Nucleic acids with region-specific modifications and methods of making the same

Abstract

Disclosed herein are modified RNAs comprising region-specific nucleotide modifications, and methods of making the same. Also disclosed are DNA-RNA-DNA (DRD) hybrid polynucleotides and methods of making the same. The modified RNAs and DRD hybrid polynucleotides may be made by phased extension of a primer annealed to one or more single stranded DNA templates using multiple distinct mixtures of nucleotide triphosphates. Also provided are compositions comprising one or more of the modified RNAs or DRD hybrid polynucleotides provided herein, and methods of using said compositions for therapeutic applications.

Description

NUCLEIC ACIDS WITH REGION-SPECIFIC MODIFICATIONS AND METHODS OF MAKING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS[00011 This application claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalPatent Application No. 63 / 549,804, filed February 5, 2024, U.S. Provisional Patent Application No. 63 / 640,592, filed April 30, 2024, and U.S. Provisional Patent Application No. 63 / 749,100, filed January 24, 2025, the entire contents of each of which is incorporated herein by reference in their entireties.BACKGROUND

[0002] The following discussion is merely provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto.10003| RNAs carry a diverse array of chemical modifications that play important roles in the regulation of their expression and pharmacological properties. For example, translation initiation, a rate-limiting step of mRNA translation, heavily depends on the 5' A7- methylguanosine (m7G) cap and its interaction with eukaryotic translation initiation factors (elFs) including the direct cap-binding eIF4E protein. Naturally occurring 2' (9-m ethyl (2'OMe) and A6-methyladenosine (m6A) modifications at the first base of 5'UTR affect cap binding of elFs and decapping enzymes, impacting downstream mRNA translation and stability. For therapeutic mRNAs, complete substitution of uridine with N1 -methylpseudouridine (m l ) allows immune evasion and increases protein production. Thus, diversifying and expanding chemical modifications within mRNAs hold remarkable therapeutic potential, yet have been largely unexplored due to limitations in synthesis methodology.

[0004] Messenger RNA (mRNA) technology is an emerging alternative to conventional small molecule, DNA, and protein therapeutics and conventional vaccine approaches because it is potent, programmable, and capable of rapid production of mRNA with desired sequences.mRNA therapy is a rapidly developing field and has been used for the expression of therapeutic proteins, ranging from vascular regeneration factors e.g., vascular endothelial growth factor A (VEGF-A), erythropoietin (EPO), GATA Binding Protein 4 (GATA4), Myocyte Enhancer Factor 2C (MEF2C), T-Box Transcription Factor 5 (TBX5), Myocardin (MYOCD)), to vaccines for COVID- 19, influenza, and Zika virus. Despite recent clinical successes, mRNA therapy still faces challenges of instability, toxicity, short-term efficacy, and potential immunological responses. Increasing the stability and translation efficiency of mRNAs to enhance their efficiency in vivo remains an important problem that must be solved to increase the feasibility of mRNA therapeutics for clinical applications.SUMMARY

[0005] The present disclosure provides nucleic acids (e.g., RNA, such as mRNA and non-coding RNA, as well as RNA-DNA hybrids) that are modified in a deliberate, step-wise manner such that particular, pre-determined regions of the nucleic acids comprise modified nucleotides while other regions can remain unmodified, even with respect to the same base that is modified in the pre-determined regions. The present disclosure provides methods for preparing the disclosed nucleic acids that comprise region-specific modifications, and for preparing desirable nucleic acid constructs comprising the disclosed regionally modified nucleic acids.

[0006] In one aspect, the present disclosure provides a method for producing a RNA, comprising: (a) annealing a RNA primer to a first single stranded DNA (ssDNA) template polynucleotide, wherein the first ssDNA template polynucleotides comprises an overlap with a second ssDNA template polynucleotide; (b) contacting the first ssDNA template polynucleotide with an engineered primer-dependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA primer, thereby producing a DNA / RNA duplex comprising a once-extended RNA; (c) separating the first ssDNA template polynucleotide from the once-extended RNA; (d) annealing the once-extended RNA to the second ssDNA template polynucleotide; and (e) contacting the second ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a second mixture ofnucleotide triphosphates (NTPs) for a time sufficient to permit extension of the once-extended RNA, thereby producing a DNA / RNA duplex comprising a twice-extended RNA. In some embodiments, the method further comprises (f) separating the second ssDNA template polynucleotide from the twice-extended RNA; (g) annealing the twice-extended RNA to a third ssDNA template polynucleotide, wherein the second ssDNA template polynucleotide comprises an overlap with the third ssDNA template polynucleotide; and (h) contacting the third ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a third mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA, thereby producing a DNA / RNA duplex comprising a thrice-extended RNA. In some embodiments, the method further comprises (i) separating the third ssDNA template polynucleotide from the thrice-extended RNA; (j) annealing the thrice-extended RNA to a fourth ssDNA template polynucleotide, wherein the third ssDNA template polynucleotide comprises an overlap with the fourth ssDNA template polynucleotide; and (k) contacting the fourth ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a fourth mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA, thereby producing a DNA / RNA duplex comprising a four-times-extended RNA. In some embodiments, the method comprises annealing, and polymerizing of at least five, at least six, at least seven, at least eight, at least nine, or at least ten ssDNA template polynucleotides. In some embodiments, each of the ssDNA template polynucleotides comprises: (a) at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 terminal nucleotides of overlap with the subsequent ssDNA template polynucleotide; or (b) no more than 20 terminal nucleotides of overlap with the subsequent ssDNA template polynucleotide. In some embodiments, the engineered primer-dependent RNA polymerase is a TGK polymerase, a T7 polymerase, or a SP6 polymerase. In some embodiments, at least one of the mixtures of NTPs does not comprise a modified nucleotide. In some embodiments, at least one of the mixtures of NTPs comprises at least one modified nucleotide. In some embodiments, each mixture of NTPs comprises at least one modified nucleotide. In some embodiments, the modified nucleotide is selected from the group consisting of 2'-O-methyladenosine (mA), alpha-phosphorothioate adenosine (PS-A), N6- methyladenosine (m6A), N6-phenyladenosine (Ph6A); N6-benzyladenosine (Bn6A), N6-cyclopentyladenosine (Cy6A), and any combination thereof. In some embodiments, separating comprises digesting the ssDNA template polynucleotide. In some embodiments, digesting comprises contacting the ssDNA template polynucleotide with an exonuclease. In some embodiments, separating does not comprise digesting the ssDNA template polynucleotide. In some embodiments, separating comprises chemical denaturation. In some embodiments, chemical denaturation comprises contacting the DNA / RNA duplex with a hydrogen-bonddisrupting agent, a chaotropic reagent, or a strong base. In some embodiments, the hydrogenbond-disrupting agent is dimethyl sulfoxide (DMSO). In some embodiments, the method further comprises heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C, preferably about 60°C. In some embodiments, the ssDNA template polynucleotide comprises a tag. In some embodiments, the tag is a hydrophobic tag or an affinity tag. In some embodiments, the tag is an FC6 hydrophobic tag. In some embodiments, the ssDNA template polynucleotide is conjugated to a solid phase. In some embodiments, the solid phase is selected from a bead or a chip. In some embodiments, separating further comprises affinity purification or reversed phase chromatography. In some embodiments, the ssDNA template oligonucleotide is recycled for subsequent use. In some embodiments, the ssDNA template polynucleotides comprise a 5 ’-untranslated region (5’UTR) ssDNA template polynucleotide, a coding sequence (CDS) ssDNA template polynucleotide, a 3’- untranslated region (3’UTR) ssDNA template polynucleotide, and a poly(A) tail ssDNA template polynucleotide. In some embodiments, the method further comprises circularizing the RNA. In some embodiments, circularizing comprises RNA ligation. In some embodiments, circularizing comprises ribozyme-mediated back splicing. In some embodiments, the RNA produced is messenger RNA (mRNA). In some embodiments, the RNA produced is non-coding RNA.

[0007] In one aspect, the present disclosure provides a method for producing a RNA, comprising: (a) annealing a RNA primer to a first single stranded DNA (ssDNA) template polynucleotide, wherein the first ssDNA template polynucleotide comprises an overlap with a second ssDNA template polynucleotide; (b) contacting the first ssDNA template polynucleotidewith an engineered primer-dependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA primer, thereby producing a DNA / RNA duplex comprising a once-extended RNA; (c) separating the first ssDNA template polynucleotide from the once-extended RNA; (d) annealing the once-extended RNA to a second ssDNA template polynucleotide, wherein the second ssDNA template polynucleotide comprises an overlap with a third ssDNA template polynucleotide; (e) contacting the second ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a second mixture of NTPs for a time sufficient to permit extension of the once-extended RNA, thereby producing a DNA / RNA duplex comprising a twice-extended RNA; (f) separating the second ssDNA template polynucleotide from the twice-extended RNA; (g) annealing the twice-extended RNA to a third ssDNA template polynucleotide, wherein the third ssDNA template polynucleotide comprises an overlap with a fourth ssDNA template polynucleotide; (h) contacting the third ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a third mixture of NTPs for a time sufficient to permit extension of the twice- extended RNA, thereby producing a DNA / RNA duplex comprising a thrice-extended RNA; (k) separating the third ssDNA template polynucleotide from the thrice-extended RNA; (1) annealing the thrice-extended RNA to a fourth ssDNA template polynucleotide; (m) contacting the fourth ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a fourth mixture of NTPs for a time sufficient to permit extension of the thrice-extended RNA, thereby producing a DNA / RNA duplex comprising a four-times-extended RNA. In some embodiments, the first ssDNA template polynucleotide is a 5 ’-untranslated region (5’UTR) ssDNA template polynucleotide; the second ssDNA template polynucleotide is a coding sequence (CDS) ssDNA template polynucleotide; the third ssDNA template polynucleotide is a 3 ’-untranslated region (3’UTR) ssDNA template polynucleotide; and the fourth ssDNA template polynucleotide is a poly(A) tail ssDNA template polynucleotide. In some embodiments, the method comprises annealing, and polymerizing of at least five, at least six, at least seven, at least eight, at least nine, or at least ten ssDNA template polynucleotides. In some embodiments, each of the ssDNA template polynucleotides comprises: (a) at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 terminal nucleotides of overlap with the subsequentssDNA template polynucleotide; or (b) no more than 20 terminal nucleotides of overlap with the terminal nucleotides of at least one other ssDNA template polynucleotide. In some embodiments, the engineered primer-dependent RNA polymerase is a TGK polymerase, a T7 polymerase, or a SP6 polymerase. In some embodiments, at least one of the mixtures of NTPs does not comprise a modified nucleotide. In some embodiments, at least one of the mixtures of NTPs comprises at least one modified nucleotide. In some embodiments, each mixture of NTPs comprises at least one modified nucleotide. In some embodiments, the modified nucleotide is selected from the group consisting of 2'-O-methyladenosine (mA); alpha-phosphorothioate adenosine (PS-A); N6- methyladenosine (m6A); N6-phenyladenosine (Ph6A); N6-benzyladenosine (Bn6A); and N6- cyclopentyladenosine (Cy6A). In some embodiments, separating comprises digesting the ssDNA template polynucleotide. In some embodiments, digesting comprises contacting the RNA with an exonuclease. In some embodiments, separating does not comprise digesting the ssDNA template polynucleotide. In some embodiments, separating comprises chemical denaturation. In some embodiments, chemical denaturation comprises contacting the DNA / RNA duplex with a hydrogen -bond-disrupting agent, a chaotropic reagent, or a strong base. In some embodiments, the hydrogen-bond-disrupting agent is dimethyl sulfoxide (DMSO). In some embodiments, the method further comprises heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C, preferably about 60°C. In some embodiments, the ssDNA template polynucleotide comprises a tag. In some embodiments, the tag is a hydrophobic tag or an affinity tag. In some embodiments, the tag is an FC6 hydrophobic tag. In some embodiments, the ssDNA template polynucleotide is conjugated to a solid phase. In some embodiments, the solid phase is selected from a bead or a chip. In some embodiments, separating further comprises affinity purification or reversed phase chromatography. In some embodiments, the ssDNA template oligonucleotide is recycled for subsequent use. In some embodiments, the method further comprises (n) separating the fourth ssDNA template polynucleotide from the four-times-extended RNA. In some embodiments, the method further comprises circularizing the RNA. In some embodiments, circularizing comprises RNA ligation. In some embodiments, circularizing comprises ribozyme-mediated back splicing.In some embodiments, the RNA produced is messenger RNA (mRNA). In some embodiments, the RNA produced is non-coding RNA.

[0008] In one aspect, the present disclosure provides a method for producing a circular RNA (circRNA), comprising: (a) annealing a RNA primer to a first single stranded DNA (ssDNA) template polynucleotide, wherein the first ssDNA template polynucleotides comprises an overlap with a second ssDNA template polynucleotide; (b) contacting the first ssDNA template polynucleotide with an engineered primer-dependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA primer, thereby producing a DNA / RNA duplex comprising a once-extended RNA; (c) separating the first ssDNA template polynucleotide from the once-extended RNA; (d) annealing the once- extended RNA to the second ssDNA template polynucleotide; (e) contacting the second ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a second mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the once- extended RNA, thereby producing a DNA / RNA duplex comprising a twice-extended RNA, (f) separating the second ssDNA template polynucleotide from the twice-extended RNA, thereby producing a linear RNA; and (g) circularizing the linear RNA, thereby producing a circRNA. In some embodiments, the method comprises annealing, and polymerizing of at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten ssDNA template polynucleotides. In some embodiments, circularizing comprises RNA ligation. In some embodiments, RNA ligation comprises splint ligation by T4 RNA ligase 2. In some embodiments, circularizing comprises ribozyme-mediated back splicing. In some embodiments, the linear RNA comprises an exon region flanked by two intron regions. In some embodiments, the exon region comprises at least 1 modified nucleotide. In some embodiments, the intron regions do not comprise a modified nucleotide. In some embodiments, the method comprises synthesizing or having synthesized a chemically capped RNA primer and at least four ssDNA template polynucleotides. In some embodiments, each of the at least two ssDNA template polynucleotides comprises: (a) at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 terminal nucleotides of overlap with the subsequent ssDNA templatepolynucleotide; or (b) no more than 20 terminal nucleotides of overlap with the terminal nucleotides of at least one other ssDNA template polynucleotide. In some embodiments, the engineered primer-dependent RNA polymerase is a TGK polymerase, a T7 polymerase, or a SP6 polymerase. In some embodiments, at least one of the mixtures of NTPs does not comprise a modified nucleotide. In some embodiments, at least one of the mixtures of NTPs comprises at least one modified nucleotide. In some embodiments, each mixture of NTPs comprises at least one modified nucleotide. In some embodiments, the modified nucleotide is selected from the group consisting of 2'-O-methyladenosine (mA); alpha-phosphorothioate adenosine (PS-A); N6- methyladenosine (m6A); N6-phenyladenosine (Ph6A); N6-benzyladenosine (Bn6A); and N6- cyclopentyladenosine (Cy6A). In some embodiments, separating comprises digesting the ssDNA template polynucleotide. In some embodiments, digesting comprises contacting the ssDNA template polynucleotide with an exonuclease. In some embodiments, separating does not comprise digesting the ssDNA template polynucleotide. In some embodiments, separating comprises chemical denaturation. In some embodiments, chemical denaturation comprises contacting the DNA / RNA duplex with a hydrogen-bond-disrupting agent, a chaotropic reagent, or a strong base. In some embodiments, the hydrogen-bond-disrupting agent is dimethyl sulfoxide (DMSO). In some embodiments, the method further comprises heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C, preferably about 60°C. In some embodiments, the ssDNA template polynucleotide comprises a tag. In some embodiments, the tag is a hydrophobic tag or an affinity tag. In some embodiments, the tag is an FC6 hydrophobic tag. In some embodiments, the ssDNA template polynucleotide is conjugated to a solid phase. In some embodiments, the solid phase is selected from a bead or a chip. In some embodiments, separating further comprises affinity purification or reversed phase chromatography. In some embodiments, the ssDNA template oligonucleotide is recycled for subsequent use. In some embodiments, the exon region is synthesized from ssDNA template polynucleotides, wherein the ssDNA template polynucleotides comprise a 5 ’-untranslated region (5’UTR) ssDNA template polynucleotide, a coding sequence (CDS) ssDNA template polynucleotide, a 3 ’-untranslated region (3’UTR) ssDNA templatepolynucleotide, and a poly(A) tail ssDNA template polynucleotide. In some embodiments, the RNA produced is messenger RNA (mRNA). In some embodiments, the RNA produced is noncoding RNA.

[0009] In one aspect, the present disclosure provides a method for producing a circular RNA (circRNA), comprising: (a) annealing a 5’ phosphorylated region 1 RNA primer to a circular single stranded DNA (ssDNA) template polynucleotide; (b) contacting the circular ssDNA template polynucleotide with an engineered primer-dependent RNA polymerase and a mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the region 1 RNA primer, thereby producing a synthesized RNA comprising the region 1 RNA primer and a region 2 RNA; (c) ligating the synthesized RNA; (d) separating the synthesized RNA from the circular ssDNA template polynucleotide, thereby producing the circRNA. In some embodiments, the 5’ phosphorylated primer comprises one or more modified nucleotides. In some embodiments, the 5’ phosphorylated primer has a length from 5 nucleotides to about 1000 nucleotides. In some embodiments, the 5’ phosphorylated primer has a length of about 800 nucleotides. In some embodiments, the region 1 RNA primer comprises at least one modified nucleotide. In some embodiments, the region 1 RNA primer comprises at least one modified nucleotide, and wherein the region 2 RNA does not comprise a modified nucleotide. In some embodiments, the region 1 RNA primer does not comprise a modified nucleotide, and wherein the region 2 RNA comprises at least one modified nucleotide. In some embodiments, separating comprises digesting the ssDNA template polynucleotide. In some embodiments, digesting comprises contacting the ssDNA template polynucleotide with an exonuclease. In some embodiments, separating does not comprise digesting the ssDNA template polynucleotide. In some embodiments, separating comprises chemical denaturation. In some embodiments, chemical denaturation comprises contacting the DNA / RNA duplex with a hydrogen-bonddisrupting agent, a chaotropic reagent, or a strong base. In some embodiments, the hydrogenbond-disrupting agent is dimethyl sulfoxide (DMSO). In some embodiments, the method further comprises heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, atleast about 85°C, at least about 90°C, or at least about 95°C, preferably about 60°C. In some embodiments, the ssDNA template polynucleotide comprises a tag. In some embodiments, the tag is a hydrophobic tag or an affinity tag. In some embodiments, the tag is an FC6 hydrophobic tag. In some embodiments, the ssDNA template polynucleotide is conjugated to a solid phase. In some embodiments, the solid phase is selected from a bead or a chip. In some embodiments, separating further comprises affinity purification or reversed phase chromatography. In some embodiments, the ssDNA template oligonucleotide is recycled for subsequent use.

[0010] In one aspect, the present disclosure provides a method for producing a DNA- RNA-DNA (DRD) hybrid polynucleotide, comprising: (a) annealing a single stranded DNA (ssDNA) primer to a first single stranded DNA (ssDNA) template polynucleotide, wherein the first ssDNA template polynucleotides comprises an overlap with a second ssDNA template polynucleotide; (b) contacting the first ssDNA template polynucleotide with an engineered primer-dependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the ssDNA primer, thereby producing a DNA / RNA duplex comprising a DNA-RNA hybrid polynucleotide; (c) separating the first ssDNA template polynucleotide from the DNA-RNA hybrid polynucleotide; (d) annealing the DNA-RNA hybrid polynucleotide to the second ssDNA template polynucleotide; and (e) contacting the second ssDNA template polynucleotide with a DNA polymerase and a second mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the DNA-RNA hybrid polynucleotide, thereby producing a DNA / RNA duplex comprising a DRD hybrid polynucleotide comprising a single stranded RNA (ssRNA) flanked by two single stranded DNAs (ssDNAs). In some embodiments, the method further comprises separating the second ssDNA template polynucleotide from the DRD hybrid polynucleotide. In some embodiments, the second ssDNA template polynucleotide comprises uridines (U) instead of thymidines (T), and separating comprises digesting the second ssDNA template polynucleotide by contacting the second ssDNA template polynucleotide with a U-specific DNase enzyme. In some embodiments, separating does not comprise digesting the ssDNA template polynucleotide. In some embodiments, separating comprises chemical denaturation. In some embodiments, chemicaldenaturation comprises contacting the DNA / RNA duplex with a hydrogen-bond-disrupting agent, a chaotropic reagent, or a strong base. In some embodiments, the hydrogen-bonddisrupting agent is dimethyl sulfoxide (DMSO). In some embodiments, the method further comprises heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C, preferably about 60°C. In some embodiments, the ssDNA template polynucleotide comprises a tag. In some embodiments, the tag is a hydrophobic tag or an affinity tag. In some embodiments, the tag is an FC6 hydrophobic tag. In some embodiments, the ssDNA template polynucleotide is conjugated to a solid phase. In some embodiments, the solid phase is selected from a bead or a chip. In some embodiments, separating further comprises affinity purification or reversed phase chromatography. In some embodiments, the ssDNA template oligonucleotide is recycled for subsequent use. In some embodiments, the two ssDNAs have sufficient homology to permit the formation of a dumbbell shape, thereby producing a dumbbell DRD unit. In some embodiments, the method further comprises ligating the DRD hybrid polynucleotide to at least one capped RNA-DNA hybrid polynucleotide. In some embodiments, the method comprises ligating the DRD hybrid polynucleotide to at least two capped RNA-DNA hybrid polynucleotides. In some embodiments, the ligating produces a multi-cistronic RNA. In some embodiments, the multi -ci stronic RNA comprises branched caps. In some embodiments, the method further comprises ligating the dumbbell DRD unit to at least one other dumbbell DRD unit. In some embodiments, the method further comprises ligating the dumbbell DRD unit to at least two other dumbbell DRD units. In some embodiments, at least one of the dumbbell DRD units comprises a branched cap. In some embodiments, the ssRNA comprises unmodified nucleotides. In some embodiments, the ssRNA comprises at least 1 modified nucleotide.

[0011] In one aspect, the present disclosure provides a kit for producing a region- specifically-modified mRNA, comprising one or more of (a) an engineered primer-dependent RNA polymerase; (b) an RNA primer; (c) a single stranded DNA (ssDNA) template polynucleotide; and (d) a mixture of nucleotide triphosphates (NTPs). In some embodiments, theengineered primer-dependent RNA polymerase is a TGK polymerase, a T7 polymerase, or a SP6 polymerase. In some embodiments, the RNA primer is a chemically capped RNA primer. In some embodiments, the RNA primer is a 5’ phosphorylated RNA primer. In some embodiments, the ssDNA template polynucleotide is a linear ssDNA template polynucleotide. In some embodiments, the ssDNA template polynucleotide is a circular ssDNA template polynucleotide. In some embodiments, the mixture of NTPs comprises at least one modified nucleotide. In some embodiments, the modified nucleotide is selected from the group consisting of 2 -0- methyladenosine (mA); alpha-phosphorothioate adenosine (PS-A); N6-methyladenosine (m6A); N6-phenyladenosine (Ph6A); N6-benzyladenosine (Bn6A); and N6-cyclopentyl adenosine (Cy6A). In some embodiments, the kit further comprises instructions for using the kit. In some embodiments, the kit further comprises one or more of: (e) a hydrogen-bond-disrupting agent; (f) a chaotropic reagent; or (g) a strong base. In some embodiments, the hydrogen-bonddisrupting agent is dimethyl sulfoxide (DMSO). In some embodiments, the kit further comprises a solid phase. In some embodiments, the solid phase is selected from a bead or a chip.[0012| In one aspect, the present disclosure provides an RNA produced by the method according to any of the foregoing embodiments.

[0013] In one aspect, the present disclosure provides a RNA comprising two or more adenosine nucleotides, two or more guanosine nucleotides, two or more cytidine nucleotides, and two or more uridine nucleotides, wherein: (i) at least one, but not all of the adenosine nucleotides are modified, (ii) at least one, but not all of the guanosine nucleotides are modified, (iii) at least one, but not all of the cytidine nucleotides are modified, (iv) at least one of the uridine nucleotides are modified, or (v) any combination of (i), (ii), (iii), or (iv), wherein all modified adenosine nucleotides, all modified guanosine nucleotides, all modified cytidine nucleotides, and all modified uridine nucleotides are localized non-randomly to one or more predetermined regions of the RNA. In some embodiments, at least one, but not all of the uridine nucleotides are modified, and wherein all modified uridine nucleotides are localized non-randomly to one or more predetermined regions of the RNA. In some embodiments, all of the uridine nucleotides are modified. In some embodiments, the uridine nucleotides that are modified are Nl-methylpseudouridine (ml'P). In some embodiments, the RNA comprises a 5' cap region, an open reading frame (ORF), and a 3' untranslated region (UTR), and wherein in each of the 5' cap region and the 3' UTR at least one adenosine nucleotide is modified, and in the ORF all adenosine nucleotides are unmodified. In some embodiments, in each of the 5' cap region and the 3' UTR all adenosine nucleotides are modified. In some embodiments, the 3' UTR comprises a poly(A) region. In some embodiments, the adenosine nucleotides that are modified are selected from 2' O-methyl (2'0Me) adenosine and N6-methyladenosine (m6A).

[0014] In one aspect, the present disclosure provides a RNA comprising at least a first region and a second region, the first region and the second region each independently comprising adenosine nucleotides, guanosine nucleotides, cytidine nucleotides, and uridine nucleotides; wherein: (i) each adenosine nucleotide in the first region is a modified adenosine and each adenosine nucleotide in the second region is an unmodified adenosine; (ii) each adenosine nucleotide in the first region is an unmodified adenosine and each adenosine nucleotide in the second region is a modified adenosine; (iii) each guanosine nucleotide in the first region is a modified guanosine and each guanosine nucleotide in the second region is an unmodified guanosine; (iv) each guanosine nucleotide in the first region is an unmodified guanosine and each guanosine nucleotide in the second region is a modified guanosine; (v) each cytidine nucleotide in the first region is a modified cytidine and each cytidine nucleotide in the second region is an unmodified cytidine; or (vi) each cytidine nucleotide in the first region is an unmodified cytidine and each cytidine nucleotide in the second region is a modified cytidine; and wherein: (i) all of the uridine nucleotides in the first region and the second region are modified; (ii) each uridine nucleotide in the first region is a modified uridine and each uridine nucleotide in the second region is an unmodified uridine; or (iii) each uridine nucleotide in the first region is an unmodified uridine and each uridine nucleotide in the second region is a modified uridine. In some embodiments, all of the uridine nucleotides in the first region and the second region are modified. In some embodiments, the uridine nucleotides that are modified are Nl- methylpseudouridine (ml ). In some embodiments, the RNA comprises a 5’UTR, an open reading frame (ORF), a 3' UTR, and a poly(A) tail, wherein: each of the adenosines in the5’UTR is a 2’OMe-modified adenosine, each of the guanosines in the 5’UTR is a 2’0Me- modified guanosine, each of the cytidines in the 5’UTR is a 2’OMe-modified cytidine, and each of the uridines in the 5’UTR is a 2’OMe-modified uridine; and wherein: each of the adenosines in the ORF and the 3’UTR is an unmodified adenosine, each of the guanosines in the ORF and the 3’UTR is an unmodified guanosine, each of the cytidines in the ORF and the 3’UTR is an unmodified cytidine, and each of the uridines in the ORF and the 3’UTR is an Nl- methylpseudouridine (ml'P). In some embodiments, the RNA comprises a 5’UTR, an open reading frame (ORF), a 3' UTR, and a poly(A) tail, wherein the 5’UTR comprises a 5’ half and a 3’ half, wherein the 5’ half comprises 50% of the nucleotides of the 5’UTR that are closest to the 5’ end of the 5’UTR, wherein the 3’ half comprises 50% of the nucleotides of the 5’UTR that are closest to the 3’ end of the 5’UTR, wherein: each of the adenosines in the 5’ half of the 5’UTR is a 2’OMe-modified adenosine, each of the guanosines in the 5’ half of the 5’UTR is a 2’0Me- modified guanosine, each of the cytidines in the 5’ half of the 5’UTR is a 2’OMe-modified cytidine, and each of the uridines in the 5’ half of the 5’UTR is a 2’OMe-modified uridine; and wherein: each of the adenosines in the 3’ half of the 5’UTR, the ORF, and the 3’UTR is an unmodified adenosine, each of the guanosines in the 3’ half of the 5’UTR, the ORF, and the 3’UTR is an unmodified guanosine, each of the cytidines in the 3’ half of the 5’UTR, the ORF, and the 3’UTR is an unmodified cytidine, and each of the uridines in the 3’ half of the 5’UTR, the ORF, and the 3’UTR is an N1 -methylpseudouridine (ml ). In some embodiments, each of the adenosines in the poly(A) tail is an N6-methyl adenosine (m6A).

[0015] In one aspect, the present disclosure provides an RNA-DNA hybrid as disclosed here (see, e.g., FIG. 4). For example, an RNA-DNA hybrid can comprise the RNA of any one of preceding aspects and at least one region of DNA. In one aspect, the RNA-DNA hybrid, comprises a first DNA region and a second DNA region with an RNA region in between the first DNA region and the second DNA region, the RNA region comprising at least a first region and a second region, the first region and the second region each independently comprising adenosine nucleotides, guanosine nucleotides, cytidine nucleotides, and uridine nucleotides; wherein: (i) each adenosine nucleotide in the first region is a modified adenosine and each adenosinenucleotide in the second region is an unmodified adenosine; (ii) each adenosine nucleotide in the first region is an unmodified adenosine and each adenosine nucleotide in the second region is a modified adenosine; (iii) each guanosine nucleotide in the first region is a modified guanosine and each guanosine nucleotide in the second region is an unmodified guanosine; (iv) each guanosine nucleotide in the first region is an unmodified guanosine and each guanosine nucleotide in the second region is a modified guanosine; (v) each cytidine nucleotide in the first region is a modified cytidine and each cytidine nucleotide in the second region is an unmodified cytidine; or (vi) each cytidine nucleotide in the first region is an unmodified cytidine and each cytidine nucleotide in the second region is a modified cytidine; and wherein: (i) all of the uridine nucleotides in the first region and the second region are modified; (ii) each uridine nucleotide in the first region is a modified uridine and each uridine nucleotide in the second region is an unmodified uridine; or (iii) each uridine nucleotide in the first region is an unmodified uridine and each uridine nucleotide in the second region is a modified uridine.

[0016] In another aspect, the present disclosure provides dual-cistronic molecules comprising two ligated RNA-DNA hybrids disclosed herein (e.g., an RNA-DNA hybrid of the preceding aspects) multi-cistronic molecules comprising at least three ligated RNA-DNA hybrids disclosed herein (e.g., an RNA-DNA hybrid of the preceding aspects). FIG. 4 provides illustrations of examples of dual cistronic mRNA and multi-cistronic mRNA comprising two or more RNA-DNA hybrids.[00171 Both the foregoing summary and the following description of the drawings and detailed description are exemplary and explanatory. They are intended to provide further details of the disclosure, but are not to be construed as limiting. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the disclosure.

[0018] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are provided as being part of the inventive subjectmatter disclosed herein and may be employed in any combination to achieve the benefits described herein.BRIEF DESCRIPTION OF THE DRAWINGS(0019] FIG. 1 depicts a comparison of traditional mRNA synthesis workflow with tandem template switching workflow. FIG. 1A shows a traditional in vitro transcription (IVT) workflow that utilizes double stranded DNA (dsDNA) template containing bacteriophage RNA polymerase (pol.) specific promoter sequence. Bacteriophage RNA polymerases, such as T7 and SP6, utilize ribonucleotide triphosphates (NTPs) as building blocks to synthesize an mRNA product, where the mRNA transcript could be functionally divided into 5' untranslated region (5'UTR), coding sequence (CDS), 3' untranslated region (3'UTR), and the poly(A) tail. FIG. IB shows IVT synthesis of modified mRNA by complete NTP substitution with modified NTPs to yield fully modified mRNA. In the example, ATP is substituted with m6ATP, and an mRNA in which all Adenosines comprised the m6A modification was generated, carrying opposite effects on stability and translation. In particular, mRNA with full / partial substitution of A by M6A throughout the transcript would have increased translation initiation by m6A reader proteins, but hampered elongation during translation due to ribosome stalling. With respect to stability, m6A, when present in the 3'UTR, is known to promote decay, but when present in the poly (A) tail is known to stabilize mRNA. FIG. 1C shows IVT synthesis of partially modified mRNA by spiking in modified NTPs, providing a heterogeneous mixture of mRNA products with different m6A patterns. FIG. ID shows IVT synthesis of capped mRNA by cap analogue spike-in. Cap analogues were added along with the NTP mixture where it competes with unmodified NTPs for transcription initiation to generate mixtures of capped / uncapped mRNA. FIG. IE shows the synthesis of region-specifically modified mRNA by tandem template switching using engineered DNA-templated RNA polymerase. The full mRNA sequence was divided into multiple segments of a single stranded DNA (ssDNA) template, each with an at least 10-20 base pair (bp) overlap with the preceding template. A 10-150 bp long chemically-capped RNA primer was prepared using a previous workflow such that it contained combinatorial patterns of chemical modifications including cap modifications, base modifications, phosphodiester linkagemodifications, and sugar backbone modifications. Modification patterns were defined by solidphase synthesis and quality-controlled by mass spectrometry. Capping efficiency was ensured >99% by HPLC purification. The chemically synthesized capped RNA primer was annealed to the first ssDNA template. Polymerization was carried out by engineered primer-dependent RNA polymerase such as TGK and a set of modified / unmodified NTP mix was used to introduce modification patterns specific to the first region. DNA template was then digested, and mRNA was annealed to the next segment of ssDNA template and polymerized using a second set of modified / unmodified NTP mix. The template switching / synthesis cycle was repeated until the full length mRNA was synthesized.

[0020] FIG. 2 shows schematics and experimental data describing the production of modified RNAs. FIG. 2A shows experimental data obtained from experiments supporting the scheme described in FIG. IE. The full length (-750 bp) NanoLuciferase (NLuc) includes 5'UTR derived from the human a-globin mRNA with an optimized Kozak sequence + codon-optimized CDS + 3'UTR consisting of two sequences derived from the amino-terminal enhancer of split mRNA and the mitochondrial encoded 12S rRNA + a 80A poly(A) tail. The full length mRNA was divided into 5 segments where each ssDNA template was chemically synthesized. Full length mRNA was synthesized from a 20-bp RNA primer after 5 rounds of template switching / polymerization. Fragments / products from each step were resolved on 15% TBE-Urea gel. FIG. 2B shows experimental data obtained from experiments supporting the significance of region-specific mRNA modifications. The same NLuc sequence as described in FIG. 2A was used except for exclusion of poly(A) tails and further inclusion of C-terminal PEST destabilization signal. NLuc-PEST (NLucP) encoding mRNA was synthesized by IVT with co- transcri phonal capping and 100% m I incorporation. The NLucP mRNA was templated tailed by annealing to a synthetic ssDNA template with 20-bp homology to the 3 'end of NLucP mRNA and 80 continuous T, resulting in an 80A poly(A) tail after transcription.None / unmodified / modified ATP was used in 100% to enable fully modified / unmodified poly(A) tail incorporation as characterized by agarose gel electrophoresis. rA: unmodified adenosine; mA: 2'-( -methyladenosine; PS-A: alpha-phosphorothioate adenosine; m6A: A6-methyladenosine; Ph6A: A^-phenyladenosine; Bn6A: A^-benzyladenosine; Cy6A: N6- cyclopentyladenosine. FIG. 2C shows a biological evaluation of tail modified NLucP mRNA by time course dual luciferase assay. Templated poly(A)-tailed NLucP mRNAs were co-transfected with FLuc mRNA as transfection control in HeLa cells. 6 hours (hrs) after transfection, culture medium was exchanged and cells treated with all conditions were re-seeded into 5 wells of a multi-well plate, in which the luminescence was measured. Ratios of NLucP to FLuc normalized to unmodified rA construct were plotted, n = 3, mean ± s.e.m.

[0021] FIG. 3 shows schematics illustrating the production of region-specifically- modified circular RNAs. FIG. 3A shows a schematic illustrating a standard method of preparing circRNA by intron-mediated ribozyme back splicing. FIG. 3B shows a schematic illustrating that upon incorporation of modified bases, intron structures are disrupted and ribozyme functions are abrogated, disabling circularization. FIG. 3C shows a schematic illustrating a method of synthesis of modified circRNA by region-specific RNA modifications. Modified bases are incorporated specifically outside of intronic regions, allowing ribozyme-mediated back splicing. FIG. 3D shows a schematic illustrating region-specifically modified RNAs prepared by the workflow in FIG. IE could be circularized by ribozyme back splicing as in FIG. 3C or using other circularization strategies such as enzymatic ligation. FIG. 3E shows synthesis of modified / unmodified circRNA using primer-dependent RNA pol. 5 'phosphorylated RNA primer (chemically synthesized, modified) is annealed to circular ssDNA template and extended using primer-dependent RNA pol (with / without modified NTPs). Synthesized RNA is then circularized by splint ligation using T4 RNA ligase 2 and the DNA strand is separated to isolate modified circRNA.

[0022] FIG. 4 shows schematics illustrating the production of DNA-RNA-DNA (DRD) hybrids. FIG. 4A shows a schematic illustrating a workflow for the preparation of DNA-RNA- DNA (DRD) building blocks. FIG. 4B shows a schematic illustrating one-pot assembly of DRD units. FIG. 4C shows a schematic illustrating that the workflow in FIG. 4B can be applied to DRD units bearing a capped RNA branch, which is synthesized by the workflow presented in FIG. 4A with a branched ssDNA-capped RNA primer. FIG. 4D shows a schematic illustratingthat the workflow in FIG. 4B can be further applied to one-pot circularization of two dumbbell DRD units with different ORF and IRES, where the 5’ overhangs of the two dumbbell DRD hybrids hybridize to each other and are ligated by T4 DNA ligase to afford circular dual-ORF mRNA. FIG. 4E shows a schematic illustrating that the scheme in FIG. 4D can be applied to multiple dumbbell DRD hybrids such that the 5’ overhangs of each forms a multi-way junction to allow one-pot circularization and assembly. FIG. 4F shows a schematic illustrating that the workflow in FIG. 4D can be similarly applied to dumbbell DRD hybrids with branched caps. FIG. 4G shows a schematic illustrating that the workflow in FIG. 4E can be similarly applied to dumbbell DRD hybrids with branched caps.

[0023] FIG. 5 depicts experimental results showing the optimization of poly(A)-tailing by template switch RNA synthesis. FIG. 5A (left) shows a schematic illustrating poly(A) over- extension. FIG. 5A (right) shows a representative TBE-Urea gel image showing poly(A) over- extension. FIG. 5B shows a representative TBE-Urea gel image showing the effect of ATP concentration (mM) on poly(A) over-extension. FIG. 5C shows a schematic illustrating the design of a polyT DNA template with a terminal dideoxynucleotide, and a representative TBE- Urea gel image showing the effect of inclusion of a terminal dideoxynucleotide on poly(A) over- extension. FIG. 5D shows a representative TBE-Urea gel image showing the effect of reaction temperature on poly(A) over-extension. FIG. 5E shows a representative TBE-Urea gel image showing the effect of NaCl concentration on poly(A) over-extension. FIG. 5F shows a representative TBE-Urea gel image showing the effect of m6ATP concentration on poly(A) over-extension. FIG. 5G (left) shows a schematic illustrating the synthesis and characterization of a poly(A)-tailed mRNA achieved by template switching. FIG. 5G (right) shows a representative TBE-Urea gel image showing the successful poly(A) tailing of an mRNA by template switch RNA synthesis.

[0024] FIG. 6 depicts experimental results showing the screening of 5’UTR modifications by template switch mRNA synthesis. FIG. 6A shows a schematic illustrating a 5’UTR-specifically modified mRNA, and a diagram of the experimental protocol for measuring translation efficiency and RNA integrity. FIG. 6B shows various base modifications screenedand evaluated for translation efficiency and RNA integrity when incorporated in the 5’UTR of a template-switch-synthesized mRNA. FIG. 6C (left) shows a schematic illustrating the various combinations of 5’UTR modification screened and evaluated for their effect of translation efficiency. FIG. 6C (right) shows a graph illustrating the effect of various 5’UTR base modifications on translation efficiency in terms of relative luminance in HeLa cells. FIG. 6D shows a graph illustrating the effect of various 5’UTR base modifications on translation efficiency in terms of relative luminance in HepG2 cells. FIG. 6E shows a graph illustrating the effect of various 5’UTR base modifications on translation efficiency in terms of relative luminescence in JAWSII cells. FIG. 6F (left) shows a schematic illustrating the various combinations of 5’UTR modification screened and evaluated for their effect of mRNA half-life. FIG. 6F (right) shows a graph illustrating the effect of various 5’UTR base modifications on mRNA half-life in terms of one-phase decay kinetics of Nluc-PEST in HeLa cells. FIG. 6G shows a graph illustrating the effect of various 5’UTR base modifications on mRNA half-life in terms of one-phase decay kinetics of Nluc-PEST in HepG2 cells. FIG. 6H shows a graph illustrating the effect of various 5’UTR base modifications on mRNA half-life in terms of one- phase decay kinetics of Nluc-PEST in JAWSII cells.

[0025] FIG. 7 depicts schematics and experimental results showing the purification of DNA / RNA hybrids without the use of nuclease digestion. FIG. 7A shows a schematic illustrating DNA / RNA hybrid separation without nuclease digestion. The single stranded DNA templates can be tagged with a hydrophobic handle (a fluorous hydrocarbon chain, for example). The DNA and the RNA components of the DNA / RNA duplex after primer extension reactions can be separated by chemical denaturation with DMSO and heat, and the hydrophobic tag renders the DNA strand to be better retained on reverse-phased columns, allowing efficient separation of the tagged / un-tagged strands. FIG. 7B shows representative chromatograms of duplex separation using denaturing HPLC. The two strands of the duplex were tagged with fluorophores (Cy3 and 6FAM) on their respective 5’ ends, where the DNA strand was further tagged with a FC6 hydrophobic tag. FIG. 7C shows representative images of the results of a gel electrophoresis characterization following separation as described regarding FIG. 7B.[0(126] FIG. 8 shows a schematic illustrating template-switching primer extension on solid-phase-immobilized DNA templates, such as bead- or chip-immobilized DNA templates.DETAILED DESCRIPTION(0027] Provided herein are modified nucleic acids, including RNAs and RNA-DNA hybrids, comprising region-specific nucleotide modifications. Also provided are methods of making the modified RNAs and RNA-DNA hybrids described herein by sequentially polymerizing a transcript (e.g., an RNA transcript) using a plurality of single stranded DNA (ssDNA) template polynucleotides, and a plurality of distinct pools of nucleotide triphosphates (NTPs) that can include modified and unmodified NTPs. The DNA templates can be utilized in a stepwise fashion to serially extend the RNA region-by-region, which allows for the incorporation of different modified nucleotides within each different region. For example, a first DNA template can be annealed to an RNA primer, which is polymerized to create a first RNA region. The first DNA template and the first RNA region are separated (e.g., via any form of denaturation, such as heat- or chemical-induced denaturation, or digestion of the template); and the first RNA region is annealed to a second template, which overlaps with the 3’ end of the first RNA region, such that the RNA can be further polymerized and extended. In between these polymerization steps, different dNTPs can be added to the reaction mixture, thereby allowing the different regions of the RNA to contain different modified nucleotides. The reaction can be continued with as many templates as needed to prepare an RNA with pre-determined, regionspecific nucleotide modifications. Further, as provided in more detail herein, the disclosed methods can be performed either in solution or using templates bound to a substrate, such that the templates can be repeatedly reused.Equivalents

[0028] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantagesdescribed herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.|0029| It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.(0030] In the claims, as well as in the specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, / .<?., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e. ,“comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

[0031] In the claims, as well as in the specification, recitation of the phrase “between X and Y”, wherein X and Y are two separate values, it should be appreciated that these ranges include the use of these end values. For example, if a claim recites a range of between 1 and 10, this includes the values of 1, 10, and any value in between (c.g., 2, 3, 4, 5, 6, 7, 8, 9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, etc.).Definitions100321 A “messenger RNA” (“mRNA”) as used herein refers to a nucleic acid comprising an open reading frame (ORF) encoding a gene product, such as a protein. An mRNA may comprise a poly-A region that is 3’ to the ORF. An mRNA may also comprise a 5’ untranslated region (5’ UTR) that is 5’ to (upstream of) the ORF, and a 3’ untranslated region (3’ UTR) that is 3’ to (downstream of) the ORF. A mRNA may also comprise a 5’ cap at the 5’ end of the mRNA.

[0033] An “open reading frame” (“ORF”), such as an ORF encoding a protein, as used herein refers to a nucleic acid sequence comprising a coding sequence that leads to the production of the protein when the ORF is translated. The nucleic acid sequence may be an RNA sequence, in which case translation of the RNA sequence produces a polypeptide with the amino acid sequence of the protein. The nucleic acid sequence may be a DNA sequence, in which case the protein is produced when an RNA polymerase uses the DNA sequence to transcribe an RNA molecule comprising an RNA sequence that is complementary to the DNA sequence, and translation of the RNA sequence produces a polypeptide with the amino acid sequence of the protein. An ORF typically begins with a START codon, such as AUG in the RNA sequence(ATG in the DNA sequence), and ends with a STOP codon, such as UAG, UAA, or UGA in the RNA sequence (TAG, TAA, or TGA in the DNA sequence), with the number of bases between the G of the start codon and the T or U of the STOP codon being a multiple of 3 (e.g., 3, 6, 9, 12, etc.).|0034| With reference to numbering of the nucleotide positions within a nucleic acid molecule, a position of +1 refers to the first nucleotide of the nucleic acid molecule (e.g., of the RNA molecule), +2 is the second nucleotide, +3 is the third nucleotide, and so on.

[0035] In some embodiments of the modified RNAs provided herein, the modified RNA is an mRNA comprising a 5' untranslated region (5' UTR) and a 3' untranslated region (3' UTR). 5' and 3' UTRs are sequences within an mRNA that do not encode amino acids of the protein encoded by the mRNA, and are thus not part of the open reading frame. The 5' UTR is 5' to (upstream of) the open reading frame. The 3' UTR is 3' to (downstream of) the open reading frame. In some embodiments, the 3' UTR comprises one or more nucleotides that are 3' to the open reading frame and 5' to (upstream of) the poly-A region of the mRNA.

[0036] In some embodiments of the modified RNAs provided herein, the modified RNA is an mRNA comprising, in 5’-to-3’ order: 1) a 5’ cap, optionally modified; 2) a modified 5’ UTR; 3) an open reading frame (ORF); 4) a 3’ UTR; and 5) a poly-A region. In some embodiments, the first nucleotide of the 5’ UTR is 3’ to (downstream of) the 5’ cap, and the last nucleotide of the 5’ UTR is 5’ to (upstream of) the first nucleotide of the ORF. In some embodiments, the first nucleotide of the ORF is 3’ to (downstream of) the last nucleotide of the 5’ UTR, and the last nucleotide of the ORF is 5’ to (upstream of) the first nucleotide of the 3’ UTR. In some embodiments, the ORF is between the last nucleotide of the 5’ UTR and the first nucleotide of the 3’ UTR. In some embodiments, the first nucleotide of the 3’ UTR is 3’ to (downstream of) the last nucleotide of the ORF, and the last nucleotide of the 3’ UTR is 5’ to (upstream of) the first nucleotide of the poly-A region. In some embodiments, the 5’ UTR is between the 5’ cap and the first nucleotide of the ORF. In some embodiments, the 3’ UTR is between the ORF and the poly-A region. In some embodiments, the 5’ cap is 5’ to (upstream of)the first nucleotide of the 5’ UTR. In some embodiments, the first nucleotide of the poly-A region is 3’ to (downstream of) the last nucleotide of the 3’ UTR.(0037] In some embodiments, the RNA is a linear RNA. A linear RNA is an RNA with a 5' terminal nucleotide and a 3' terminal nucleotide. The 5' terminal nucleotide of a linear RNA is covalently bonded to only one adjacent nucleotide of the RNA, with the adjacent nucleotide occurring 3' to the 5' terminal nucleotide in the nucleic acid sequence of the RNA. The 3' terminal nucleotide of a linear RNA is covalently bonded to only one adjacent nucleotide of the RNA, with the adjacent nucleotide occurring 5' to the 3' terminal nucleotide in the nucleic acid sequence of the RNA. In a nucleic acid sequence comprising every nucleotide of a linear RNA in 5'-to-3' order, the 5' terminal nucleotide is the first nucleotide in the sequence, and the 3' terminal nucleotide is the last nucleotide in the sequence.[0038| In some embodiments, the RNA is a circular RNA. A circular RNA is an RNA with no 5' terminal nucleotide or 3' terminal nucleotide. Every nucleotide in a circular mRNA is covalently bonded to both 1) a 5' adjacent nucleotide; and 2) a 3' adjacent nucleotide. In a circular RNA with a nucleic acid sequence comprising every nucleotide of the circular RNA in 5'-to-3' order, the last nucleotide of the nucleic acid sequence is covalently bonded to the first nucleotide of the nucleic acid sequence. In some embodiments of circular RNAs with a 5' cap region, a 5' UTR, a 3' UTR, and a poly-A region, the poly-A region is 3' to (downstream from) the 3' UTR and 5' to (upstream of) the 5' cap region.|0039| An RNA molecule that can be translated is referred to as a messenger RNA, or mRNA. A DNA or RNA sequence encodes a gene through codons. A codon refers to a group of three nucleotides within a nucleic acid, such as DNA or RNA, sequence. An anticodon refers to a group of three nucleotides within a nucleic acid, such as a transfer RNA (tRNA), that are complementary to a codon, such that the codon of a first nucleic acid associates with the anticodon of a second nucleic acid through hydrogen bonding between the bases of the codon and anticodon. For example, the codon 5'-AUG-3' on an mRNA has the corresponding anticodon 3'-UAC-5' on a tRNA. During translation, a tRNA with an anticodon complementary to thecodon to be translated associates with the codon on the mRNA, generally to deliver an amino acid that corresponds to the codon to be translated, or to facilitate termination of translation and release of a translated polypeptide from a ribosome.

[0040] Translation is the process in which the RNA coding sequence is used to direct the production of a polypeptide. The first step in translation is initiation, in which a ribosome associates with an mRNA, and a first transfer RNA (tRNA) carrying a first amino acid associates with the first codon, or START codon. The next phase of translation, elongation, involves three steps. First, a second tRNA with an anticodon that is complementary to codon following the START codon, or second codon, and carrying a second amino acid, associates with the mRNA. Second, the carbon atom of terminal, non-side chain carboxylic acid moiety of the first amino acid reacts with the nitrogen of the terminal, non-side chain amino moiety of the second amino acid carried, forming a peptide bond between the two amino acids, with the second amino acid being bound to the second tRNA, and the first amino acid bound to the second amino acid, but not the first tRNA. Third, the first tRNA dissociates from the mRNA, and the ribosome advances along the mRNA, such that the position at which the first tRNA associated with the ribosome is now occupied by the second tRNA, and the position previously occupied by the second tRNA is now free for an additional tRNA carrying an additional amino acid to associate with the mRNA. These three steps of 1) association of a tRNA carrying amino acid, 2) formation of a peptide bond, which adds an additional amino acid to a growing polypeptide, and 3) advancement of the ribosome along the mRNA, continue until the ribosome reaches a STOP codon, which results in termination of translation. Generally, tRNAs that associate with STOP codons do not carry an amino acid, so the association of a tRNA that does not carry an amino acid during the elongation step results in cleavage of the bond between the polypeptide and the tRNA carrying the final amino acid in the polypeptide, such that the polypeptide is released from the ribosome.Alternatively, ribosomes may dissociate from the mRNA and release the polypeptide if no tRNA associates with the STOP codon.

[0041] A “nucleic acid,” or “polynucleotide,” as used herein, refers to an organic molecule comprising two or more covalently bonded nucleotides. A “nucleotide,” as used herein,refers to an organic molecule comprising a 1) a nucleoside comprising a sugar covalently bonded to a nitrogenous base (nucleobase); and 2) a phosphate group that is covalently bonded to the sugar of the nucleoside. Nucleotides in a polynucleotide are typically joined by a phosphodiester bond, in which the 3' carbon of the sugar of a first nucleotide is linked to the 5' carbon of the sugar of a second nucleic acid by a bridging phosphate group. Typically, the bridging phosphate comprises two non-bridging oxygen atoms, which are bonded only to a phosphorus atom of the phosphate, and two bridging oxygen atoms, each of which connects the phosphorus atom to either the 3' carbon of the first nucleotide or the 5' carbon of the second nucleotide. In a nucleic acid sequence describing the order of nucleotides in a nucleic acid, a first nucleotide is said to be 5' to (upstream of) a second nucleotide if the 3' carbon of first nucleotide is connected to the 5' carbon of the second nucleotide. Similarly, a second nucleotide is said to be 3' to (downstream of) a first nucleotide if the 5' carbon of the second nucleotide is connected to the 3' carbon of the first nucleotide. Nucleic acid sequences are typically read in 5 '->3 ' order, starting with the 5' nucleotide and ending with the 3' nucleotide.[0042| A “modified nucleotide,” as used herein, refers to a nucleotide with a structure that is not the canonical structure of an adenosine nucleotide, cytidine nucleotide, guanine nucleotide, or uracil nucleotide. A canonical structure of a molecule refers to a structure that is generally known in the art to be the structure referred to by the name of the molecule. A canonical structure of an adenosine nucleotide, which comprises an adenine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of adenosine monophosphate:[00431 The canonical structure of AMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar aredeprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0044] The canonical structure of a cytosine nucleotide which comprises a cytosine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of cytidine monophosphate:The canonical structure of CMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.10045] The canonical structure of a guanine nucleotide which comprises a guanine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of guanosine monophosphate:The canonical structure of GMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0046] The canonical structure of a uracil nucleotide which comprises a uracil base, ribose sugar, and one or more phosphate groups, is shown below, in the form of uridine monophosphate:The canonical structure of UMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.100471 The structure of a modified nucleotide may differ from the structure of a canonical nucleotide due to one or more modifications in the sugar, nitrogenous base, or phosphate of the nucleotide. In some embodiments, the modified nucleotide comprises a modified nucleoside that is not the canonical structure of an adenine nucleoside, cytosine nucleoside, guanine nucleoside, or uracil nucleoside.|0048] An example of a canonical structure of adenosine, an adenine nucleoside, is reproduced below:(adenosine). The canonical structure of adenosine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acid sequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.|0049] An example of a canonical structure of cytidine, a cytosine nucleoside, is reproduced below:(cytidine). The canonical structure of cytidine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acid sequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0050] An example of a canonical structure of guanosine, a guanine nucleoside, is reproduced below:(guanosine). The canonical structure of guanosine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acid sequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.[00511 An example of a canonical structure of uridine, a uracil nucleoside, is reproduced below:(uridine). The canonical structure of uridine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acidsequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0052] A “ligase,” as used herein, refers to an enzyme that is capable of forming a covalent bond between two nucleotides, and the process of “ligation” refers to the formation of the covalent bond between the two nucleotides.

[0053] A “poly-A tail,” as used herein, refers to a nucleic acid sequence comprising adenosine nucleotides that is attached to the 3' end of a nucleic acid, such as an RNA. A poly-A tail or poly-A region may consist of nucleotides that are 25-100%, 30-100%, 40-100%, 50- 100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99- 100% adenosine nucleotides. As used herein, the terms “poly-A tail” and “poly-A region” are used interchangeably. The adenosine nucleotides comprised by a poly-A tail may be canonical adenosine nucleotides or modified (non-canonical) adenosine nucleotides.

[0054] A “5' cap,” as used herein, refers to one or more nucleotides that are covalently attached to the 5' end of a nucleic acid, such as an RNA molecule. A “5' cap region,” as used herein, refers to a nucleic acid comprising a 5' nucleotide cap and one or more modified nucleotides. A 5' cap may comprise a 5' capping nucleotide that is attached to the 5' end of a mRNA by a 5' to 5' triphosphate internucleotide linkage. In some embodiments, a nucleotide attached to a mRNA by a 5' to 5' triphosphate intemucleotide linkage is referred to as a “native” 5' capping nucleotide. In some embodiments, a native 5' capping nucleotide is a 7- methylguanosine (m7G) nucleotide. In some embodiments, a 5' cap is a modified 5' cap, comprising one or more modified nucleotides, such as the 5' capping nucleotide, or one or more modified intemucleotide modifications, such as modifications to the 5' to 5' triphosphate internucleotide linkage. In some embodiments, a 5' cap comprises one or more nucleotides with a sugar modification, such as 2'-O-methylation.

[0055] An example of a canonical structure of 7-methylguanosine (m7G) attached to a ribonucleic acid sequence (e.g., a mRNA) by a 5' to 5' triphosphate intemucleotide linkage isreproduced below:|0056| A “counterion” or “anionic counterion” is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality. In some embodiments, an anionic counterion is monovalent (e.g., including one formal negative charge). An anionic counterion may also be multivalent (e.g., including more than one formal negative charge), such as divalent or trivalent. Exemplary counterions include halide ions (e.g., F , CE, Br, I"), NO3 , CIO4 , OH , H2PO4 , HCO3 , HSO4 , sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthal ene-2-sulfonate, naphthalene- 1 -sulfonic acid-5-sulfonate, ethan-1 -sulfonic acid-2- sulfonate, and the like), carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, and the like), BF4 , PF4", PFe", AsFe", SbFe ", B[3,5 (CFs^CeHs ] , B(C6FS)4-, BPh4 , A1(OC(CF3)3)4- and carborane anions (e.g., CB11H12- or (HCBi iMesBrs) ). Exemplary counterions which may be multivalent include CO32, HPO42, PO43, B4O72, SO42, S2O32, carboxylate anions (e.g, tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like), and carboranes.[0057 | Use of the phrase “at least one instance” refers to 1, 2, 3, 4, or more instances, but also encompasses a range, e.g., for example, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive.Modified RNAs

[0058] In some aspects, the present disclosure provides modified RNAs a region-specific nucleotide modification. In some embodiments, a modified RNA comprises two or more adenosine nucleotides, two or more guanosine nucleotides, two or more cytidine nucleotides, and two or more uridine nucleotides, wherein all modified adenosine nucleotides, all modified guanosine nucleotides, all modified cytidine nucleotides, and all modified uridine nucleotides are localized non-randomly to one or more predetermined regions of the RNA. In some embodiments, (i) at least one, but not all of the adenosine nucleotides are modified, (ii) at least one, but not all of the guanosine nucleotides are modified, (iii) at least one, but not all of the cytidine nucleotides are modified, (iv) at least one of the uridine nucleotides are modified, or (v) any combination of (i), (ii), (iii), or (iv),

[0059] The “5' cap region”, as used herein, refers to a region of an RNA that is 5' to (upstream of) the ORF. In some embodiments, the 5’ cap region comprises a 5’ untranslated region (5’ UTR). In some embodiments, the 5’ cap region comprises a 5’ cap. In eukaryotic cells, mRNAs possess a cap structure in which an N7-methylguanine (m7G) moiety is linked to the first transcribed nucleotide by a 5 ’-5 ’-triphosphate bridge. The 5' cap plays multiple roles in pre- mRNA splicing, mRNA export, RNA stability through blocking degradation by the 5 ’-3’ exoribonuclease (ExoN), escaping recognition of the cellular innate immune system, and the production of proteins encoded by mRNAs. The presence of a 5' cap in an mRNA facilitates the initiation of translation (see, e.g., Gallie. Genes & Dev. 1991. 5:2108-2116, and Munroe et al. Mol Cell Biol. 1990. 10(7):3441-3455). The 5' cap is added by a 5' capping enzyme, such as mRNA guanylyltransferase. Translation initiation is a rate-limiting step of mRNA translation and heavily depends on the 5’ N7-methylguanosine (m7G) cap and its interaction with eukaryotic translation initiation factors (elFs), including the cap-binding eIF4E protein. Chemical modification on or near the 5’ cap influence binding of elFs and decapping enzymes, which subsequently impact downstream mRNA translation and stability. For example, the presence of 2’ O-methyl (2’0Me) groups on the first and second transcribed nucleotides (known as Cap- 0 / 1 / 2, referring to zero, one, or two 2’0Me groups) reduces mRNA immunogenicity and increases protein expression. Additionally, N6-methyladenosine (m6A) on the first base controlsmRNA stability through increased resistance to decapping by Dcp2. Furthermore, the 5' cap stabilizes the mRNA by protecting the ORF from the activity of exonucleases, such as polynucleotide phosphorylase (PNPase), which can remove 3' and 5' nucleotides from an mRNA. As an exonuclease removes nucleotides, the mRNA becomes progressively shorter, and once all the nucleotides downstream of the open reading frame are removed, the nucleotides removed by the exonuclease will be nucleotides of the ORF. Removal of nucleotides from the ORF prevents translation of the encoded protein. Additionally, the association of an exonuclease with the mRNA near the ORF can inhibit translation by sterically hindering ribosomes and tRNAs from associating with the mRNA. The composition of a 5' cap typically comprises a 5' m7G attached to the mRNA by a 5' to 5' triphosphate intemucleotide linkage.

[0060] In some embodiments of the modified RNAs provided herein, the modified RNA comprises one or more modified nucleotides. In some embodiments of the modified RNAs provided herein, the modified RNA is an mRNA comprising one or more modified nucleotides in the 5' cap region, the 5' UTR, the ORF, the 3’ UTR, and / or the poly(A) tail. In some embodiments, the modified RNA includes one or more nucleotides that are not canonical adenosine, cytidine, guanosine, or uridine nucleotides. In some embodiments, the modified RNA is an mRNA in which the 5' cap region, the 5' UTR, the ORF, or the 3’ UTR includes one or more nucleotides that are not canonical adenosine, cytidine, guanosine, or uridine nucleotides. In some embodiments, the modified RNA is a non-coding RNA that does not comprise an ORF, and that includes one or more nucleotides that are not canonical adenosine, cytidine, guanosine, or uridine nucleotides. Non-limiting examples of non-coding RNAs that can be incorporated into the disclosed compositions and used for the disclosed purposes include guide RNA, sgRNA (single guide RNA), pegRNA (prime editing guide RNA), and other guide / template donor RNA species for gene editing.

[0061] In some embodiments, the modified RNA is a linear RNA. In some embodiments, the modified RNA is a circular RNA.(0(162) In some embodiments, the modified RNA comprises two or more adenosine nucleotides, two or more guanosine nucleotides, two or more cytidine nucleotides, and two or more uridine nucleotides. In some embodiments, at least one of the adenosine nucleotides are modified. In some embodiments, at least one, but not all of the adenosine nucleotides are modified. In some embodiments, all of the adenosine nucleotides are modified. In some embodiments, at least one of the guanosine nucleotides are modified. In some embodiments, at least one, but not all of the guanosine nucleotides are modified. In some embodiments, all of the guanosine nucleotides are modified. In some embodiments, at least one of the cytidine nucleotides are modified. In some embodiments, at least one, but not all of the cytidine nucleotides are modified. In some embodiments, all of the cytidine nucleotides are modified. In some embodiments, at least one of the uridine nucleotides are modified. In some embodiments, at least one, but not all of the uridine nucleotides are modified. In some embodiments, all of the uridine nucleotides are modified.

[0063] In some embodiments, all modified adenosine nucleotides, all modified guanosine nucleotides, all modified cytidine nucleotides, and all modified uridine nucleotides are localized non-randomly to one or more predetermined regions of the RNA. For example, a modified RNA can include a first region comprising a 5’ UTR, an ORF, and a 3’ UTR, and a second region comprising a poly(A) tail, wherein all of the adenosines in the first region are unmodified, and wherein all of the adenosines in the second region are modified. In another illustrative example, a modified RNA can include a first region and a second region, wherein all of the adenosines in the first region are modified, wherein all of the adenosines in the second region are unmodified, wherein all of the uridines in the first region are unmodified, and wherein all of the uridines in the second region are modified.|0064| In some embodiments, the modified RNA comprises two or more adenosine nucleotides, two or more guanosine nucleotides, two or more cytidine nucleotides, and two or more uridine nucleotides, wherein: (i) at least one, but not all of the adenosine nucleotides are modified, (ii) at least one, but not all of the guanosine nucleotides are modified, (iii) at least one, but not all of the cytidine nucleotides are modified, (iv) at least one of the uridine nucleotides aremodified, or (v) any combination of (i), (ii), (iii), or (iv), wherein all modified adenosine nucleotides, all modified guanosine nucleotides, all modified cytidine nucleotides, and all modified uridine nucleotides are localized non-randomly to one or more predetermined regions of the RNA. In some embodiments, the modified RNA comprises a 5' cap region, an open reading frame (ORF), and a 3' untranslated region (UTR), wherein in each of the 5' cap region and the 3' UTR at least one adenosine nucleotide is modified, and in the ORF all adenosine nucleotides are unmodified. In some embodiments, the adenosine nucleotides that are modified are selected from 2' O-methyl (2'0Me) adenosine and N6-methyladenosine (m6A).

[0065] In some embodiments of a modified RNA, all of the uridine nucleotides are unmodified. In some embodiments of a modified RNA, all of the uridine nucleotides are modified. In some embodiments of a modified RNA, at least one of the uridine nucleotides are modified. In some embodiments of a modified RNA, the uridine nucleotides that are modified are N1 -methylpseudouridine (ml ).

[0066] In some embodiments, the modified RNA comprises at least two distinct regions. Accordingly, a modified RNA can include a first region and a second region. In some embodiments, the modified RNA comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 or more distinct regions. In some embodiments a distinct region comprises at least 3 nucleotides, at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, at least 2000 nucleotides, at least 5000 nucleotides. In some embodiments, each of the at least two distinct regions comprises a different collection of modified and unmodified nucleotides. For example, in some embodiments, all of the adenosines and guanosines of the first region are modified, and no other nucleotides of the first region are modified, whereas all of the cytidines of the second region are modified, and no other nucleotides of the second region are modified.

[0067] In some embodiments, the modified RNA comprises at least a first region and a second region, the first region and the second region each independently comprising adenosinenucleotides, guanosine nucleotides, cytidine nucleotides, and uridine nucleotides; wherein: (i) each adenosine nucleotide in the first region is a modified adenosine and each adenosine nucleotide in the second region is an unmodified adenosine; (ii) each adenosine nucleotide in the first region is an unmodified adenosine and each adenosine nucleotide in the second region is a modified adenosine; (iii) each guanosine nucleotide in the first region is a modified guanosine and each guanosine nucleotide in the second region is an unmodified guanosine; (iv) each guanosine nucleotide in the first region is an unmodified guanosine and each guanosine nucleotide in the second region is a modified guanosine; (v) each cytidine nucleotide in the first region is a modified cytidine and each cytidine nucleotide in the second region is an unmodified cytidine; or (vi) each cytidine nucleotide in the first region is an unmodified cytidine and each cytidine nucleotide in the second region is a modified cytidine.(0068] In some embodiments, (i) all of the uridine nucleotides in the first region and the second region are modified; (ii) each uridine nucleotide in the first region is a modified uridine and each uridine nucleotide in the second region is an unmodified uridine; or (iii) each uridine nucleotide in the first region is an unmodified uridine and each uridine nucleotide in the second region is a modified uridine. In some embodiments, all of the uridine nucleotides in the first region and the second region are modified. In some embodiments, the uridine nucleotides that are modified are N1 -methylpseudouridine (m l ).

[0069] In some embodiments, the RNA comprises a 5’UTR, an open reading frame (ORF), a 3' UTR, and a poly(A) tail. In some embodiments, the RNA comprises a 5’UTR, an open reading frame (ORF), a 3' UTR, and a poly(A) tail, wherein: each of the adenosines in the 5’UTR is a 2’OMe-modified adenosine, each of the guanosines in the 5’UTR is a 2’OMe- modified guanosine, each of the cytidines in the 5’UTR is a 2’OMe-modified cytidine, and each of the uridines in the 5’UTR is a 2’OMe-modified uridine; and wherein: each of the adenosines in the ORF and the 3 ’UTR is an unmodified adenosine, each of the guanosines in the ORF and the 3 ’UTR is an unmodified guanosine, each of the cytidines in the ORF and the 3 ’UTR is an unmodified cytidine, and each of the uridines in the ORF and the 3 ’UTR is an Nl- methylpseudouridine (ml'P).

[0070] In some embodiments, the RNA comprises a 5’UTR, an open reading frame (ORF), a 3' UTR, and a poly(A) tail, wherein the 5’UTR comprises a 5’ half and a 3’ half, wherein the 5’ half comprises 50% of the nucleotides of the 5’UTR that are closest to the 5’ end of the 5’UTR, wherein the 3’ half comprises 50% of the nucleotides of the 5’UTR that are closest to the 3’ end of the 5’UTR. In some embodiments, the RNA comprises a 5’UTR, an open reading frame (ORF), a 3' UTR, and a poly(A) tail, wherein the 5’UTR comprises a 5’ half and a 3’ half, wherein the 5’ half comprises 50% of the nucleotides of the 5’UTR that are closest to the 5’ end of the 5’UTR, wherein the 3’ half comprises 50% of the nucleotides of the 5’UTR that are closest to the 3’ end of the 5’UTR, wherein: each of the adenosines in the 5’ half of the 5’UTR is a 2’OMe-modified adenosine, each of the guanosines in the 5’ half of the 5’UTR is a 2’0Me- modified guanosine, each of the cytidines in the 5’ half of the 5’UTR is a 2’OMe-modified cytidine, and each of the uridines in the 5’ half of the 5’UTR is a 2’OMe-modified uridine; and wherein: each of the adenosines in the 3’ half of the 5’UTR, the ORF, and the 3 ’UTR is an unmodified adenosine, each of the guanosines in the 3’ half of the 5’UTR, the ORF, and the 3 ’UTR is an unmodified guanosine, each of the cytidines in the 3’ half of the 5’UTR, the ORF, and the 3 ’UTR is an unmodified cytidine, and each of the uridines in the 3’ half of the 5’UTR, the ORF, and the 3 ’UTR is an N1 -methylpseudouridine (ml'P). In some embodiments, each of the adenosines in the poly(A) tail is an N6-methyladenosine (m6A).

[0071] In some embodiments, the modified RNA comprises a modified nucleotide comprising a modified phosphate, resulting in a modified internucleotide linkage. Modified phosphates used in the present invention may be, but are not limited to, phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3 '-O-methylphosphonate, 5 '-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate. In some embodiments, more than one modified phosphate is used. In some embodiments, the modified RNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more modified phosphates. In someembodiments, the modified RNA comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified phosphates. In some embodiments, the modified phosphates of the modified RNA comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total phosphates in the modified RNA.

[0072] In some embodiments, the modified RNA comprises a modified nucleotide comprising a modified sugar. Modified sugars used in the present invention may be, but are not limited to, 2'-deoxy fluoro (2FA), Z-adenosine (ZA), 2 '-deoxy adenosine (dA), locked nucleic acid (LNA), 2'-methoxy (20Me), 2 '-methoxy ethoxy (2M0E), 2'-thioribose, 2', 3 '-dideoxyribose, 2'-amino-2'-deoxyribose, 2' deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'- O-methylribose, 2'-O-methyldeoxyribose, 3 '-amino-2', 3 '-di deoxyribose, 3'-azido-2',3'- dideoxyribose, 3 ’-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'- aminoribose, 5 '-thioribose, 5-nitro-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C- methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose. Z-adenosine (ZA) refers to the enantiomer of Z>-adenosine. A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2’ and 4’ carbons. This structure effectively “locks” the ribose in the 3’-endo structural conformation. In some embodiments, the modified RNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more modified sugars. In some embodiments, the modified RNA comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified sugars. In some embodiments, the modified sugars of the modified RNA comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total sugars in the modified RNA.

[0073] In some embodiments, the modified RNA comprises a modified nucleotide comprising a modified nucleobase. Modified nucleobases used in the present invention may be,but are not limited to, inosine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenosine, methylpseudouracil, 2-thiocytosine, 2- thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6- dihydrouracil, 5-[(3-Indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5- aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5- carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5- hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5- iodocytosine, 5-iodouracil, 5 -meth oxy cytosine, 5-methoxyuracil, 5 -methyl cytosine, 5- methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5- propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7- deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8- azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin- 16-7-deaza-7-propargylaminoguanine, biotin- 16- aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3- 6-propargylaminouracil, cyanine 3 -aminoallylcytosine, cyanine 3 -aminoallyluracil, cyanine 5-6- propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, Nl- ethylpseudouracil, N1 -methoxymethylpseudouracil, N1 -methyladenine, N1 -methylpseudouracil, N1 -propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6- methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3 -deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl- thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis- hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2- methylthio-N6-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, the modified RNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more modified nucleobases. In some embodiments, the modified RNA comprises between 1 and 3, between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 modified nucleobases. In some embodiments, the modified nucleobases of the modified RNA comprise about 3%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% of the total nucleobases in the modified RNA.

[0074] In some embodiments, the modified RNA comprises a 5’ cap. The 5’ cap used in accordance with the present disclosure may be, but is not limited to, 7-methyguanosine (m7G), N7, 3 ’ -O-dimethyl-guanosine-5 ’ -triphosphate-5 ’ -guanosine (m7 G-3 ’ m-ppp-G), N7,2 ’ -O- dimethyl-guanosine-5’ -triphosphate-5 ’-guanosine (m7Gm-ppp-G), 7-benzylguanosine (Bn7G), chlorobenzylguanosine (ClBn7G), m7G bearing an LNA sugar (m7G-LNA), chlorobenzyl-O- ethoxyguanosine (ClBnOEt7G), 7-(4-chlorophenoxyethyl)-guanosine, 7-ethyl guanosine (e7G), 7-propyl guanosine (p7G), 7-isopropyl guanosine (ip7G), 7-butyl guanosine (b7G), 7-isobutyl guanosine (ib7G), 7-cyclopentyl guanosine (cp7G), 7-(carboxymethyl) guanosine (cm7G), 7-(2- phenylethyl) guanosine [7-(2-PhEt)G], 7-(l -phenylethyl) guanosine [7-(l-PhEt)G], m7GpppBH3G (DI and D2 stereoisomers), m7GppBH3G (DI and D2 stereoisomers), m7GpBH3G (DI and D2 stereoisomers), m7GppBH3pm7G, m272'°GpppBH3G (DI and D2 stereoisomers), m27’2'^GppBmpG (DI and D2 diastereomers), m27,2’°GppspG (DI and D2 diastereomers), N- Arylmethyl analogs, glyceryl, 4',5'-methylene nucleotide, l-(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4- dihydroxybutyl nucleotide, acyclic 3, 5 -dihydroxy pentyl nucleotide, 3 '-3 '-inverted nucleotide moiety, 3 '-3 '-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, capl, cap2, cap3, cap4, ARCA, modifiedARCA, inosine, N1 -methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

[0075] In some embodiments, the modified RNA comprises an open reading frame (ORF). In some embodiments, the ORF encodes a therapeutic protein. As used herein, a “therapeutic protein” refers to a protein that prevents, reduces, or alleviates one or more signs or symptoms of a disease or disorder when expressed in a subject, such as a human subject that has, for example, an essential enzyme, clotting factor, transcription factor, growth factor, cytokine, chemokine, antibody (or antibody fragment thereof), protein hormone, signaling protein, structural protein, or cell surface receptor encoded by a gene that is mutated in a subject. A mutation in a gene encoding such a protein may cause diminished levels of the protein to be expressed in one or more cells of the subject. Expression of an essential enzyme, clotting factor, transcription factor, growth factor, cytokine, chemokine, antibody (or antibody fragment thereof), protein hormone, signaling protein, structural protein, or cell surface receptor from an RNA may therefore compensate for a mutation in the gene encoding such a protein in a subject. In some embodiments, the therapeutic protein is a protein that is expressed in one or more cells of a subject a level that is less than (e.g., significantly less than) that of a reference value, such as the level of expression of the protein that is typical in cells of one or more healthy subjects (i.e., subjects who do not have and are not at risk for developing the disease or disorder). Non-limiting examples of therapeutic proteins include base editors (e.g., adenine base editors or RNA base editors), CRISPR-associated proteins (Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Casl2 [Cpfl], or Casl3 [C2c2] endonuclease), RNase proteins e.g., RNase III), hormones (e.g., insulin, renin, parathyroid hormone, thyroid hormone), thrombin, fibrinogen, metabolic enzymes, erythropoietin (EPO), growth hormone (e.g., GSH), interferons, antibodies (e.g., monoclonal antibodies), colony-stimulating factors (CSFs, e.g., granulocyte colonystimulating factor [G-CSF]), tissue plasminogen activator (tPA), Factor VIII, Factor IX, enzymes (e.g., for conditions such as Gaucher’s disease or Fabry disease), interleukins, bone morphogenic proteins (BMPs), relaxin, alpha- 1 antitrypsin, filgrastim, oxytocin, somatostatin, calcitonin, glucagon, liraglutide, vasopressin, epigenetic modulating proteins, and growth factors.

[0076] In some embodiments, the ORF encodes an antigen. As used herein, “antigen” refers to a molecule (e. ., a protein) that, when expressed in a subject, elicits the generation of antibodies in the subject that bind to the antigen. In some embodiments, the antigen is a protein derived from a pathogen, such as a pathogenic virus, bacterium, protozoan, or fungus. In some embodiments, the antigen is a protein derived from a virus (viral antigen) or a fragment thereof. In some embodiments, the antigen is a protein derived from a bacterium (bacterial antigen) or a fragment thereof. In some embodiments, the antigen is a protein derived from a protozoan (protozoal antigen) or a fragment thereof. In some embodiments, the antigen is a protein derived from a fungus (fungal antigen) or a fragment thereof. A fragment of a full-length protein refers to a protein with an amino acid sequence that is present in, but shorter than, the amino acid sequence of the full-length protein. Thus, in some embodiments, the RNA transcripts produced by the methods provided herein may be used for prophylactic purposes, such as for vaccination of a subject.Producing modified Nucleic Acids comprising region-specific nucleotide modifications

[0077] For the purposes of the present disclosure, the modified nucleic acids may be RNAs (e.g., mRNAs or non-coding RNAs) or RNA-DNA hybrids.

[0078] In some aspects, the methods provided herein produce an RNA with regionspecific modifications.

[0079] In some aspects, provided is a method for producing a RNA, comprising sequential and phased annealing and extension of an RNA primer to at least two single stranded DNA (ssDNA) template polynucleotides. A ssDNA template polynucleotide can be synthesized or obtained using any known technique. In certain embodiments, at least two ssDNA template polynucleotides are obtained that have an overlap with at least one other ssDNA template polynucleotide. An overlap can include terminal nucleotides of the at least two ssDNA template polynucleotides. As used herein, the term “terminal nucleotides” means nucleotides positioned at one end of a polynucleotide. For example, 10 terminal nucleotides of a polynucleotide caninclude the 10 nucleotides closest to the 5’ end of the polynucleotide, or the 10 nucleotides closest to the 3’ end of the polynucleotide. In some embodiments, the at least two ssDNA template polynucleotides comprise an overlap. In certain embodiments, at least two ssDNA template polynucleotides are obtained that have at least 3 nucleotides of overlap with at least one other ssDNA template polynucleotide, based on a reference sequence. For example, the ssDNA template polynucleotides can have at least 1, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1000 overlapping nucleotides. In some embodiments, the ssDNA template polynucleotides have between about 10 to about 20 overlapping nucleotides. In some embodiments, the ssDNA template polynucleotides have no more than about 20 overlapping nucleotides. The number of ssDNA template polynucleotides used in the method for producing an RNA is not particularly limited. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 or more ssDNA template polynucleotides are obtained.10080| In some embodiments of a method for producing a RNA, an RNA primer is annealed to a first ssDNA template polynucleotide. The RNA primer and the first ssDNA template polynucleotide can then be contacted with an engineered primer-dependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA primer. Engineered primer-dependent RNA polymerases are known in the art. Examples of engineered primer-dependent RNA polymerases include but are not limited to TGK polymerase, a T7 polymerase, or a SP6 polymerase. Numerous TGK polymerase variants are suitable for use in accordance with this disclosure. The polymerase from which TGK polymerase is derived is Tgo DNA polymerase. TGK is Tgo DNA polymerase having the following amino acid substitutions: V93Q, D141A, E143A, Y409G, A485L, and E664K. TGK polymerase is described in Cozens et al. (2012), A short adaptive path from DNA to RNA polymerases, PNAS, 109(21), 8067-8072, which is incorporated herein by reference in its entirety. Other TGK polymerase variants include TGLLK (Tgo DNA polymerase having the following amino acid substitutions: V93Q, D141A, E143A, Y409G, A485L, I521L, F545L, and E664K), also described in Cozens et al. Other engineered primer-dependent RNA polymerasesinclude TGLLK having the amino acid substitutions T541G and K592A, Tgo-D4K (Tgo having the amino acid substitutions V93Q, D141A, E143A, L403P, A485L, K569H, P657T, E658Q, T676I, Y663H, E664K, D669A, and K671N), Tgo-6G12 / 152 IL (Tgo having the amino acid substitutions V93Q, D141A, E143A, A485L, I521L, V589A, E609K, I610M, K659Q, E664Q, Q665P, R668K, D669A, T676R, A681S, K671N, K674R, L704P, and E730G), Tgo-EPFLH (Tgo having the amino acid substitutions V93Q, D141A, E143A, H147E, L403P, L408F, A485L, I521L, E664H), and Kod-RSGA (Kod having the amino acid substitutions D141A, E143A, A485R, N491S, R606G, and T723A). Tgo-D4k, Tgo-6G12 / I521L, Tgo-EPFLH, and Kod-RSGA are described in Medina et al. (2021), Functional Comparison of Laboratory- Evolved XNA Polymerases for Synthetic Biology, ACS Synth. Bio., 2021, 10(6), 1429-1437, which is incorporated herein by reference in its entirety.(0081 ] In some embodiments, the RNA primer and the first ssDNA template polynucleotide are contacted with an engineered primer-dependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA primer. The first mixture of NTPs may contain unmodified NTPs or modified NTPs. Extension of the RNA primer results in the production of a DNA / RNA duplex comprising a once-extended RNA, wherein the once-extended RNA has a sequence complementary to that of the first ssDNA template polynucleotide.[00821 Following production of the once-extended RNA, the once-extended RNA can be separated from the first ssDNA template polynucleotide. In some embodiments, separation is accomplished by enzymatic digestion. For example, the ssDNA template polynucleotide can be digested by an exonuclease enzyme. In some embodiments, the ssDNA template polynucleotide can be digested by a DNase enzyme. In some embodiments, separation is accomplished by chemical denaturation. In some embodiments, chemical denaturation comprises contacting the DNA / RNA duplex with a hydrogen-bond-disrupting agent, a chaotropic reagent, or a strong base. In some embodiments, the hydrogen-bond-disrupting agent is dimethyl sulfoxide (DMSO). In some embodiments, chemical denaturation further comprises heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C. In some embodiments, chemical denaturation further comprises heating the DNA / RNA duplex to 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, or 99°C. In some preferred embodiments, chemical denaturation further comprises heating the DNA / RNA duplex to about 60°C.

[0083] In some embodiments, the ssDNA template polynucleotide comprises a tag. In some embodiments, the tag is a hydrophobic tag or an affinity tag. In some embodiments, the tag is an FC6 hydrophobic tag. In some embodiments, the ssDNA template polynucleotide is conjugated to a solid phase. In some embodiments, the solid phase is selected from a bead or a chip. In some embodiments, separating further comprises affinity purification or reversed phase chromatography. In some embodiments, the ssDNA template oligonucleotide is recycled for subsequent use.

[0084] The once-extended RNA can be annealed to a second ssDNA template polynucleotide. In some embodiments, the second ssDNA template polynucleotide has an overlap with the first ssDNA template polynucleotide. In some embodiments, the second ssDNA template polynucleotide and the first ssDNA template polynucleotide can have at least 1, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1000 overlapping nucleotides. In some embodiments, the ssDNA template polynucleotides have between about 10 to about 20 overlapping nucleotides. In some embodiments, the ssDNA template polynucleotides have no more than about 20 overlapping nucleotides. For example, in some embodiments, the 3’ end of the first ssDNA template polynucleotide has 3 nucleotides of overlap with the 5’ end of the second ssDNA template polynucleotide. The once-extended RNA and the second ssDNA template polynucleotide can then be contacted with an engineered primer-dependent RNA polymerase and a second mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the once-extended RNA. The second mixture of NTPs may contain unmodified NTPs or modified NTPs. In someembodiments, at least one of the first mixture of NTPs and the second mixture of NTPs comprises at least one modified NTP. In some embodiments, the second mixture of NTPs is not identical to the first mixture of NTPs. In the context of mixtures of NTPs, “not identical” means that the mixtures of NTPs do not contain the same collection of classes of NTPs. Two mixtures of NTPs are “not identical” when, for example, each of the mixtures contain adenosine, guanosine, uridine and cytosine, but one mixture contains modified adenosine, and the other mixture contains unmodified adenosine. Extension of the once-extended RNA results in the production of a DNA / RNA duplex comprising a twice-extended RNA, wherein the twice- extended RNA has a sequence complementary to that of the first ssDNA template polynucleotide and the second ssDNA template polynucleotide, and overlapping nucleotides thereof. In some embodiments, the RNA produced is a region-specifically-modified RNA.(0085] In some embodiments, the method for producing an RNA further comprises separating the twice-extended RNA from the second ssDNA template polynucleotide.

[0086] In some embodiments, the method for producing a RNA further comprises annealing the twice-extended RNA to a third ssDNA template polynucleotide, wherein the second ssDNA template polynucleotide comprises an overlap with the third ssDNA template polynucleotide; and contacting the third ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a third mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the twice-extended RNA. In certain embodiments, the second ssDNA template polynucleotide and the third ssDNA template polynucleotide have an overlap, based on a reference sequence. For example, the second ssDNA template polynucleotide and the third ssDNA template polynucleotide can have at least 1, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1000 overlapping nucleotides. In some embodiments, the second ssDNA template polynucleotide and the third ssDNA template polynucleotide have between about 10 to about 20 overlapping nucleotides. In some embodiments, the second ssDNA template polynucleotide and the third ssDNA template polynucleotide have no more than about 20 overlapping nucleotides. In some embodiments, the first mixture of NTPs, the second mixture of NTPs, and the third mixture ofNTPs are not identical. In some embodiments, only two of the three mixtures of NTPs are identical. In some embodiments, each of the mixtures of NTPs comprises a different collection of classes of NTPs. In some embodiments, each of the mixtures of NTPs comprises a different collection of modified and unmodified NTPs. In some embodiments, at least one of the mixtures of NTPs comprises a different collection of modified and unmodified NTPs than at least one other mixture of NTPs. Extension of the twice-extended RNA results in the production of a DNA / RNA duplex comprising a thrice-extended RNA, wherein the thrice-extended RNA has a sequence complementary to that of the first ssDNA template polynucleotide, the second ssDNA template polynucleotide, the third ssDNA template polynucleotide, and overlapping nucleotides thereof.

[0087] In some embodiments, the method for producing an RNA further comprises separating the thrice-extended RNA from the third ssDNA template polynucleotide.

[0088] In some embodiments, the method for producing a RNA further comprises annealing the thrice-extended RNA to a fourth ssDNA template polynucleotide, wherein the third ssDNA template polynucleotide comprises an overlap with the fourth ssDNA template polynucleotide; and contacting the fourth ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a fourth mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the thrice-extended RNA. In some embodiments, the first mixture of NTPs, the second mixture of NTPs, the third mixture of NTPs, and the fourth mixture of NTPs are not identical. In certain embodiments, the third ssDNA template polynucleotide and the fourth ssDNA template polynucleotide have an overlap, based on a reference sequence. For example, the third ssDNA template polynucleotide and the fourth ssDNA template polynucleotide can have at least 1, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1000 overlapping nucleotides. In some embodiments, the third ssDNA template polynucleotide and the fourth ssDNA template polynucleotide have between about 10 to about 20 overlapping nucleotides. In some embodiments, the third ssDNA template polynucleotide and the fourth ssDNA template polynucleotide have no more than about 20 overlapping nucleotides. In some embodiments,only two of the three mixtures of NTPs are identical. In some embodiments, only three of the three mixtures of NTPs are identical. In some embodiments, each of the mixtures of NTPs comprises a different collection of classes of NTPs. In some embodiments, each of the mixtures of NTPs comprises a different collection of modified and unmodified NTPs. In some embodiments, at least one of the mixtures of NTPs comprises a different collection of modified and unmodified NTPs than at least one other mixture of NTPs. Extension of the region thrice- extended RNA results in the production of a DNA / RNA duplex comprising a four-times- extended RNA, wherein the four-times-extended RNA has a sequence complementary to that of the first ssDNA template polynucleotide, the second ssDNA template polynucleotide, the third ssDNA template polynucleotide, the fourth ssDNA template polynucleotide, and overlapping nucleotides thereof.(0089] In some embodiments, the method for producing an RNA further comprises separating the four-times-extended RNA from the fourth ssDNA template polynucleotide.

[0090] In some embodiments, the method comprises annealing, and polymerizing of at least five, at least six, at least seven, at least eight, at least nine, or at least ten ssDNA template polynucleotides.

[0091] A modified nucleotide can be a modified nucleotide comprising a modified nucleobase, a modified sugar, a modified phosphate, and / or a modified 5’ cap, such as but not limited to those described herein.

[0092] In some embodiments, the ssDNA template polynucleotides comprise a 5’- untranslated region (5’UTR) ssDNA template polynucleotide. In some embodiments, the ssDNA template polynucleotides comprise a coding sequence (CDS) ssDNA template polynucleotide. In some embodiments, the ssDNA template polynucleotides comprise a 3 ’-untranslated region (3’UTR) ssDNA template polynucleotide. In some embodiments, the ssDNA template polynucleotides comprise a poly(A) tail ssDNA template polynucleotide. In some embodiments, the ssDNA template polynucleotides comprise a 5 ’-untranslated region (5’UTR) ssDNAtemplate polynucleotide, a coding sequence (CDS) ssDNA template polynucleotide, a 3’- untranslated region (3’UTR) ssDNA template polynucleotide, and a poly(A) tail ssDNA template polynucleotide.

[0093] In some embodiments, the method for producing an RNA comprises circularizing the RNA. Technologies for the circularization of RNAs are well understood in the art. For example, circularization can be accomplished by RNA ligation. When a ligase forms a covalent bond between two ends of a linear RNA, a circular RNA is produced. Ligation of the 3’ terminal nucleotide of a linear nucleic acid to the 5’ terminal nucleotide of the linear nucleic acid produces a circular nucleic acid. Ligation by an RNA ligase occurs in several steps. First, an amino (-NH2) group of an amino acid (e.g., a lysine) of the ligase bonds to a phosphate group of adenosine triphosphate (ATP), such that an adenosine monophosphate (AMP) group is bound to the RNA ligase. Second, a 5' terminal phosphate of the second nucleic acid displaces the phosphate of the RNA ligase-bound AMP. Finally, an oxygen of the 3' terminal hydroxyl group of the first nucleic acid binds to the phosphorus atom of the 5' terminal phosphate of the second nucleic acid. This final step forms a phosphodiester bond between terminal nucleotides of the nucleic acids, thereby forming a single nucleic acid with a continuous sugar-phosphate backbone. In some embodiments, the ligase is T4 RNA Ligase I, T4 RNA Ligase II, or RtcB. In some embodiments, the ligation is performed using a split ribozyme (see, e.g., Gambill et al., “A split ribozyme that links detection of a native RNA to orthogonal protein outputs.” Nat Commun 14, 543 (2023)). Circularization can also be accomplished by back splicing, such as ribozyme- mediated back splicing.(0094] In some aspects, provided is a method for producing a circular RNA (circRNA), comprising annealing an RNA primer to a single-stranded circular DNA template polynucleotide, and extension of the RNA primer. In some embodiments, an RNA primer is an RNA produced by a method described herein. In some embodiments, all of the nucleotides of the RNA primer are unmodified. In some embodiments, all of the adenosine nucleotides of the RNA primer are modified. In some embodiments, all of the cytosine nucleotides of the RNA primer are modified. In some embodiments, all of the guanosine nucleotides of the RNA primer aremodified. In some embodiments, all of the uridine nucleotides of the RNA primer are modified. In some embodiments, all of the adenosine nucleotides of the RNA primer are unmodified. In some embodiments, all of the cytosine nucleotides of the RNA primer are unmodified. In some embodiments, all of the guanosine nucleotides of the RNA primer are unmodified. In some embodiments, all of the uridine nucleotides of the RNA primer are unmodified. In some embodiments, at least one of the nucleotides of the RNA primer is modified. In some embodiments, at least one of the adenosine nucleotides of the RNA primer is modified. In some embodiments, at least one of the cytosine nucleotides of the RNA primer is modified. In some embodiments, at least one of the guanosine nucleotides of the RNA primer is modified. In some embodiments, at least one of the uridine nucleotides of the RNA primer is modified. Optionally, the RNA primer may be capped (e.g., when preparing an mRNA) or uncapped (e.g., when preparing a non-coding RNA).

[0095] In some embodiments of a method for producing a circular RNA, an RNA has a length of at least 5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 500 nucleotides, at least 800 nucleotides, or at least 1000 nucleotides. The size of the RNA primer is not particularly limited.(0096] In some embodiments, the RNA primer is a capped RNA primer. In some embodiments, the RNA primer is a 5’ phosphorylated RNA primer.

[0097] In some embodiments, an RNA produced by methods described herein is a linear RNA. In some embodiments, a linear RNA comprises an exon region and at least one intron region. In some embodiments, a linear RNA comprises an exon region flanked by two intron regions. In some embodiments, a linear RNA comprises an exon region flanked by two intron regions, wherein the intron regions do not contain any modified nucleotides, and wherein the exon region comprises at least one modified nucleotide.

[0098] In some embodiments, a linear RNA produced by methods described herein can be circularized. In some embodiments, circularization comprises RNA ligation. In some embodiments, circularization comprises split ligation, such as splint ligation by T4 RNA ligase II. In some embodiments, circularizing comprises ribozyme-mediated back splicing.|0099| In some aspects, provided is a method for producing a RNA, comprising: (a) annealing a RNA primer to a first single stranded DNA (ssDNA) template polynucleotide, wherein the first ssDNA template polynucleotides comprises an overlap with a second ssDNA template polynucleotide; (b) contacting the first ssDNA template polynucleotide with an engineered primer-dependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA primer, thereby producing a once- extended RNA; (c) separating the once-extended RNA from the first ssDNA template polynucleotide; (d) annealing the once-extended RNA to the second ssDNA template polynucleotide; and (e) contacting the second ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a second mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the once-extended RNA, thereby producing a DNA / RNA duplex comprising a twice-extended RNA. In certain embodiments, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide have an overlap, based on a reference sequence. For example, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide can have at least 1, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1000 overlapping nucleotides. In some embodiments, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide have between about 10 to about 20 overlapping nucleotides. In some embodiments, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide have no more than about 20 overlapping nucleotides.Optionally, the RNA primer may be capped (e.g., when preparing an mRNA) or uncapped (e.g., when preparing a non-coding RNA).

[0100] In some aspects, provided is a method for producing a circular RNA (circRNA), comprising: (a) annealing a RNA primer to a first single stranded DNA (ssDNA) templatepolynucleotide, wherein the first ssDNA template polynucleotides comprises an overlap with a second ssDNA template polynucleotide; (b) contacting the first ssDNA template polynucleotide with an engineered primer-dependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA primer, thereby producing a DNA / RNA duplex comprising a once-extended RNA; (c) separating the once- extended RNA from the first ssDNA template polynucleotide; (d) annealing the once-extended RNA to the second ssDNA template polynucleotide; (e) contacting the second ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a second mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the once-extended RNA, thereby producing a DNA / RNA duplex comprising a twice-extended RNA, (f) separating the twice-extended RNA from the second ssDNA template polynucleotide, thereby producing a linear RNA; and (g) circularizing the linear RNA, thereby producing a circRNA. In certain embodiments, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide have an overlap, based on a reference sequence. For example, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide can have at least 1, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1000 overlapping nucleotides. In some embodiments, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide have between about 10 to about 20 overlapping nucleotides. In some embodiments, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide have no more than about 20 overlapping nucleotides. Optionally, the RNA primer may be capped (e.g., when preparing an mRNA) or uncapped (e.g., when preparing a non-coding RNA).[01011 In some aspects, provided is a method for producing a circular RNA (circRNA), comprising: (a) annealing a 5’ phosphorylated region 1 RNA or once-extended RNA primer to a circular single stranded DNA (ssDNA) template polynucleotide; (b) contacting the circular ssDNA template polynucleotide with an engineered primer-dependent RNA polymerase and a mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the region 1 RNA primer, thereby producing a synthesized RNA comprising the region 1 RNA primer and aregion 2 RNA; (c) ligating the synthesized RNA; and (d) separating the synthesized RNA from the circular ssDNA template polynucleotide, thereby producing the circRNA.

[0102] In some aspects, provided is a method for producing a DNA-RNA-DNA (DRD) hybrid polynucleotide. As used herein, the terms “DNA-RNA-DNA hybrid polynucleotide” and “DRD hybrid polynucleotide” refers to a polynucleotide composed of both DNA and RNA. In some embodiments, the DRD hybrid polynucleotide is a single stranded polynucleotide. In some embodiments, the DRD hybrid polynucleotide comprises an RNA polynucleotide flanked by two DNA polynucleotides. In some embodiments, the DRD hybrid polynucleotide comprises at least one 5’ cap. In some embodiments, the DRD hybrid polynucleotide comprises branched caps.

[0103] In some embodiments of a DRD hybrid polynucleotide comprising an RNA polynucleotide flanked by two DNA polynucleotides, the two DNA polynucleotides have sufficient homology to permit the formation of a dumbbell DRD unit. The dumbbell DRD unit can include a double stranded DNA portion and a circular RNA portion. In some embodiments, a dumbbell DRD unit can be ligated to another dumbbell DRD unit to form a DRD comprising a double stranded DNA portion and two circular RNA portions. In some embodiments, a dumbbell DRD unit can be ligated to two other dumbbell DRD units to form a DRD hybrid comprising three double stranded DNA portions and three circular RNA portions. The circular RNA portions can include non-coding RNA or protein-coding RNA. In some embodiments, the DRD hybrid includes at least one cap. In some embodiments, the DRD hybrid includes branched caps.

[0104] In some embodiments of a method of producing a DRD hybrid polynucleotide comprises: (a) annealing a single stranded DNA (ssDNA) primer to a first single stranded DNA (ssDNA) template polynucleotide; (b) contacting the first ssDNA template polynucleotide with an engineered primer-dependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the ssDNA primer, thereby producing a DNA / RNA duplex comprising a DNA-RNA hybrid polynucleotide; (c) separating the first ssDNA template polynucleotide from the DNA-RNA hybrid polynucleotide; (d) annealing the DNA-RNA hybrid polynucleotide to the second ssDNA template polynucleotide; and (e)contacting the second ssDNA template polynucleotide with a DNA polymerase and a second mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the DNA- RNA hybrid polynucleotide, thereby producing a DNA / RNA duplex comprising a DRD hybrid polynucleotide comprising a single stranded RNA (ssRNA) polynucleotide flanked by two single stranded DNA (ssDNA) polynucleotides. In some embodiments, the DNA polymerase is the same as the RNA polymerase from (b) or an RNA-primer dependent polymerase such as TGK. In some embodiments, the first ssDNA template polynucleotides comprises an overlap with a second ssDNA template polynucleotide. In certain embodiments, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide have an overlap, based on a reference sequence. For example, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide can have at least 1, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1000 overlapping nucleotides. In some embodiments, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide have between about 10 to about 20 overlapping nucleotides. In some embodiments, the first ssDNA template polynucleotide and the second ssDNA template polynucleotide have no more than about 20 overlapping nucleotides.

[0105] Separating can include digesting the first ssDNA template polynucleotide. Digesting the first ssDNA template polynucleotide can be accomplished using a 3' to 5' DNA exonuclease that does not digest DNA-RNA chimeras having RNA on the 3' end, such as but not limited to those described at neb.com / en-us / tools-and-resources / selection-charts / properties-of- exonucleases-and-nonspecific-endonucleases. In some embodiments, the RNA-primer-dependent DNA polymerase is a polymerase that is capable of polymerizing DNA from an RNA primer, such as TGK polymerase. In some embodiments, the method further comprises separating the second ssDNA template polynucleotide from the DRD hybrid polynucleotide. Separating can include digesting the second ssDNA template polynucleotide. In some embodiments, the second ssDNA template polynucleotide can contain uridines (U) instead of thymidines (T). Digesting the second ssDNA template polynucleotide can be accomplished by contacting the second ssDNA template polynucleotide with a U-specific DNase enzyme, if the second ssDNA templatepolynucleotide contains uridines (U) instead of thymidines (T). In some embodiments, separating can be accomplished by chemical denaturation as described herein.

[0106] In some embodiments, the two ssDNAs have sufficient homology to permit the formation of a dumbbell shape, thereby producing a dumbbell DRD unit. In some embodiments, the method further comprises ligating the DRD hybrid polynucleotide to at least one capped RNA-DNA hybrid polynucleotide. In some embodiments, the method comprises ligating the DRD hybrid polynucleotide to at least two capped RNA-DNA hybrid polynucleotides. In some embodiments, the ligating produces a multi-cistronic RNA. In some embodiments, the multi- cistronic RNA comprises branched caps. In some embodiments, the method further comprises ligating the dumbbell DRD unit to at least one other dumbbell DRD unit. In some embodiments, the method further comprises ligating the dumbbell DRD unit to at least two other dumbbell DRD units.

[0107] In some embodiments, at least one of the dumbbell DRD units comprises a branched cap. In some embodiments, the ssRNA comprises unmodified nucleotides. In some embodiments, the ssRNA comprises at least 1 modified nucleotide.|0108| The disclosed template switching methods may include alteration of various parameter, particularly when preparing the poly(A)-tailing of a modified RNA. Poly(A)-tailing of RNA by direct extension of an RNA primer along a polyT DNA template can lead to over- extension of the poly(A) tail, but over-extension can be eliminated or minimized by appropriately adjusting the parameters.10109] For example, over-extension of the poly(A) tail may be reduced or eliminated by reducing the concentration of ATP in the reaction solution. Thus, the concentration of ATP may be about 5 mM or less, about 4.5 mM or less, about 4 mM or less, about 3.5 mM or less, about 3 mM or less, about 2.5 mM or less, about 2 mM or less, about 1.5 mM or less, about 1 mM or less, about 0.95 mM or less, about 0.9 mM or less, about 0.85 mM or less, about 0.8 mM or less, about 0.75 mM or less, about 0.7 mM or less, about 0.65 mM or less, about 0.6 mM or less,about 0.55 mM or less, about 0.5 mM or less, about 0.45 mM or less, about 0.4 mM or less, about 0.35 mM or less, about 0.3 mM or less, about 0.25 mM or less, about 0.2 mM or less, about 0.15 mM or less, or about 0.1 mM or less. In some embodiments, the concentration of AT may be 0.1 mM to 5 mM, 0.2 mM to 5 mM, 0.3 mM to 5 mM, 0.4 mM to 5 mM, 0.5 mM to 5 mM, 0.6 mM to 5 mM, 0.7 mM to 5 mM, 0.8 mM to 5 mM, 0.9 mM to 5 mM, 0.1 mM to 4 mM, 0.2 mM to 4 mM, 0.3 mM to 4 mM, 0.4 mM to 4 mM, 0.5 mM to 4 mM, 0.6 mM to 4 mM, 0.7 mM to 4 mM, 0.8 mM to 4 mM, 0.9 mM to 4 mM, 0.1 mM to 3 mM, 0.2 mM to 3 mM, 0.3 mM to 3 mM, 0.4 mM to 3 mM, 0.5 mM to 3 mM, 0.6 mM to 3 mM, 0.7 mM to 3 mM, 0.8 mM to 3 mM, 0.9 mM to 3 mM, 0.1 mM to 2 mM, 0.2 mM to 2 mM, 0.3 mM to 2 mM, 0.4 mM to 2 mM, 0.5 mM to 2 mM, 0.6 mM to 2 mM, 0.7 mM to 2 mM, 0.8 mM to 2 mM, 0.9 mM to 2 mM, 0.1 mM to 1 mM, 0.2 mM to 1 mM, 0.3 mM to 1 mM, 0.4 mM to 1 mM, 0.5 mM to 1 mM, 0.6 mM to 1 mM, 0.7 mM to 1 mM, 0.8 mM to 1 mM, 0.9 mM to 1 mM, 0.1 mM to 0.9 mM, 0.2 mM to 0.9 mM, 0.3 mM to 0.9 mM, 0.4 mM to 0.9 mM, 0.5 mM to 0.9 mM, 0.6 mM to 0.9 mM, 0.1 mM to 0.8 mM, 0.2 mM to 0.8 mM, 0.3 mM to 0.8 mM, 0.8 mM to 0.8 mM, 0.8 mM to 0.8 mM, 0.6 mM to 0.8 mM, 0.1 mM to 0.7 mM, 0.2 mM to 0.7 mM, 0.3 mM to 0.7 mM, 0.4 mM to 0.7 mM, 0.5 mM to 0.7 mM, 0.6 mM to 0.7 mM, 0.1 mM to 0.6 mM, 0.2 mM to 0.6 mM, 0.3 mM to 0.6 mM, 0.4 mM to 0.6 mM, or 0.5 mM to 0.6 mM.|0110| Similarly, chain-terminating dideoxynucleotides can be incorporated into a DNA template to mitigate poly(A) tail overextension. A single G / C / A can be included at the end of the polyT DNA template, and a corresponding dideoxyNTP (ddNTP) can be included in the reaction mixture.(0111 ] Similarly, manipulation of reaction temperature can reduce or eliminate over- extension. Reduction of reaction temperature may reduce over-extension, but also reduce RNA yield. Nevertheless, in some embodiments, may be about 50°C, about 51 °C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, or about 65°C.

[0112] Finally, manipulation of salt concentration may also reduce or eliminate poly(A) over-extension. In particular, increasing the concentration of salt (e.g., NaCl) in the reaction mixture can reduce over-extension without compromising RNA yield. Thus, the salt content may be about 1 mM or more, about 2 mM or more, about 3 mM or more, about 4 mM or more, about 5 mM or more, about 6 mM or more, about 7 mM or more, about 8 mM or more, about 9 mM or more, about 10 mM or more, about 15 mM or more, about 20 mM or more, about 25 mM or more, about 30 mM or more, about 35 mM or more, about 40 mM or more, about 45 mM or more, about 50 mM or more, about 75 mM or more, about 100 mM or more, about 125 mM or more, about 150 mM or more, about 175 mM or more, about 200 mM or more, about 225 mM or more, about 250 mM or more, about 275 mM or more, about 300 mM or more, about 325 mM or more, about 350 mM or more, about 375 mM or more, about 400 mM or more, about 425 mM or more, about 450 mM or more, about 475 mM or more, or about 500 mM or more. 011.3] In some embodiments of a method of producing an RNA, the method does not comprise single nucleotide incorporation (SNI). In some embodiments of a method of producing an RNA, the method does not comprise single nucleotide incorporation (SNI) of a modified nucleotide. In some embodiments of a method of producing an RNA, the method does not comprise stepwise SNI. In some embodiments of a method of producing an RNA, the method does not comprise stepwise SNI of modified nucleotides.Compositions comprising modified Nucleic Acids with region-specific nucleotide modifications|0114| For the purposes of the present disclosure, the modified nucleic acids may be RNAs (e.g., mRNAs or non-coding RNAs) or RNA-DNA hybrids.

[0115] In some aspects, the present disclosure provides a delivery reagent comprising any of the modified RNAs or DRD hybrid polynucleotides provided herein. In some embodiments, any of the modified RNAs or DRD hybrid polynucleotides provided herein are conjugated to a delivery agent. Any of the modified RNA or DRD hybrid polynucleotidesprovided herein may be conjugated to a delivery agent that includes, for example, to a lipid, a peptide, a protein, an antibody, or a carbohydrate. Lipids used in the conjugation and delivery of RNAs are generally known in the art, and include, for example, cholesterol. Peptides, proteins, antibodies, and carbohydrates used in the conjugation and delivery of modified RNAs or DRD hybrid polynucleotides are generally known in the art and include, for example, any peptide, protein, antibody, or carbohydrate known to bind specifically to a moiety (e.g., a protein) on the surface of a target cell type. Methods for conjugating a lipid, peptide, protein, antibody, or carbohydrate to a modified RNA include, for example, methods of conjugating a lipid, peptide, protein, antibody, or carbohydrate to a modified RNA at a 5’ or 3’ terminus, and are generally known in the art.

[0011] In some embodiments, any of the modified RNAs or DRD hybrid polynucleotides provided herein are conjugated to or encapsulated by a delivery agent that includes, for example, a nanoparticle, a microparticle, or an exosome. A nanoparticle refers to a particle having a diameter between approximately 10 nm and 1000 nm. A microparticle is defines as a particle having a diameter greater than 1000 nm (1 pm), such as a particle having a diameter between approximately 1 pm and 100 pm. In some embodiments, a nanoparticle or microparticle is approximately spherical. In some embodiments, a nanoparticle or microparticle is hollow, comprising an internal core. In some embodiments, a nanoparticle or microparticle is a lipid nanoparticle or lipid microparticle, respectively. A lipid nanoparticle or lipid microparticle refers to a composition comprising one or more lipids that form an aggregate of lipids, or an enclosed structure with an interior surface and an exterior surface. In some embodiments, a lipid nanoparticle or lipid microparticle comprises a lipid bilayer that encloses an aqueous core. Lipids used in the formulation of lipid nanoparticles and lipid microparticles for delivering RNAs are generally known in the art, and include, but are not limited to, ionizable amino lipids, noncationic lipids, sterols, and polyethylene glycol-modified lipids. See, e.g., Buschmann et al.Vaccines. 2021. 9(1):65. In some embodiments, the modified RNA is surrounded by the lipids of the lipid nanoparticle or the lipid microparticle and are present in the interior of the lipid nanoparticle or lipid microparticle. In some embodiments, the modified RNA is dispersedthroughout the lipids of the lipid nanoparticle or lipid microparticle. In some embodiments, the lipid nanoparticle or lipid microparticle comprises an ionizable amino lipid, a non-cationic lipid, a sterol, and / or a polyethylene glycol (PEG)-modified lipid. Lipid nanoparticles and lipid microparticles comprising modified mRNAs may be prepared by any means generally known in the art, such as, for example, detergent dialysis, emulsion, centrifugation, evaporation, thin film hydration, or ethanol dilution. See, e.g., Barba et al. Pharmaceutics. 2019. 11(8):360. An exosome refers to a type of lipid nanoparticle produced by eukaryotic cells as a result of the inward budding of vesicles within multivesicular bodies and are generally between 30 nm and 150 nm in diameter. Exosomes comprise a heterogenous mixture of endogenous lipids, such as phospholipids, membrane-anchored proteins, and carbohydrates present in eukaryotic cells, and enclose an aqueous core. Exosomes may have beneficial features that are difficult to achieve with synthetically produced lipid nanoparticles, such as, for example, the ability to pass through the blood brain barrier and deliver RNAs to tissues within the brain. Exosomes comprising RNAs may be produced by any means generally known in the art, such as, for example, by sonicating or electroporating isolated exosomes in the presence of a modified RNA, or mixing exosomes with a lipid-conjugated modified RNA, such as, for example, a modified RNA that has been conjugated to cholesterol. See, c. ., Roberts et al. Nat Rev Drug Discov. 2020. 19(10):673- 694.

[0117] In some embodiments, a nanoparticle or microparticle is a polymeric nanoparticle or polymeric microparticle, respectively. A polymeric nanoparticle or polymeric microparticle refers to a nanoparticle or microparticle composition, respectively, comprising one or more polymers that form an aggregate of polymers, or an enclosed structure with an interior surface and an exterior surface. In some embodiments, a polymeric nanoparticle or polymeric microparticle comprises a polymeric layer that encloses an aqueous core. Polymers used in the formulation of polymeric nanoparticles and polymeric microparticles for delivering RNA are generally known in the art, and include cationic polymers such as, but are not limited to, poly ethyl enimine (PEI), poly-amido-amine (PAA), poly-beta amino-esters (PBAEs), polylysine (PLL), spermine, chitosan, polyurethane, and derivatives thereof (e.g., PEI stearic acid (PSA)copolymer). See, e.g., Liu et al. Front Bioeng Biotechnol. 2021. 9:718753. In some embodiments, the modified RNA is surrounded by the polymers of the polymeric nanoparticle or the polymeric microparticle and are present in the interior of the polymeric nanoparticle or polymeric microparticle. In some embodiments, the modified RNA is dispersed throughout the polymers of the polymeric nanoparticle or polymeric microparticle.

[0118] In some embodiments, a nanoparticle or microparticle is a protein nanoparticle or protein microparticle, respectively. A protein nanoparticle or protein microparticle refers to a nanoparticle or microparticle composition, respectively, comprising one or more proteins that form an aggregate of proteins, or an enclosed structure with an interior surface and an exterior surface. In some embodiments, a protein nanoparticle or protein microparticle comprises a protein layer that encloses an aqueous core. Proteins used in the formulation of protein nanoparticles and protein microparticles for delivering RNA are generally known in the art, and include but are not limited to, viral coat proteins and ferritin. See, e.g., Wang et al. Nat Nanotechnol. 2020. 15(5):406-416. In some embodiments, the modified RNA is surrounded by the proteins of the protein nanoparticle or the protein microparticle and are present in the interior of the protein nanoparticle or protein microparticle. In some embodiments, the modified RNA is external to the proteins of the protein nanoparticle or the protein microparticle and are attached to the exterior surface of the protein nanoparticle or protein microparticle. In some embodiments, the modified RNA is conjugated to proteins of the protein nanoparticle or protein microparticle through a covalent linkage, such as, for example, that formed by a click chemistry reaction, or by fusing the modified RNA and protein each to a protein or peptide of a protein / peptide pair known to react to form a covalent linkage.

[0119] In some embodiments, a nanoparticle or microparticle is a solid nanoparticle or solid microparticle. A solid nanoparticle or solid microparticle refers to a nanoparticle or microparticle composition, respectively, comprising one or more materials that form a solid structure, which has an external surface and may or may not comprise an internal surface. A solid nanoparticle or solid microparticle may comprise any suitable material that is generally known in the art, such as, for example, gold, silver, or silicon dioxide (silica). In someembodiments, a modified RNA is conjugated to the external surface of a solid nanoparticle or solid microparticle. Solid nanoparticles and solid microparticles comprising modified RNAs or DRD hybrid polynucleotides may be produced by any means generally known in the art, such as, for example, by linking the modified RNAs or DRD hybrid polynucleotides to the surface of the solid nanoparticle or solid microparticle through thiol linkages (e.g., modifying the DNA to comprise cyclic disulfide-anchoring groups), or by modifying the external surface of the solid nanoparticle or solid microparticle with one or more cationic materials (e.g., PEI) within which modified RNAs or DRD hybrid polynucleotides are present. See, e.g, Roberts et al. Nat Rev DrugDiscov. 2020. 19(10):673-694, Lee et al. Nano Lett. 2007, 7(7):2112-2115, and Paris and Vallet-Regi. Pharmaceutics. 2020, 12(6):526.

[0120] In some aspects, the present disclosure provides cells comprising any of the modified RNAs or DRD hybrid polynucleotides provided herein. In some embodiments, the cell is a human cell comprising any one of the modified RNAs or DRD hybrid polynucleotides provided herein. A “cell” is the basic structural and functional unit of all known independently living organisms. It is the smallest unit of life that is classified as a living thing. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as plants, fungi, and animals, including cattle, horses, chickens, turkeys, sheep, swine, dogs, cats, and humans, are multicellular. In some embodiments, the half-life of the modified RNA in the cell is 15-900 minutes. In some embodiments, the half-life of the modified RNA in the cell is 30-600 minutes. In some embodiments, the half-life of the modified RNA in the cell is 60-300 minutes. In some embodiments, the half-life of the modified RNA is at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 minutes. In some embodiments, the half-life of the modified RNA in the cell is at least 30, at least 60, at least 90, at least 120, at least 150, at least 180, at least 210, at least 240, at least 270, at least 300, at least 330, at least 360, at least 390, at least 420, at least 450, at least 480, at least 510, at least 540, at least 570, at least 600, at least 630, at least 660, at least 690, at least 720, at least 750, at least 780, at least 810, at least 840, or at least 870 minutes. In some aspects, the present disclosure provides compositions comprising any of the modified mRNAs, delivery agents, or cellsprovided herein. In some embodiments, the composition further comprises one or more additional agents, such as a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a small molecule, an aptamer, a lipid, or a carbohydrate. In some embodiments, the additional agent has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is an agent for use in modulating the expression and / or activity of one or more gene products (e.g., proteins) in a subject. In some embodiments, the additional agent is a nucleic acid for use in decreasing the expression and / or activity of one or more gene products (e.g, proteins), such as a short hairpin RNA (shRNA), small interfering RNA (siRNA), or an antisense oligonucleotide (ASO). In some embodiments, the additional agent is an inhibitor for decreasing the activity of one or more gene products (e.g., proteins). In some embodiments, the agent is a small molecular inhibitor. In some embodiments, the additional agent is an agent for enhancing an immune response in a subject. In some embodiments, the additional agent is an antigen, such as a nucleic acid antigen, a protein antigen, or a phospholipid antigen. In some embodiments, the additional agent is an adjuvant, such as, for example, aluminum hydroxide or potassium aluminum sulfate (alum), monophosphoryl lipid A (MPL), an oil-in-water emulsion (e.g., a squalene emulsion), a cytosine phosphoguanine (CpG) oligodeoxynucleotide, or another adjuvant that is known in the art. See, e.g, Di Pasquale, A et al. Vaccines. 2015. 3(2):320— 343. In some embodiments, the composition is a pharmaceutical composition comprising any one of the modified RNAs or DRD hybrid polynucleotides, delivery agents, or cells provided herein, and a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients, carriers, buffers, stabilisers, isotonicising agents, preservatives or antioxidants, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g., parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal.(0121] In some aspects, the present disclosure provides a method of administering to a subject any of the modified RNAs or DRD hybrid polynucleotides, delivery agents, cells, compositions, or pharmaceutical compositions provided herein. In some embodiments, thesubject is a human. In some embodiments, the administration is parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal. In some embodiments, the composition is to be stored below 50°C, below 40 °C, below 30 °C, below 20 °C, below 10 °C, below 0 °C, below -10 °C, below -20 °C, below -30 °C, below -40 °C, below -50 °C, below -60°C, below -70 °C, or below -80 °C, such that the nucleic acids are relatively stable over time. In some embodiments, the modified RNA is introduced into a cell in a subject by in vivo electroporation. In vivo electroporation is the process of introducing nucleic acids or other molecules into a cell of a subject using a pulse of electricity, which promote passage of the nucleic acids or other molecules through the cell membrane and / or cell wall. See, c'. . , Somiari et al. Molecular Therapy. 2000. 2(3): 178— 187. The modified RNA to be delivered is administered to the subject, such as by injection, and a pulse of electricity is applied to the injection site, whereby the electricity promotes entry of the nucleic acid into cells at the site of administration. In some embodiments, the modified RNA is delivered to and taken up by cells of the subject (e.g., cells local to the site of administration or throughout the subject) via a delivery agent that is associated with (e.g., conjugated to) the modified RNA. In some embodiments, the modified RNA is administered with other elements, such as buffers and / or excipients, that increase the efficiency of electroporation.{01221 In some aspects, the present disclosure provides a kit comprising any of the modified RNAs or DRD hybrid polynucleotides provided herein. In some embodiments, the kit comprises an engineered primer-dependent RNA polymerase. In some embodiments, the kit comprises an RNA primer, such as but not limited to a capped RNA primer or a phosphorylated RNA primer. In some embodiments, the kit comprises a single stranded DNA (ssDNA) template polynucleotide. In some embodiments, the kit comprises a circular ssDNA template polynucleotide. In some embodiments, the kit comprises a mixture of nucleotide triphosphates (NTPs). In some embodiments, the mixture of NTPs comprises at least one modified nucleotide. In some embodiments, the kit comprises a ligase. In some embodiments, the kit comprises an RNA ligase. In some embodiments, the kit comprises a T4 RNA ligase. In some embodiments, a kit comprises a T4 RNA ligase 1. In some embodiments, a kit comprises a T4 RNA ligase 2. Insome embodiments, the kit comprises an RtcB RNA ligase. In some embodiments, a kit comprises a hydrogen-bond-disrupting agent, such as but not limited to dimethyl sulfoxide (DMSO). In some embodiments, a kit comprises a chaotropic agent. In some embodiments, a kit comprises a strong base. In some embodiments, a kit comprises a solid phase, such as but not limited to a bead or chip.

[0123] In some embodiments, the kit comprises a buffer for carrying out a ligation reaction. In some embodiments, the kit further comprises a nucleotide triphosphate, such as ATP, to provide energy required by the ligase. In some embodiments, the kit is to be stored below 50 °C, below 40 °C, below 30 °C, below 20 °C, below 10 °C, below 0 °C, below -10 °C, below -20 °C, below -30 °C, below -40 °C, below -50 °C, below -60°C, below -70 °C, or below -80 °C, such that the nucleic acids are relatively stable over time.

[0124] In some aspects, the present disclosure provides a kit comprising any of the pharmaceutical compositions provided herein and a delivery device. A delivery device refers to machine or apparatus suitable for administering a composition to a subject, such as a syringe or needle. In some embodiments, the kit is to be stored below 50 °C, below 40 °C, below 30 °C, below 20 °C, below 10 °C, below 0 °C, below -10 °C, below -20 °C, below -30 °C, below -40 °C, below -50 °C, below -60°C, below -70 °C, or below -80 °C, such that the nucleic acids of the pharmaceutical composition are relatively stable over time. In some embodiments, the kit comprises instructions for administering any of the pharmaceutical compositions provided herein to a subject.EXAMPLES

[0125] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.Example 1: Synthesis of mRNA with region-specific modifications by primer dependentRNA polymerase and tandem template switching

[0126] Chemical synthesis of RNA oligonucleotides allows incorporation of diverse modifications at medicinal level precisions, while the upper limit of synthetic RNA strand is about 150 bases. Existing methods for preparing full-length mRNA relies on in vitro transcription (IVT) where a double-stranded DNA (dsDNA) template containing promoter sequences that is recognized and transcribed by bacteriophage RNA polymerases such as T7 and SP6 utilizing mixture of ribonucleotide triphosphates (NTP) mixture as synthetic building blocks (FIG. 1A). Incorporation of chemical modifications could be achieved by complete substitution of one NTP with its modified version as in the case of ml'P. Complete substitution during IVT, however, fails to evaluate most other modifications due to differential effects of mRNA modifications at different regions of the transcripts. One such example is m6A, which is known to promote translation and stabilize mRNA transcript when it’s in the 5'UTR and poly(A) tail, but negatively impacts tRNA decoding in protein coding sequences (CDS), and destabilizes mRNAs when present in the 3'UTR. Substitution with m6ATP during traditional IVT therefore leads to nonfunctional products due to its detrimental effects in the CDS, while missing the opportunity for harnessing the translation enhancing properties when present at other regions of a transcript (FIG. IB). In some cases, a modified NTP is partially spiked in into the original NTPs, but it produces an inseparable and uncharacterizable mixture of differentially modified mRNA (FIG. 1C). Similarly, partial spike-in remains the state-of-the-art for synthetic mRNA capping where cap analogues are added to IVT reaction yielding an inseparable mixture of capped / uncapped products (FIG. ID).

[0127] To achieve controlled, specific modifications at defined sites within mRNA transcripts, we developed a chemoenzymatic synthesis workflow utilizing chemical oligo synthesis and capping, followed by tandem DNA-templated RNA primer extension using an engineered RNA polymerase (FIG. IE). We define the concept of “region-specifically-modified mRNA,” where a certain type of modification occurs only in a set of defined region(s) within the mRNA transcript. One example of a region specifically-modified mRNA is an mRNA in which homogeneously all the adenines within the 5'UTR and poly(A) tails are m6A modified, but in which the remaining portions of the transcript contain unmodified adenines. One example of anon-region-specifically-modified mRNA is present in FIG. 1C, where m6A patterns are heterogeneous within the product mixture and occur randomly from transcript to transcript. A special case of region-specifically-modified mRNA is shown in FIG. IB, in which all adenines are substituted with m6A throughout the entire transcript.| 0128| The synthesis workflow for the generation of region-specifically-modified mRNA transcripts involves the following steps. The full mRNA sequence is first divided into multiple segments of a single stranded DNA (ssDNA) template, each with an at least 10-20 base pair (bp) overlap with the preceding template. ssDNA template could be prepared using solid-phase DNA synthesis for shorter segments (< 200 bp). For longer segments, ssDNA templates could be prepared using PCR with biotinylated primers followed by depletion of the undesired strand by streptavidin beads pull-down; alternatively, ssDNA template could be prepared by sequential in vitro transcription-reverse transcription-RNase H workflow, such as that described in Miura et al., (2018) Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat. Protoc. 13, 195-215, which is incorporated herein by reference in its entirety. A 10-150 bp long chemically capped RNA primer was prepared by a divergent construction workflow, as described in U.S. Provisional Patent Application No. 63 / 578300 and U.S. Provisional Patent Application No. 63 / 61089, such that it contains combinatorial patterns of chemical modifications including cap modifications, base modifications, phosphodiester linkage modifications, and sugar backbone modifications. Modification patterns are defined by solidphase synthesis and quality-controlled by mass spectrometry. Capping efficiency is ensured >99% by HPLC purification. The chemically synthesized capped RNA primer is annealed to the first ssDNA template. Polymerization is carried out by engineered primer-dependent RNA polymerase such as TGK (or other engineered DNA-dependent RNA polymerase capable of elongating RNA primer); a set of modified / unmodified NTP mix is used to introduce modification patterns specific to the first region. The DNA template was then digested, and mRNA was annealed to the next segment of ssDNA template and polymerized using a second set of modified / unmodified NTP mix. The template switching / synthesis cycle was repeated until the full length mRNA was synthesized.Example 2: Generation of region-specifically-modified mRNA constructs

[0129] To test the feasibility of the tandem template switching synthesis scheme described in FIG. IE, full length (-750 bp) NanoLuciferase (NLuc) sequences including 5'UTR derived from the human a-globin mRNA with an optimized Kozak sequence + codon-optimized CDS + 3'UTR consisting of two sequences derived from the amino-terminal enhancer of split mRNA and the mitochondrial encoded 12S rRNA + a 80A poly(A) tail was divided into 5 segments, where each ssDNA template was chemically synthesized. Full length mRNA was synthesized from a 20-bp RNA primer after 5 rounds of template switching / polymerization where products with desired lengths after each round were observed using gel electrophoresis (FIG. 2A). For each round of synthesis, 1 equivalent (100 pmol) of RNA was annealed to 1.2-2.0 equivalents (120-200 pmol) of ssDNA template by heating to 94 °C for 15s and cooling to 4 °C at 0.1 °C / s in l x Thermopol buffer [NEB, B9004S] with 3 mM MgSO4, 2.5 mM each NTP, and 1 U / pL Superaseln RNase inhibitor [Invitrogen, AM2694], TGK polymerase was then added on ice to a final concentration of 150 nM, and the reactions were incubated at 65 °C for 20 mins, followed by another two cycles of 94 °C for 15s, 50 °C for 1 min, and 65 °C for 20 mins. ssDNA template was then digested by TURBO DNase [Invitrogen, AM2238] and the RNA was purified by Monarch RNA cleanup kit [NEB, T2040S],

[0130] To further showcase the significance of region-specific mRNA modifications, the same NLuc sequence as described in FIG. 2A was used except for exclusion of poly(A) tails and further inclusion of C-terminal PEST destabilization signal, which effectively reduced the halflife of protein reporter to -20 mins to allow accurate readout of mRNA activity at each time point. NLuc-PEST (NLucP) encoding mRNA was synthesized by IVT with co-transcriptional capping and 100% ml incorporation. The NLuc-PEST nucleotide sequence is shown in SEQ ID NO: 1, and the NLuc-PEST amino acid sequence is shown in SEQ ID NO: 2.ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAAC CTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTG TCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCG AAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGC ACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGAC GGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACC CTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCT GCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCT GGCGTCTCACGGCTTTCCGCCTGAGGTTGAAGAGCAAGCCGCCGGTACATTGCCTAT GTCCTGCGCACAAGAAAGCGGTATGGACCGGCACCCAGCCGCTTGTGCTTCAGCTC GCATCAACGTCTAAta (SEQ ID NO: 1).MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIH VIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIA VFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASHGFPPEVEE QAAGTLPMSCAQESGMDRHPAACASAR1NV* (SEQ ID NO: 2).

[0131] The NLucP mRNA was templated tailed by annealing to a synthetic ssDNA template with 20-bp homology to the 3'end of NLucP mRNA and 80 continuous T, resulting in an 80A poly(A) tail after transcription. None / unmodified / modified ATP was used in 100% to enable fully modified / unmodified poly(A) tail incorporation as characterized by agarose gel electrophoresis (FIG. 2B). Successfully tailed mRNA including unmodified rA, PS-A, m6A, Ph6A, and Cy6A along with untailed mock control were transfected to HeLa cells with FLuc mRNA as transfection control. 6 hours (hrs) after transfection, cell culture medium was exchanged and cells treated with all conditions were re-seeded into 5 wells of a multi-well plate, which the luciferase-based luminescence was measured. Ratios of NLucP to FLuc normalized to unmodified rA construct were plotted (FIG. 2C). Satisfactorily, enhanced expression of tail- modified mRNA was observed over time with PS-A tailed mRNA expressing 8-fold higher and m6A / Ph6A tailed mRNA expressing >24-fold higher than the unmodified A control after 36 hours.Example 3: Synthesis of region-specifically modified circRNA by DNA templated RNA primer extension

[0132] Typical workflows for circRNA synthesis rely on the presence of flipped introns, where a ribozyme undergoes back splicing to yield circRNA. (FIG. 3A). Such methods are incompatible with most RNA modifications as substituted bases would disrupt intron structures and abrogate ribozyme function (FIG. 3B). Synthesis of heavily modified circRNA could be achieved through a region-specifically modified RNA intermediate prepared from workflow described in FIG. IE, where modified bases are incorporated specifically outside of intronic regions, allowing ribozyme-mediated back splicing (FIG. 3C). The RNA circularized in FIG. 3C could either contain one type of modification or multiplexed sets of modifications, and circularization could be achieved by either back splicing or other ligation based methods using RNA ligases (FIG. 3D). Alternatively, modified circRNA could be synthesized from a circular ssDNA template using primer-dependent RNA polymerases such as TGK (FIG. 3E). A 5 'phosphorylated RNA primer (chemically synthesized, modified) can be annealed to circular ssDNA template and extended using primer-dependent RNA pol (with / without modified NTPs). Synthesized RNA can then be circularized by splint ligation using T4 RNA ligase 2, and the DNA strand can be separated to isolate modified circRNA.Example 4: Synthesis and assembly of DNA-RNA-DNA (DRD) hybrids

[0133] The present Example illustrates the synthesis and assembly of DNA-RNA-DNA (DRD) hybrid polynucleotides.

[0134] Assembly of DRD hybrid polynucleotides requires the preparation of DRD building blocks. As shown in FIG. 4A, such building blocks can be prepared by subjecting a 5’- phosphrylated ssDNA primer to two rounds of tempi ate- switch synthesis, where, in the second round, a uridine(U)-containing DNA template is used and selectively digested by U-specific DNases. Examples of U-specific DNases can be found at neb.com / en-us / products / m5505-user- enzyme#Product%20Information. In some cases, the 573 ’-sequences of the DRD units can betailed such that they contain homology to form a “dumbbell” shape, thereby forming a dumbbell DRD unit. Dumbbell DNA circularization is described, for example, in Wei et al. (2016), Production of dumbbell probe through hairpin cleavage-ligation and increasing RCA sensitivity and specificity by circle to circle amplification, Sci. Rep., 6:29229.|0135| Following preparation of DRD building blocks, the building blocks can be assembled by one-pot assembly. As shown in FIG. 4B, multiple DRD units could be assembled in one-pot on to a capped RNA-DNA by T4 DNA ligase assisted by U-DNA splint with homology to the 573 ’-DNA of the DRD units, followed by digestion of the splints using U- specific DNases. In some cases, the multiple DRD units encode multiple RNA open reading frames (ORFs) and internal ribosomal entry site (IRES) elements. The RNA sequences in the DRD can be further chemically or region-specifically modified. As shown in FIG. 4C, the workflow in FIG. 4B can be applied to DRD units containing a capped RNA branch, such as that synthesized by the workflow in FIG. 4A with a branched ssDNA-capped RNA primer. After one pot assembly, a multi -ci str onic mRNA with multiple branched caps is synthesized.|0136| As shown in FIG. 4D, the workflow in FIG. 4B can be further applied to one-pot circularization of two dumbbell DRD units with different ORF and IRES sequences, where the 5’ overhangs of the two dumbbell DRD hybrids hybridize to each other and are ligated by T4 DNA ligase to afford circular dual-ORF mRNA. The result is the formation of a circular dual- cistronic mRNA (IRES driven).

[0137] As shown in FIG. 4E, the principles of FIG. 4D can be applied to multiple dumbbell DRD hybrids such that the 5’ overhangs of each forms a multi-way junction to allow one-pot circularization and assembly. The result is the formation of a circular multi-ci stronic mRNA (IRES driven).

[0018] As shown in FIG. 4F and FIG. 4G, the principles of FIG. 4D and FIG. 4E can be applied to dumbbell DRD hybrids with branched caps. The result is the formation of a circulardual-cistronic mRNA (branch-capped) and a circular multi -ci stronic mRNA (branch-capped), respectively.

[0139] Each of the above schemes can be applied to RNA sequences that are proteincoding or non-coding. Such RNA sequences can have modified nucleotides or unmodified nucleotides, and / or can be region-specifically modified RNAs as described herein.Example 5: Methodological optimization for templated poly(A)-tailing|0140| The present Example illustrates the optimization of poly(A)-tailing of RNA by template switching.[01411 Poly(A)-tailing of RNA by direct extension of an RNA primer along a polyT DNA template can lead to over-extension of the poly(A) tail, possibly due to partial denaturation under thermal conditions (FIG. 5A). To observe this, a 20 nucleotide primer (5 pM) was mixed with a 30T DNA template (plus a 20 nucleotide annealing region, for a total of 50 nucleotides in length) (5 pM). The RNA primer was then extended by TGK at 65° C for 30 mins with 10 mM of ATP or ATPaS. Extension was characterized using a TBE-Urea gel. As shown in FIG. 5A, an over-extension product was observed, evidenced by the high-molecular weight (>50 nt) species present in the +ATP and +ATPaS lanes.

[0142] To test whether over-extension could be mitigated by optimizing the reaction conditions, Applicant then systematically varied the experimental protocol and assessed their impact on poly(A) tail over-extension.

[0143] As shown in FIG. 5B, reducing the concentration of ATP in the reaction solution significantly reduced over-extension. Reactions were carried out in 5 pM of primer / template at 65° C for 30 mins. Over-extension was reduced with lower ATP concentrations.

[0144] As shown in FIG. 5C, chain-terminating dideoxynucleotides can be incorporated into a DNA template to mitigate poly(A) tail overextension. A single G / C / A was included at theend of the polyT DNA template, and a corresponding dideoxyNTP (ddNTP) was included in the reaction mixture. Inclusion of ddG appeared to more efficiently mitigate over-extension than ddC or ddT, although a complete block of over-extension was not achieved.

[0145] As shown in FIG. 5D, manipulation of reaction temperature impacted over- extension. Reduction of reaction temperature lowered reduced over-extension, but also reduced RNA yield.

[0146] As shown in FIG. 5E, manipulation of salt concentration significantly impacted poly(A) over-extension. In particular, increasing the concentration of NaCl in the reaction mixture reduced over-extension without compromising RNA yield. The most robust reduction of over-extension was observed when NaCl was present in the reaction mixture at 400 mM.

[0147] As shown in FIG. 5F, modified ATP such as m6ATP could be effectively added with increased m6ATP concentration (to > 30 mM) without over-extension or compromised yield.

[0148] As shown in FIG. 5G, a full-length mRNA containing a completely modified poly(A) tail could be synthesized under the following conditions: template concentration = 20 pM, mRNA concentration = 1 pM, TGK polymerase concentration = 1 pM, temperature = 65 °C, ATP (or modified ATP) concentration = 5 mM ~ 30 mM, Reaction buffer = 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 10 mM KC1, 5 mM MgSO4, 0.1% Triton® X-100, 400 mM NaCl, 1 U / pL Rnase Inhibitor. The reaction was conducted for 30 min and then templates were digested with Turbo DNase. Tail integrity was characterized by Rnase H assay. Briefly, the following reaction was prepared to anneal mRNA to the aforementioned ssDNA probe: 100 ng of purified mRNA sample, 2 pmol of RNaseH DNA probe complementary to the 3’ UTR region of mRNA, 2 pL of annealing stock solution (50 mM KC1, 2.5 mM EDTA, 1 : 100 Superase-In), and nuclease-free water up to a total volume of 10 pL. Reactions were denatured at 70 °C for 5 min, followed by cooling to RT at a rate of 0.2 °C / s in a benchtop thermocycler. Following probe annealing, 1 pL of Thermostable RNase H (NEB: M0523S) and 1 pL of the 10* buffer wereadded to each reaction, which was incubated at 50 °C for 30 min. Subsequently, samples were mixed with one volume of Gel Loading Buffer II (ThermoFisher Scientific: AM8546G), which had been supplemented with EDTA to a final concentration of 50 mM. Samples in I * loading buffer were denatured at 70 °C for 3-5 min prior to loading and resolution on 15% Novex TBE- Urea Gels (ThermoFisher Scientific: EC68655BOX), run in 0.5* Tris-borate-EDTA (TBE) buffer.Example 6: Screening of 5’UTR modifications by template switching mRNA synthesis

[0149] The present Example illustrates a screening method for assessing 5’UTR modifications using template switching mRNA synthesis.

[0150] Briefly, 5’ UTR-specifically modified mRNA encoding a degron-tagged Nano luciferase (Nluc-PEST) was synthesized by template-switch mRNA synthesis such that the defined bases were fully modified only within the 5’UTR; all bases beyond the 5’UTR were regular A / G / C or N'-methyl-pseudouridine (m h| / ). mRNAs were transfected into HeLa / HepG2 / JAWSII cells, and luminescence was measured at 10 / 31 / 52 / 73 hours after transfection (FIG. 6A). The short intracellular half-life of Nluc-PEST (~20 mins) allows accurate characterization of mRNA half-life. Various different base modifications were assessed, as shown in FIG. 6B.

[0151] NanoLuc expression was measured at 10 hours post transfection as a proxy for translation efficiency in cellulo. Relative luminescence was calculated by NanoLuc luminescence normalized by co-transfected Firefly luciferase luminescence control, and then normalized to the unmodified 5’UTR construct, n = 3. Mean ± s.e.m. 5’UTR m6A and 5moU modifications lead to translation reduction in all three cell lines: HeLa cells (FIG. 6C), HepG2 cells (FIG. 6D), and JAWSII cells (FIG. 6E). ml\| / , Ac4C, and PSC appeared to best promote translation efficiency. Phosphorothioate modifications led to increased expression when introduced in single bases but reduced translation when more than two bases were modified. 2’F did not appear to afford substantial benefit to translation efficiency. 2’0Me exhibited site-dependent effects; when only the first half of 5’UTR was 2’OMe-modified, increased translation efficiency was observed in all three cell lines. When the whole 5’UTR was 2’Ome-modified, no increase in translation efficiency was observed. In JAWSII cells, a decreased in translation efficiency was observed (FIG. 6E).|01521 mRNA half-life was then calculated from the Nluc-PEST luminescence using one-phase decay kinetics. Mean ± std. mRNA half-life was calculated in all three tested cell lines: HeLa cells (FIG. 6F), HepG2 cells (FIG. 6G), and JAWSII cells (FIG. 6H). Fully 2’OMe-modified 5’UTRs exhibited increased mRNA half-life in all cell lines tested.Example 7: Purification of DNA / RNA hybrids without nuclease digestion

[0153] The present Example illustrates a method for accomplishing purification of DNA / RNA hybrids without the need to digest DNA templates via a nuclease enzyme. Also illustrated is a method for achieving template-switch synthesis using a ssDNA template immobilized on a solid phase.

[0154] Nuclease digestion of ssDNA templates may in some instances represent a potential barrier to the scalability of the template-switch synthesis method described herein. Applicant thus hypothesized that direct separation of the DNA / RNA duplex could allow recovery of the precious long single stranded DNA (ssDNA) templates and avoid the cost and potential damage caused by nuclease digestion. Such a direct DNA / RNA duplex separation process may require: (1) efficient duplex denaturation at the beginning stage; (2) no duplex renaturation during the separation process; and (3) a separation technique that could distinguish oligonucleotide molecules of the same or similar lengths. On requirements (1) and (2), DNA denaturation could be achieved by a hydrogen bond disrupter (e.g., an organic solvent, chaotropic reagents such as urea, or strong bases such as NaOH) and heat. Strong bases could not be applied in the setting of DNA / RNA duplex as RNA is base-labile. The ability of hydrogen bond disrupters to denature oligonucleotides are also extremely sensitive to salt concentrations, which promote duplex formation. Among all reagents screened, DMSO was found to be the mosteffective denaturation reagent in Applicant’s hands and others’(pmc. ncbi.nlm.nih.gov / articles / PMC4168728 / ). Regarding requirement (3), given that oligonucleotides are inherently hydrophilic, use of hydrophobic tags (such as aromatic rings or other hydrocarbons) could separate tagged / un-tagged strands on reversed phase columns or other hydrophobic stationary phases. Among the hydrophobic tags, fluorous hydrocarbon molecules are unique in both their strong hydrophobicity and fluorophilicity, and have been used in long oligonucleotide purification (pubs.acs.org / doi / 10.1021 / jo050795y). Applicant therefore envisioned that after primer extension, a hydrophobically-tagged DNA template could be efficiently separated from the RNA strand by chemical denaturation, followed by affinity purification or reversed phase chromatography to allow both isolation of the RNA product and recycling of the DNA template (FIG. 7A).(0155] To demonstrate such a method, Applicant synthesized complementary DNA / RNA strands, where the DNA strand was tagged with the FC6 hydrophobic tag and the Cy3 fluorophore (FIG. 7A) and the RNA strand was tagged with a 6FAM fluorophore only. The DNA strand contained the following nucleotide sequence: / FC6 / / Cy3 / GCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTT AACTTGCTATTTCTAGCTCTAAAACATCCTGGTCGAGCTGGACGG (SEQ ID NO: 3). The RNA strand contained the following nucleotide sequence: CCGTCCAGCTCGACCAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC / 6FAM / (SEQ ID NO: 4).

[0156] The DNA / RNA duplex was first denatured by contacting the DNA / RNA duplex with dimethyl sulfoxide (DMSO) / water (1: 1), heated to 60°C, and then subjected to high performance liquid chromatography (HPLC) purification. The HPLC conditions were as follows: 10 mM Tri ethyl ammonium acetate (TEAA) in water (buffer A), Acetonitrile (buffer B); 95% A + 5% B (0 min) ~ 70% A + 30% B (10 min) ~ 30% A + 70% B (15 min) ~ 5% A + 95% B (20 min); flow rate = 1 mL / min; stationary phase = PLRP-S Polymeric Reversed-Phase Column (Agilent).

[0157] When separately run on a standard reversed phase column (PLRP-S, Agilent), the FC6 tag already leads to >30% acetonitrile percentage difference in terms of its HPLC retention (FIG. 7B, trace 1 and 2). When the DNA / RNA duplex were run together, renaturation of the duplex was observed at room temperature (FIG. 7B, trace 3) and effective separation was achieved when heated to 60°C (FIG. 7B, trace 4). Separation was further confirmed by gel electrophoresis and imaging of the corresponding fluorophores (FIG. 7C).

[0158] Similarly, using the chemical denaturation method described above, a template switching primer extension reaction could be performed using a ssDNA template immobilized on a solid phase (beads or chips). (FIG. 8) The RNA / DNA duplex could be chemically denatured using DMSO / water as described above, where the RNA strand could be eluted from the DNA template to allow recycling of the ssDNA template. The eluted RNA could then be captured by the next strand of immobilized template by partial homology to facilitate subsequent cycles of primer extension.

[0159] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.[016O[ While certain embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.[0161 J The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimedtechnology. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.|01621 The present disclosure is not to be limited in terms of the particular embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and compositions within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, or compositions, which can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0163] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0164] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, inclusive of the endpoints. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer toranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0165] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0016] Other embodiments are set forth in the following claims.

Claims

WHAT IS CLAIMED IS:CLAIMS1. A method for producing a RNA, comprising:(a) annealing an RNA primer to a first single stranded DNA (ssDNA) template polynucleotide, wherein the first ssDNA template polynucleotides comprises an overlap with a second ssDNA template polynucleotide;(b) contacting the first ssDNA template polynucleotide with an engineered primerdependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA primer, thereby producing a DNA / RNA duplex comprising a once-extended RNA;(c) separating the first ssDNA template polynucleotide from the once-extended RNA;(d) annealing the once-extended RNA to the second ssDNA template polynucleotide; and(e) contacting the second ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a second mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the once-extended RNA, thereby producing a DNA / RNA duplex comprising a twice-extended RNA.

2. The method of claim 1, further comprising:(f) separating the second ssDNA template polynucleotide from the twice-extended RNA;(g) annealing the twice-extended RNA to a third ssDNA template polynucleotide, wherein the second ssDNA template polynucleotide comprises an overlap with the third ssDNA template polynucleotide; and(h) contacting the third ssDNA template polynucleotide with the engineered primerdependent RNA polymerase and a third mixture of nucleotide triphosphates (NTPs) for a timesufficient to permit extension of the RNA, thereby producing a DNA / RNA duplex comprising a thrice-extended RNA.

3. The method of claim 2, further comprising:(i) separating the third ssDNA template polynucleotide from the thrice-extended RNA;(j) annealing the thrice-extended RNA to a fourth ssDNA template polynucleotide, wherein the third ssDNA template polynucleotide comprises an overlap with the fourth ssDNA template polynucleotide; and(k) contacting the fourth ssDNA template polynucleotide with the engineered primerdependent RNA polymerase and a fourth mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA, thereby producing a DNA / RNA duplex comprising a four-times-extended RNA.

4. The method of any one of claims 1-3, wherein the method comprises annealing, and polymerizing of at least five, at least six, at least seven, at least eight, at least nine, or at least ten ssDNA template polynucleotides.

5. The method of any one of claims 1-4, wherein each of the ssDNA template polynucleotides comprises:(a) at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 terminal nucleotides of overlap with the subsequent ssDNA template polynucleotide; or(b) no more than 20 terminal nucleotides of overlap with the subsequent ssDNA template polynucleotide.

6. The method of any one of claims 1-5, wherein the engineered primer-dependent RNA polymerase is a TGK polymerase, a T7 polymerase, or a SP6 polymerase.

7. The method of any one of claims 1-6, wherein at least one of the mixtures of NTPs does not comprise a modified nucleotide.

8. The method of any one of claims 1-6, wherein at least one of the mixtures of NTPs comprises at least one modified nucleotide.

9. The method of any one of claims 1-6, wherein each mixture of NTPs comprises at least one modified nucleotide.

10. The method of claim 8 or 9, wherein the modified nucleotide is selected from the group consisting of 2'-O-methyladenosine (mA), alpha-phosphorothioate adenosine (PS-A), N6- methyladenosine (m6A), N6-phenyladenosine (Ph6A); N6-benzyladenosine (Bn6A), N6- cyclopentyladenosine (Cy6A), and any combination thereof.

11. The method of any one of claims 1-10, wherein separating comprises digesting the ssDNA template polynucleotide.

12. The method of claim 11, wherein digesting comprises contacting the ssDNA template polynucleotide with an exonuclease.

13. The method of any one of claims 1-10, wherein separating does not comprise digesting the ssDNA template polynucleotide.

14. The method of any one of claims 1-10, wherein separating comprises chemical denaturation.

15. The method of claim 14, wherein chemical denaturation comprises contacting the DNA / RNA duplex with a hydrogen-bond-disrupting agent, a chaotropic reagent, or a strong base.

16. The method of claim 15, wherein the hydrogen-bond-disrupting agent is dimethyl sulfoxide (DMSO).

17. The method of claim 15 or 16, further comprising heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C, preferably about 60°C.

18. The method of any one of claims 14-17, wherein the ssDNA template polynucleotide comprises a tag.

19. The method of claim 18, wherein the tag is a hydrophobic tag or an affinity tag.

20. The method of claim 19, wherein the tag is an FC6 hydrophobic tag.

21. The method of any one of claims 1-20, wherein the ssDNA template polynucleotide is conjugated to a solid phase.

22. The method of claim 21, wherein the solid phase is selected from a bead or a chip.

23. The method of any one of claims 1-22, wherein separating further comprises affinity purification or reversed phase chromatography.

24. The method of any one of claims 1-23, wherein the ssDNA template oligonucleotide is recycled for subsequent use.

25. The method of any one of claims 1-24, wherein the ssDNA template polynucleotides comprise a 5 ’-untranslated region (5’UTR) ssDNA template polynucleotide, a coding sequence(CDS) ssDNA template polynucleotide, a 3 ’-untranslated region (3’UTR) ssDNA template polynucleotide, and a poly(A) tail ssDNA template polynucleotide.

26. The method of any one of claims 1-25, further comprising circularizing the RNA.

27. The method of claim 26, wherein circularizing comprises RNA ligation.

28. The method of claim 26, wherein circularizing comprises ribozyme-mediated back splicing.

29. The method of any one of claims 1-28, wherein the RNA produced is messenger RNA (mRNA).

30. The method of any one of claims 1-28, wherein the RNA produced is non-coding RNA.

31. A method for producing a RNA, comprising:(a) annealing an RNA primer to a first single stranded DNA (ssDNA) template polynucleotide, wherein the first ssDNA template polynucleotide comprises an overlap with a second ssDNA template polynucleotide;(b) contacting the first ssDNA template polynucleotide with an engineered primerdependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the RNA primer, thereby producing a DNA / RNA duplex comprising a once-extended RNA;(c) separating the first ssDNA template polynucleotide from the once-extended RNA;(d) annealing the once-extended RNA to a second ssDNA template polynucleotide, wherein the second ssDNA template polynucleotide comprises an overlap with a third ssDNA template polynucleotide;(e) contacting the second ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a second mixture of NTPs for a time sufficient to permitextension of the once-extended RNA, thereby producing a DNA / RNA duplex comprising a twice-extended RNA;(f) separating the second ssDNA template polynucleotide from the twice-extended RNA;(g) annealing the twice-extended RNA to a third ssDNA template polynucleotide, wherein the third ssDNA template polynucleotide comprises an overlap with a fourth ssDNA template polynucleotide;(h) contacting the third ssDNA template polynucleotide with the engineered primerdependent RNA polymerase and a third mixture of NTPs for a time sufficient to permit extension of the twice-extended RNA, thereby producing a DNA / RNA duplex comprising a thrice- extended RNA;(k) separating the third ssDNA template polynucleotide from the thrice-extended RNA;(l) annealing the thrice-extended RNA to a fourth ssDNA template polynucleotide;(m) contacting the fourth ssDNA template polynucleotide with the engineered primerdependent RNA polymerase and a fourth mixture of NTPs for a time sufficient to permit extension of the thrice-extended RNA, thereby producing a DNA / RNA duplex comprising a four-times-extended RNA.

32. The method of claim 31, wherein the first ssDNA template polynucleotide is a 5’- untranslated region (5’UTR) ssDNA template polynucleotide; the second ssDNA template polynucleotide is a coding sequence (CDS) ssDNA template polynucleotide; the third ssDNA template polynucleotide is a 3 ’-untranslated region (3’UTR) ssDNA template polynucleotide; and the fourth ssDNA template polynucleotide is a poly(A) tail ssDNA template polynucleotide.

33. The method of claim 31 or 32, wherein the method comprises annealing, and polymerizing of at least five, at least six, at least seven, at least eight, at least nine, or at least ten ssDNA template polynucleotides.

34. The method of any one of claims 31-33, wherein each of the ssDNA template polynucleotides comprises:(a) at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 terminal nucleotides of overlap with the subsequent ssDNA template polynucleotide; or(b) no more than 20 terminal nucleotides of overlap with the terminal nucleotides of at least one other ssDNA template polynucleotide.

35. The method of any one of claims 31-34, wherein the engineered primer-dependent RNA polymerase is a TGK polymerase, a T7 polymerase, or a SP6 polymerase.

36. The method of any one of claims 31-35, wherein at least one of the mixtures of NTPs does not comprise a modified nucleotide.

37. The method of any one of claims 31-35, wherein at least one of the mixtures of NTPs comprises at least one modified nucleotide.

38. The method of any one of claims 31-35, wherein each mixture of NTPs comprises at least one modified nucleotide.

39. The method of claim 35 or 36, wherein the modified nucleotide is selected from the group consisting of 2'-O-methyladenosine (mA); alpha-phosphorothioate adenosine (PS-A); N6- methyladenosine (m6A); N6-phenyladenosine (Ph6A); N6-benzyladenosine (Bn6A); and N6- cyclopentyladenosine (Cy6A).

40. The method of any one of claims 31-39, wherein separating comprises digesting the ssDNA template polynucleotide.

41. The method of claim 40, wherein digesting comprises contacting the RNA with an exonuclease.

42. The method of any one of claims 31-39, wherein separating does not comprise digesting the ssDNA template polynucleotide.

43. The method of any one of claims 31-39, wherein separating comprises chemical denaturation.

44. The method of claim 43, wherein chemical denaturation comprises contacting the DNA / RNA duplex with a hydrogen-bond-disrupting agent, a chaotropic reagent, or a strong base.

45. The method of claim 44, wherein the hydrogen-bond-disrupting agent is dimethyl sulfoxide (DMSO).

46. The method of claim 44 or 45, further comprising heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C, preferably about 60°C.

47. The method of any one of claims 43-46, wherein the ssDNA template polynucleotide comprises a tag.

48. The method of claim 47, wherein the tag is a hydrophobic tag or an affinity tag.

49. The method of claim 48, wherein the tag is an FC6 hydrophobic tag.

50. The method of any one of claims 31-49, wherein the ssDNA template polynucleotide is conjugated to a solid phase.

51. The method of claim 50, wherein the solid phase is selected from a bead or a chip.

52. The method of any one of claims 31-51, wherein separating further comprises affinity purification or reversed phase chromatography.

53. The method of any one of claims 31-52, wherein the ssDNA template oligonucleotide is recycled for subsequent use.

54. The method of nay one of claims 31-53, further comprising:(n) separating the fourth ssDNA template polynucleotide from the four-times- extended RNA.

55. The method of any one of claims 31-54, further comprising circularizing the RNA.

56. The method of claim 55, wherein circularizing comprises RNA ligation.

57. The method of claim 55, wherein circularizing comprises ribozyme-mediated back splicing.

58. The method of any one of claims 31-57, wherein the RNA produced is messenger RNA (mRNA).

59. The method of any one of claims 31-57, wherein the RNA produced is non-coding RNA.

60. A method for producing a circular RNA (circRNA), comprising:(a) annealing an RNA primer to a first single stranded DNA (ssDNA) template polynucleotide, wherein the first ssDNA template polynucleotides comprises an overlap with a second ssDNA template polynucleotide;(b) contacting the first ssDNA template polynucleotide with an engineered primerdependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a timesufficient to permit extension of the RNA primer, thereby producing a DNA / RNA duplex comprising a once-extended RNA;(c) separating the first ssDNA template polynucleotide from the once-extended RNA;(d) annealing the once-extended RNA to the second ssDNA template polynucleotide;(e) contacting the second ssDNA template polynucleotide with the engineered primer-dependent RNA polymerase and a second mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the once-extended RNA, thereby producing a DNA / RNA duplex comprising a twice-extended RNA,(f) separating the second ssDNA template polynucleotide from the twice-extended RNA, thereby producing a linear RNA; and(g) circularizing the linear RNA, thereby producing a circRNA.

61. The method of claim 60, wherein the method comprises annealing, and polymerizing of at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten ssDNA template polynucleotides.

62. The method of claim 60 or 61, wherein circularizing comprises RNA ligation.

63. The method of claim 62, wherein RNA ligation comprises splint ligation by T4 RNA ligase 2.

64. The method of claim 62, wherein circularizing comprises ribozyme-mediated back splicing.

65. The method of any one of claims 60-64, wherein the linear RNA comprises an exon region flanked by two intron regions.

66. The method of claim 65, wherein the exon region comprises at least 1 modified nucleotide.

67. The method of claim 65 or 66, wherein the intron regions do not comprise a modified nucleotide.

68. The method of any one of claims 60-67, wherein the method comprises synthesizing or having synthesized a chemically capped RNA primer and at least four ssDNA template polynucleotides.

69. The method of any one of claims 60-68, wherein each of the at least two ssDNA template polynucleotides comprises:(a) at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 terminal nucleotides of overlap with the subsequent ssDNA template polynucleotide; or(b) no more than 20 terminal nucleotides of overlap with the terminal nucleotides of at least one other ssDNA template polynucleotide.

70. The method of any one of claims 60-69, wherein the engineered primer-dependent RNA polymerase is a TGK polymerase, a T7 polymerase, or a SP6 polymerase.

71. The method of any one of claims 60-70, wherein at least one of the mixtures of NTPs does not comprise a modified nucleotide.

72. The method of any one of claims 60-70, wherein at least one of the mixtures of NTPs comprises at least one modified nucleotide.

73. The method of any one of claims 60-70, wherein each mixture of NTPs comprises at least one modified nucleotide.

74. The method of claim 72 or 73, wherein the modified nucleotide is selected from the group consisting of 2'-O-methyladenosine (mA); alpha-phosphorothioate adenosine (PS-A); N6-methyladenosine (m6A); N6-phenyladenosine (Ph6A); N6-benzyladenosine (Bn6A); and N6- cyclopentyladenosine (Cy6A).

75. The method of any one of claims 60-74, wherein separating comprises digesting the ssDNA template polynucleotide.

76. The method of claim 75, wherein digesting comprises contacting the ssDNA template polynucleotide with an exonuclease.

77. The method of any one of claims 60-74, wherein separating does not comprise digesting the ssDNA template polynucleotide.

78. The method of any one of claims 60-74, wherein separating comprises chemical denaturation.

79. The method of claim 78, wherein chemical denaturation comprises contacting the DNA / RNA duplex with a hydrogen-bond-disrupting agent, a chaotropic reagent, or a strong base.

80. The method of claim 79, wherein the hydrogen-bond-disrupting agent is dimethyl sulfoxide (DMSO).

81. The method of claim 79 or 80, further comprising heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C, preferably about 60°C.

82. The method of any one of claims 78-81, wherein the ssDNA template polynucleotide comprises a tag.

83. The method of claim 82, wherein the tag is a hydrophobic tag or an affinity tag.

84. The method of claim 83, wherein the tag is an FC6 hydrophobic tag.

85. The method of any one of claims 60-84, wherein the ssDNA template polynucleotide is conjugated to a solid phase.

86. The method of claim 85, wherein the solid phase is selected from a bead or a chip.

87. The method of any one of claims 60-86, wherein separating further comprises affinity purification or reversed phase chromatography.

88. The method of any one of claims 60-87, wherein the ssDNA template oligonucleotide is recycled for subsequent use.

89. The method of any one of claims 60-88, wherein the exon region is synthesized from ssDNA template polynucleotides, wherein the ssDNA template polynucleotides comprise a 5’- untranslated region (5’UTR) ssDNA template polynucleotide, a coding sequence (CDS) ssDNA template polynucleotide, a 3 ’-untranslated region (3’UTR) ssDNA template polynucleotide, and a poly(A) tail ssDNA template polynucleotide.

90. The method of any one of claims 60-89, wherein the RNA produced is messenger RNA (mRNA).

91. The method of any one of claims 60-89, wherein the RNA produced is non-coding RNA.

92. A method for producing a circular RNA (circRNA), comprising:(a) annealing a 5’ phosphorylated region 1 RNA primer to a circular single stranded DNA (ssDNA) template polynucleotide;(b) contacting the circular ssDNA template polynucleotide with an engineered primer-dependent RNA polymerase and a mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the region 1 RNA primer, thereby producing a synthesized RNA comprising the region 1 RNA primer and a region 2 RNA;(c) ligating the synthesized RNA;(d) separating the synthesized RNA from the circular ssDNA template polynucleotide, thereby producing the circRNA.

93. The method of claim 92, wherein the 5’ phosphorylated primer comprises one or more modified nucleotides.

94. The method of claim 92 or 93, wherein the 5’ phosphorylated primer has a length from 5 nucleotides to about 1000 nucleotides.

95. The method of claim 94, wherein the 5’ phosphorylated primer has a length of about 800 nucleotides.

96. The method of any one of claims 92-95, wherein the region 1 RNA primer comprises at least one modified nucleotide.

97. The method of any one of claims 92-95, wherein the region 1 RNA primer comprises at least one modified nucleotide, and wherein the region 2 RNA does not comprise a modified nucleotide.

98. The method of any one of claims 92-95, wherein the region 1 RNA primer does not comprise a modified nucleotide, and wherein the region 2 RNA comprises at least one modified nucleotide.

99. The method of any one of claims 92-98, wherein separating comprises digesting the ssDNA template polynucleotide.

100. The method of claim 99, wherein digesting comprises contacting the ssDNA template polynucleotide with an exonuclease.

101. The method of any one of claims 92-98, wherein separating does not comprise digesting the ssDNA template polynucleotide.

102. The method of any one of claims 92-98, wherein separating comprises chemical denaturation.

103. The method of claim 102, wherein chemical denaturation comprises contacting the DNA / RNA duplex with a hydrogen-bond-disrupting agent, a chaotropic reagent, or a strong base.

104. The method of claim 103, wherein the hydrogen-bond-disrupting agent is dimethyl sulfoxide (DMSO).

105. The method of claim 103 or 104, further comprising heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C, preferably about 60°C.

106. The method of any one of claims 102-105, wherein the ssDNA template polynucleotide comprises a tag.

107. The method of claim 106, wherein the tag is a hydrophobic tag or an affinity tag.

108. The method of claim 107, wherein the tag is an FC6 hydrophobic tag.

109. The method of any one of claims 92-108, wherein the ssDNA template polynucleotide is conjugated to a solid phase.

110. The method of claim 109, wherein the solid phase is selected from a bead or a chip.

111. The method of any one of claims 92-110, wherein separating further comprises affinity purification or reversed phase chromatography.

112. The method of any one of claims 92-111, wherein the ssDNA template oligonucleotide is recycled for subsequent use.

113. A method for producing a DNA-RNA-DNA (DRD) hybrid polynucleotide, comprising:(a) annealing a single stranded DNA (ssDNA) primer to a first single stranded DNA (ssDNA) template polynucleotide, wherein the first ssDNA template polynucleotides comprises an overlap with a second ssDNA template polynucleotide;(b) contacting the first ssDNA template polynucleotide with an engineered primerdependent RNA polymerase and a first mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the ssDNA primer, thereby producing a DNA / RNA duplex comprising a DNA-RNA hybrid polynucleotide;(c) separating the first ssDNA template polynucleotide from the DNA-RNA hybrid polynucleotide;(d) annealing the DNA-RNA hybrid polynucleotide to the second ssDNA template polynucleotide; and(e) contacting the second ssDNA template polynucleotide with a DNA polymerase and a second mixture of nucleotide triphosphates (NTPs) for a time sufficient to permit extension of the DNA-RNA hybrid polynucleotide, thereby producing a DNA / RNA duplex comprising aDRD hybrid polynucleotide comprising a single stranded RNA (ssRNA) flanked by two single stranded DNAs (ssDNAs).

114. The method of claim 113, further comprising separating the second ssDNA template polynucleotide from the DRD hybrid polynucleotide.

115. The method of claim 113 or 114, wherein the second ssDNA template polynucleotide comprises uridines (U) instead of thymidines (T), and wherein separating comprises digesting the second ssDNA template polynucleotide by contacting the second ssDNA template polynucleotide with a U-specific DNase enzyme.

117. The method of claim 113 or 114, wherein separating does not comprise digesting the ssDNA template polynucleotide.

118. The method of claim 113 or 114, wherein separating comprises chemical denaturation.

119. The method of claim 118, wherein chemical denaturation comprises contacting the DNA / RNA duplex with a hydrogen-bond-disrupting agent, a chaotropic reagent, or a strong base.

120. The method of claim 119, wherein the hydrogen-bond-disrupting agent is dimethyl sulfoxide (DMSO).

121. The method of claim 119 or 120, further comprising heating the DNA / RNA duplex to at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C, preferably about 60°C.

122. The method of any one of claims 118-121, wherein the ssDNA template polynucleotide comprises a tag.

123. The method of claim 122, wherein the tag is a hydrophobic tag or an affinity tag.

124. The method of claim 123, wherein the tag is an FC6 hydrophobic tag.

125. The method of any one of claims 113-124, wherein the ssDNA template polynucleotide is conjugated to a solid phase.

126. The method of claim 125, wherein the solid phase is selected from a bead or a chip.

127. The method of any one of claims 113-126, wherein separating further comprises affinity purification or reversed phase chromatography.

128. The method of any one of claims 113-127, wherein the ssDNA template oligonucleotide is recycled for subsequent use.

129. The method of any one of claims 113-128, wherein the two ssDNAs have sufficient homology to permit the formation of a dumbbell shape, thereby producing a dumbbell DRD unit.

130. The method of any one of claims 113-128, further comprising ligating the DRD hybrid polynucleotide to at least one capped RNA-DNA hybrid polynucleotide.

131. The method of claim 130, comprising ligating the DRD hybrid polynucleotide to at least two capped RNA-DNA hybrid polynucleotides.

132. The method of claim 130 or 131, wherein the ligating produces a multi-cistronic RNA.

133. The method of claim 132, wherein the multi-cistronic RNA comprises branched caps.

134. The method of claim 129, further comprising ligating the dumbbell DRD unit to at least one other dumbbell DRD unit.

135. The method of claim 129, further comprising ligating the dumbbell DRD unit to at least two other dumbbell DRD units.

136. The method of claim 134 or 135, wherein at least one of the dumbbell DRD units comprises a branched cap.

137. The method of any one of claims 113-136, wherein the ssRNA comprises unmodified nucleotides.

138. The method of any one of claims 113-137, wherein the ssRNA comprises at least 1 modified nucleotide.

139. A kit for producing a region-specifically-modified mRNA, comprising one or more of:(a) an engineered primer-dependent RNA polymerase;(b) an RNA primer;(c) a single stranded DNA (ssDNA) template polynucleotide; and(d) a mixture of nucleotide triphosphates (NTPs).

140. The kit of claim 139, wherein the engineered primer-dependent RNA polymerase is a TGK polymerase, a T7 polymerase, or a SP6 polymerase.

141. The kit of claim 139 or 140, wherein the RNA primer is a chemically capped RNA primer.

142. The kit of claim 139 or 140, wherein the RNA primer is a 5’ phosphorylated RNA primer.

143. The kit of any one of claims 139-142, wherein the ssDNA template polynucleotide is a linear ssDNA template polynucleotide.

144. The kit of any one of claims 139-143, wherein the ssDNA template polynucleotide is a circular ssDNA template polynucleotide.

145. The kit of any one of claims 139-144, wherein the mixture of NTPs comprises at least one modified nucleotide.

146. The kit of claim 145, wherein the modified nucleotide is selected from the group consisting of 2'-O-methyladenosine (mA); alpha-phosphorothioate adenosine (PS-A); N6- methyladenosine (m6A); N6-phenyladenosine (Ph6A); N6-benzyladenosine (Bn6A); and N6- cyclopentyladenosine (Cy6A).

147. The kit of any one of claims 139-146, further comprising instructions for using the kit.

148. The kit of any one of claims 139-147, further comprising one or more of:(e) a hydrogen-bond-disrupting agent;(f) a chaotropic reagent; or(g) a strong base.

149. The kit of claim 148, wherein the hydrogen-bond-disrupting agent is dimethyl sulfoxide (DMSO).

150. The kit of any one of claims 113-149, further comprising a solid phase.

151. The kit of claim 150, wherein the solid phase is selected from a bead or a chip.

152. An RNA produced by the method of any one of claims 1-138.

153. An RNA comprising two or more adenosine nucleotides, two or more guanosine nucleotides, two or more cytidine nucleotides, and two or more uridine nucleotides, wherein:(i) at least one, but not all of the adenosine nucleotides are modified,(ii) at least one, but not all of the guanosine nucleotides are modified,(iii) at least one, but not all of the cytidine nucleotides are modified,(iv) at least one of the uridine nucleotides are modified, or(v) any combination of (i), (ii), (iii), or (iv), wherein all modified adenosine nucleotides, all modified guanosine nucleotides, all modified cytidine nucleotides, and all modified uridine nucleotides are localized non-randomly to one or more predetermined regions of the RNA.

154. The RNA of claim 153, wherein at least one, but not all of the uridine nucleotides are modified, and wherein all modified uridine nucleotides are localized non-randomly to one or more predetermined regions of the RNA.

155. The RNA of claim 153, wherein all of the uridine nucleotides are modified.

156. The RNA of any one of claims 153-155, wherein the uridine nucleotides that are modified are N1 -methylpseudouridine (m I T).

157. The RNA of any one of claims 153-156, wherein the RNA comprises a 5' cap region, an open reading frame (ORF), and a 3' untranslated region (UTR), and wherein in each of the 5' cap region and the 3' UTR at least one adenosine nucleotide is modified, and in the ORF all adenosine nucleotides are unmodified.

158. The RNA of claim 157, wherein in each of the 5' cap region and the 3' UTR all adenosine nucleotides are modified.

159. The RNA of claims 157 or 158, wherein the 3' UTR comprises a poly(A) region.

160. The RNA of any one of claims 157-159, wherein the adenosine nucleotides that are modified are selected from 2' O-methyl (2'0Me) adenosine and N6-methyladenosine (m6A).

161. An RNA comprising at least a first region and a second region, the first region and the second region each independently comprising adenosine nucleotides, guanosine nucleotides, cytidine nucleotides, and uridine nucleotides; wherein:(i) each adenosine nucleotide in the first region is a modified adenosine and each adenosine nucleotide in the second region is an unmodified adenosine;(ii) each adenosine nucleotide in the first region is an unmodified adenosine and each adenosine nucleotide in the second region is a modified adenosine;(iii) each guanosine nucleotide in the first region is a modified guanosine and each guanosine nucleotide in the second region is an unmodified guanosine;(iv) each guanosine nucleotide in the first region is an unmodified guanosine and each guanosine nucleotide in the second region is a modified guanosine;(v) each cytidine nucleotide in the first region is a modified cytidine and each cytidine nucleotide in the second region is an unmodified cytidine; or(vi) each cytidine nucleotide in the first region is an unmodified cytidine and each cytidine nucleotide in the second region is a modified cytidine; and wherein:(i) all of the uridine nucleotides in the first region and the second region are modified;(ii) each uridine nucleotide in the first region is a modified uridine and each uridine nucleotide in the second region is an unmodified uridine; or(iii) each uridine nucleotide in the first region is an unmodified uridine and each uridine nucleotide in the second region is a modified uridine.

162. The RNA of claim 161, wherein all of the uridine nucleotides in the first region and the second region are modified.

163. The RNA of claims 161 or 162, wherein the uridine nucleotides that are modified are Nl- methylpseudouridine (ml'P).

164. The RNA of any one of claims 152-163, wherein the RNA comprises a 5’UTR, an open reading frame (ORF), a 3' UTR, and a poly(A) tail, wherein: each of the adenosines in the 5’UTR is a 2’OMe-modified adenosine, each of the guanosines in the 5’UTR is a 2’OMe-modified guanosine, each of the cytidines in the 5’UTR is a 2’OMe-modified cytidine, and each of the uridines in the 5’UTR is a 2’OMe-modified uridine; and wherein: each of the adenosines in the ORF and the 3 ’UTR is an unmodified adenosine, each of the guanosines in the ORF and the 3 ’UTR is an unmodified guanosine, each of the cytidines in the ORF and the 3 ’UTR is an unmodified cytidine, and each of the uridines in the ORF and the 3 ’UTR is an N1 -methylpseudouridine (ml'P).

165. The RNA of any one of claims 162-163, wherein the RNA comprises a 5’UTR, an open reading frame (ORF), a 3' UTR, and a poly(A) tail, wherein the 5’UTR comprises a 5’ half and a 3’ half, wherein the 5’ half comprises 50% of the nucleotides of the 5’UTR that are closest to the 5’ end of the 5’UTR, wherein the 3’ half comprises 50% of the nucleotides of the 5’UTR that are closest to the 3’ end of the 5’UTR, wherein: each of the adenosines in the 5’ half of the 5’UTR is a 2’OMe-modified adenosine,each of the guanosines in the 5’ half of the 5’UTR is a 2’OMe-modified guanosine, each of the cytidines in the 5’ half of the 5’UTR is a 2’OMe-modified cytidine, and each of the uridines in the 5’ half of the 5’UTR is a 2’OMe-modified uridine; and wherein: each of the adenosines in the 3’ half of the 5’UTR, the ORF, and the 3’UTR is an unmodified adenosine, each of the guanosines in the 3’ half of the 5’UTR, the ORF, and the 3’UTR is an unmodified guanosine, each of the cytidines in the 3’ half of the 5’UTR, the ORF, and the 3’UTR is an unmodified cytidine, and each of the uridines in the 3’ half of the 5’UTR, the ORF, and the 3’UTR is an N1 -methylpseudouridine (ml'P).

166. The RNA of claim 164 or 165, wherein each of the adenosines in the poly(A) tail is an N6-methyladenosine (m6A).

167. An RNA-DNA hybrid, comprising the RNA of any one of claims 152-166 and at least one region of DNA.

168. An RNA-DNA hybrid, comprising a first DNA region and a second DNA region with an RNA region in between the first DNA region and the second DNA region, the RNA region comprising at least a first region and a second region, the first region and the second region each independently comprising adenosine nucleotides, guanosine nucleotides, cytidine nucleotides, and uridine nucleotides; wherein:(i) each adenosine nucleotide in the first region is a modified adenosine and each adenosine nucleotide in the second region is an unmodified adenosine;(ii) each adenosine nucleotide in the first region is an unmodified adenosine and each adenosine nucleotide in the second region is a modified adenosine;(iii) each guanosine nucleotide in the first region is a modified guanosine and each guanosine nucleotide in the second region is an unmodified guanosine;(iv) each guanosine nucleotide in the first region is an unmodified guanosine and each guanosine nucleotide in the second region is a modified guanosine;(v) each cytidine nucleotide in the first region is a modified cytidine and each cytidine nucleotide in the second region is an unmodified cytidine; or(vi) each cytidine nucleotide in the first region is an unmodified cytidine and each cytidine nucleotide in the second region is a modified cytidine; and wherein:(i) all of the uridine nucleotides in the first region and the second region are modified;(ii) each uridine nucleotide in the first region is a modified uridine and each uridine nucleotide in the second region is an unmodified uridine; or(iii) each uridine nucleotide in the first region is an unmodified uridine and each uridine nucleotide in the second region is a modified uridine.

169. A dual-cistronic molecule comprising two ligated RNA-DNA hybrids according to claim 167 or 168.

170. A multi-cistronic molecule comprising at least three ligated RNA-DNA hybrids according to claim 167 or 168.

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