA-5’ (NNNN = 4-8 base pair restriction site) SEQ ID NO: 3 5’-NNNNGAAGAGC-3’ (NNNN = 4-8 base pair restriction site) SEQ ID NO: 4 3’-NNNNCTTCTCG-5’ (NNNN = 4-8 base pair restriction site) SEQ ID NO: 5 5’-GCTCTTCCTTATAATACGATCACTATAGGAAATAAG-3’ SEQ ID NO: 6 5’-AAAAAAAAAAAAAAAAAACGAAGAGC-3’ SEQ ID NO: 7 3’-CGAGAAGGAATATTATGCTGAGTATATCCTTTATTC-5’ SEQ ID NO: 8 3’-TTTTTTTTTTTTTTTTTTGCTTCTCG-5’ SEQ ID NO: 9 5’-TTATAATACGATCACTATAGGAAATAAG-3’
Docket No.11555-013WO1 SEQ ID NO: 10 5’-AAAAAAAAAAAAAAAAAA-3’ SEQ ID NO: 11 3’-ATTATGCTGAGTATATCCTTTATTC-5’ SEQ ID NO: 12 3’-TTTTTTTTTTTTTTTTTTGCT-5’ SEQ ID NO: 13 5’-AAAAAAAAAAAAAAAAAACGA-3’ (C = biotin-16-dCTP, A = biotin-dATP) SEQ ID NO: 14 3’-AATTATGCTGAGTATATCCTTTATTC-5’ (T = aminoallyl dUTP or alkyl-Cl dUTP) SEQ ID NO: 15 5’-GCTAGCTTATAATACGATCACTATAGGAAATAAG-3’ SEQ ID NO: 16 5’-AAAAAAAAAAAAAAAAAAGCGGCCGC-3’ SEQ ID NO: 17 3’-CGATCGAATATTATGCTGAGTATATCCTTTATTC-5’ SEQ ID NO: 18 3’-TTTTTTTTTTTTTTTTTTCGCCGGCG-5’ SEQ ID NO: 19 5’-CTTATAATACGATCACTATAGGAAATAAG-3’ SEQ ID NO: 20 5’-AAAAAAAAAAAAAAAAAAGC-3’
Docket No.11555-013WO1 SEQ ID NO: 21 3’-GATCGAATATTATGCTGAGTATATCCTTTATTC-5’ SEQ ID NO: 22 3’-TTTTTTTTTTTTTTTTTTCGCCGG-5’ SEQ ID NO: 23 5’-AAAAAAAAAAAAAAAAAAGCGH-3’ (C = biotin-dCTP, GH = 3’deoxyGTP) SEQ ID NO: 24 3’-dUGATCGAATATTATGCTGAGTATATCCTTTATTC-5’ (dU = NH2-dUTP) SEQ ID NO: 25 5’-GCTCTTCC-3’ SEQ ID NO: 26 3’-CGAGAAGGAAT-5’ (T = aminoallyl dUTP or alkyl-Cl dUTP) SEQ ID NO: 27 3’-CGAGAAGGAATATTATGCTGAGTATATCCTTTATTC-5’ (T = aminoallyl dUTP or alkyl-Cl dUTP) SEQ ID NO: 28 5’-TAAGGAAGAGCACGTGCTCTTCC-3’ (T = alkyl-Cl dUTP) SEQ ID NO: 29 5’-CTCTTCC-3’ SEQ ID NO: 30 3’-GAGAAGGAAT-5’
Docket No.11555-013WO1 SEQ ID NO: 31 5’-GAATTCACTGCTTCTCGAGTTCACTGCAATTAATACGACTCACTATAGGAGGT AGTAGAGGTGAAGATTTA-3’ SEQ ID NO: 32 3’-CTTAAGTGACGAAGAGCACAAGTGACGTTAATTATGCTGAGTGATATCCTCCA TCATCTCCACTTCTAAAT-5’ SEQ ID NO: 33 5’-CACTGCAATTAATACGACTCACTATAGGAGGTAGTAGAGGTGAAGATTTA-3’ SEQ ID NO: 34 5’-GAATTCACTGCTTCTCGAGTT-3’
Docket No.11555-013WO1