Compositions and methods for inducing RNA translation

Abstract

Disclosed herein are methods of inducing RNA translation by contacting untranslatable RNAs or RNAs with low translatability with trans-acting intermolecular elements, including capped and polyA-tailed oligonucleotides. Also provided are compositions comprising one or more of the capped or tailed RNA oligonucleotides provided herein, and methods of using said compositions for therapeutic applications.

Description

COMPOSITIONS AND METHODS FOR INDUCING RNATRANSLATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 655,790, filed June 4, 2024, the entire contents of which are incorporated herein by reference in their entireties.BACKGROUND

[0001] 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.

[0002] 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

[0003] In one aspect, provided herein is a method for producing a translatable RNA, comprising contacting a cap acceptor RNA with a cap donor RNA under conditions sufficient to permit binding of the cap acceptor RNA and the cap donor RNA, wherein before binding of the cap acceptor RNA and the cap donor RNA, the cap acceptor RNA does not comprise a 5 ’cap and is untranslatable. In some embodiments, the method further comprises contacting the cap acceptorRNA and the cap donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme. In some embodiments, the cap donor RNA is an endogenous mRNA comprising an m7G cap. In some embodiments, the cap donor RNA is an exogenous mRNA comprising an m7G cap. In some embodiments, the cap donor RNA is a capped oligonucleotide generated through a nucleolytic reaction. In some embodiments, the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage. In some embodiments, the cap acceptor RNA is an endogenous mRNA that does not comprise an m7G cap. In some embodiments, the cap acceptor RNA is an exogenous mRNA that does not comprise an m7G cap. In some embodiments, the exogenous mRNA is encoded by a DNA vector. In some embodiments, the cap acceptor RNA is an endogenous uncapped RNA. In some embodiments, the cap acceptor RNA is an endogenous circRNA. In some embodiments, the cap acceptor RNA is an exogenous circRNA. In some embodiments, the exogenous circRNA is encoded by a DNA vector. In some embodiments, binding of the cap acceptor RNA and the cap donor RNA comprises one or more non-covalent interactions. In some embodiments, the cap acceptor RNA and the cap donor RNA comprises one or more covalent interactions. In some embodiments, binding of the cap acceptor RNA and the cap donor RNA comprises direct hybridization. In some embodiments, the one or more RBPs comprises a CRISPR protein. In some embodiments, the one or more RBPs comprises an Ago complex protein. In some embodiments, the one or more RBPs comprises an Rtcb enzyme. In some embodiments, the cap donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA. In some embodiments, the cap donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA.

[0004] In one aspect, the present disclosure provides a method for producing a translatable RNA, comprising contacting a tail acceptor RNA with a tail donor RNA under conditions sufficient to permit binding of the tail acceptor RNA and the tail donor RNA, wherein before binding of the tail acceptor RNA and the tail donor RNA, the tail acceptor RNA does not comprise a polyA tail and is untranslatable or has low translatability. In some embodiments, the method further comprises contacting the tail acceptor RNA and the tail donor RNA with one or more RNAbinding proteins (RBPs) and / or a ribozyme. In some embodiments, the tail donor RNA is an endogenous mRNA comprising polyA tail. In some embodiments, the tail donor RNA is an exogenous mRNA comprising a polyA tail. In some embodiments, the tail donor RNA is a polyA-tailed oligonucleotide generated through a nucleolytic reaction. In some embodiments, the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage. In some embodiments, the tail acceptor RNA is an endogenous mRNA that does not comprise polyA tail. In some embodiments, the tail acceptor RNA is an exogenous mRNA that does not comprise a polyA tail. In some embodiments, the exogenous mRNA is encoded by a DNA vector. In some embodiments, the tail acceptor RNA is an endogenous un-tailed RNA. In some embodiments, the tail acceptor RNA is an endogenous circRNA. In some embodiments, the tail acceptor RNA is an exogenous circRNA. In some embodiments, the exogenous circRNA is encoded by a DNA vector. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises one or more non-covalent interactions. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises one or more covalent interactions. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises direct hybridization. In some embodiments, the one or more RBPs comprises a CRISPR protein. In some embodiments, the one or more RBPs comprises an Ago complex protein. In some embodiments, the one or more RBPs comprises an Rtcb enzyme. In some embodiments, the tail donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA. In some embodiments, the tail donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA.

[0005] In one aspect, provided herein is an RNA produced by any of the methods described above.

[0006] In one aspect, provided herein is a delivery agent comprising the RNA described above, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle. In some embodiments, the nanoparticle or microparticle is a lipid nanoparticle or microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle.

[0007] In one aspect, provided herein is a cell comprising the RNA described above. In some embodiments, the cell is a mammalian cell.

[0008] In one aspect, provided herein is a composition comprising the RNA described above, the delivery agent described above, or the cell described above. In some embodiments, the composition further comprises an additional agent. In some embodiments, the additional agent is an agent which has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is chosen from the list consisting of 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 is a shRNA, a siRNA, or an antisense oligonucleotide (ASO). In some embodiments, the additional agent is an antigen or adjuvant. In some embodiments, the composition is a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0009] In one aspect, provided herein is the RNA described above, the delivery agent described above, the cell described above, or the composition described above for use in preventing or treating a disease in a subject.

[0010] In one aspect, provided herein is a kit comprising one or more of the RNA described above, the delivery agent described above, the cell described above, or the composition described above. In some embodiments, the kit further comprises one or more RNA binding proteins (RBPs) or polynucleotides encoding the one or more RBPs.

[0011] In one aspect, provided is a kit comprising the composition described above, a device for administering the composition to a subject, and instructions for administering the composition to the subject.

[0012] In one aspect, provided herein is a method for inducing translation of a target cap acceptor RNA, comprising contacting the cap acceptor RNA with a cap donor RNA under conditions sufficient to permit binding of the cap acceptor RNA and the cap donor RNA, wherein before binding of the cap acceptor RNA and the cap donor RNA, the cap acceptor RNA does not comprise a 5’cap and is untranslatable. In some embodiments, the method further comprises contacting the cap acceptor RNA and the cap donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme. In some embodiments, the cap donor RNA is an endogenous mRNA comprising an m7G cap. In some embodiments, the cap donor RNA is an exogenous mRNA comprising an m7G cap. In some embodiments, the cap donor RNA is acapped oligonucleotide generated through a nucleolytic reaction. In some embodiments, the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage. In some embodiments, the cap acceptor RNA is an endogenous mRNA that does not comprise an m7G cap. In some embodiments, the cap acceptor RNA is an exogenous mRNA that does not comprise an m7G cap. In some embodiments, the exogenous mRNA is encoded by a DNA vector. In some embodiments, the cap acceptor RNA is an endogenous uncapped RNA. In some embodiments, the cap acceptor RNA is an endogenous circRNA. In some embodiments, the cap acceptor RNA is an exogenous circRNA. In some embodiments, the exogenous circRNA is encoded by a DNA vector. In some embodiments, binding of the cap acceptor RNA and the cap donor RNA comprises one or more non-covalent interactions. In some embodiments, the cap acceptor RNA and the cap donor RNA comprises one or more covalent interactions. In some embodiments, binding of the cap acceptor RNA and the cap donor RNA comprises direct hybridization. In some embodiments, the one or more RBPs comprises a CRISPR protein. In some embodiments, the one or more RBPs comprises an Ago complex protein. In some embodiments, the one or more RBPs comprises an Rtcb enzyme. In some embodiments, the cap donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA. In some embodiments, the cap donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA. In some embodiments, translation occurs in cellulo.

[0013] In one aspect, the present disclosure provides a method for inducing translation of a target tail acceptor RNA, comprising contacting the tail acceptor RNA with a tail donor RNA under conditions sufficient to permit binding of the tail acceptor RNA and the tail donor RNA, wherein before binding of the tail acceptor RNA and the tail donor RNA, the tail acceptor RNA does not comprise a polyA tail and is untranslatable or has low translatability. In some embodiments, the method further comprises contacting the tail acceptor RNA and the tail donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme. In some embodiments, the tail donor RNA is an endogenous mRNA comprising polyA tail. In some embodiments, the tail donor RNA is an exogenous mRNA comprising a polyA tail. In some embodiments, the taildonor RNA is a polyA-tailed oligonucleotide generated through a nucleolytic reaction. In some embodiments, the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage. In some embodiments, the tail acceptor RNA is an endogenous mRNA that does not comprise polyA tail. In some embodiments, the tail acceptor RNA is an exogenous mRNA that does not comprise a polyA tail. In some embodiments, the exogenous mRNA is encoded by a DNA vector. In some embodiments, the tail acceptor RNA is an endogenous un-tailed RNA. In some embodiments, the tail acceptor RNA is an endogenous circRNA. In some embodiments, the tail acceptor RNA is an exogenous circRNA. In some embodiments, the exogenous circRNA is encoded by a DNA vector. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises one or more non-covalent interactions. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises one or more covalent interactions. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises direct hybridization. In some embodiments, the one or more RBPs comprises a CRISPR protein. In some embodiments, the one or more RBPs comprises an Ago complex protein. In some embodiments, the one or more RBPs comprises an Rtcb enzyme. In some embodiments, the tail donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA. In some embodiments, the tail donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA. In some embodiments, translation occurs in cellulo.

[0014] The foregoing general description and following detailed description are examples and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.

[0015] 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 subject matter disclosed herein and may be employed in any combination to achieve the benefits described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1A-1D show conceptualization of mRNA cap snatching. FIG. 1A is a schematic showing a system in which three components are included in the mRNA cap snatching scheme: (1) a reporter mRNA (which does not include a cap, and is untranslatable), (2) a cap donor RNA (which includes a cap), and (3) one or more RNA binding proteins. An untranslatable reporter mRNA can be activated after cap snatching and introducing a proximal cap structure and express the protein encoded by reporter. FIG. IB is a schematic showing that cap donor RNA can include various RNA species with cap structure that can target the reporter mRNA: e.g., an endogenous mRNA with a m7G cap, exogenous mRNA or oligonucleotide with a m7G cap, or a capped oligonucleotide generated through a nucleolytic reaction (CRISPR, siRNA, miRNA etc. mediated cleavage). FIG. 1C is a schematic showing that the reporter mRNA can include untranslatable endogenous mRNA and / or exogenous mRNA that does not include a m7G cap: e.g., endogenous or transfected uncapped mRNA, endogenous or exogenous circRNA or mRNA / circRNA encoded by DNA vector. FIG. ID is a schematic showing that the cap donor RNA / cap acceptor RNA interaction mode can include non-covalent interactions and / or covalent linkages: e.g., direct hybridization of the reporter mRNA to the cap donor RNA through homology arm can induce the translation of reporter mRNA; RNA binding proteins (e.g. CRISPR protein, Ago complex etc.) can assist the binding between reporter mRNA and donor RNA, which activates translation; for the cap donor generated through nucleolytic reactions, endogenous mRNA repairing pathway (e.g. Rtcb enzyme) can be utilized to re-ligate the capped oligo with reporter mRNA to form covalent linkage.

[0017] FIGS. 2A-2D show conceptualization of mRNA tail snatching. FIG. 2A is a schematic showing a system in which three components are included in the mRNA tail snatching scheme: (1) a reporter mRNA (without a polyA tail and thus likely having low translatability), (2) a tail donor RNA (which includes a polyA tail), and (3) one or more RNA binding proteins. Expression of a reporter mRNA can be activated and enhanced after tail snatching and introducing a proximal / covalent tail. FIG. 2B is a schematic showing that the tail donor RNA include various RNA species with polyA that can be target the reporter mRNA: e.g., an endogenous mRNA with a polyA tail, an exogenous mRNA or oligonucleotide with a polyA tail, or a polyA oligonucleotide generated through a nucleolytic reaction (CRISPR, siRNA, miRNA etc. mediated cleavage). FIG. 2C is a schematic showing that a reporter mRNA can includeendogenous and / or exogenous mRNA without a polyA tail, and / or mRNA encoded by DNA vector. FIG. 2D is a schematic showing that the cap donor RNA / cap acceptor RNA interaction mode can include non-covalent interactions and / or covalent linkages: e.g., direct hybridization of the reporter mRNA to the tail donor RNA through homology arm can induce the translation of reporter mRNA; RNA binding proteins (e.g. CRISPR protein, Ago complex etc.) can assist the binding between reporter mRNA and donor RNA, which activates translation; for the tail donor generated through nucleolytic reactions, endogenous mRNA repairing pathway (e.g. Rtcb enzyme) can be utilized to re-ligate the polyA oligonucleotide with reporter mRNA to form covalent linkage.

[0018] FIG. 3A-3C shows experimental data demonstrating the induction of translation by cap snatching. FIG. 3A shows induction of NLuc circRNA translation by covalent or noncovalent internal caps, m IT-modified circRNA were prepared by LEGO and co-transfected with varying amounts of a capped oligo complementary to the ligated sequence at the circRNA 5'-UTR.QRNA with m7G (construct 2) was compared, n = 3, biological replicates. Mean ± sem. P values were calculated by ordinary one-way ANOVA with multiple comparisons against construct 1 without any co-transfected capped oligos (Dunnett’s multiple comparison test). FIG. 3B shows that an internal branched cap effectively induces translation of both the upstream open reading frame (ORF) and the downstream open reading frame (ORF). A linear mRNA was designed to contain a 5’ full length FLuc ORF and a 3' 3><HiBiT ORF with an out of frame start codon from the FLuc ORF (construct 3). The dual-ORF mRNA was circularized (construct 4) and contained a branched cap (construct 5) where the LNAm7G cap was located upstream of the FLuc ORF and downstream of the 3 HiBiT ORF. n = 3, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided t-test. FIG. 3C shows a schematic illustrating “cap-proximal” mechanisms of translation initiation through the lateral slot-in mode, which permits cap and 5' UTR sequences connected to mRNA via either natural phosphodiester backbone or unnatural covalent linkages (proximal cv.s-acting caps). Furthermore, translation initiation could be driven by non-covalent cap structures in proximity of the 5' end of mRNA via intramolecular (a distal cv.s-acting “3' UTR-cap”) or intermolecular (a / ra / rs-acting, non-covalent 5 '-cap) base-pairing.DETAILED DESCRIPTION

[0019] Provided herein are methods of inducing RNA translation by contacting untranslatable RNAs or RNAs with low translatability with trans-acting intermolecular elements, including capped and polyA-tailed oligonucleotides. Also provided are methods of producing translatable RNAs by contacting untranslatable RNAs or RNAs with low translatability with trans-acting intermolecular elements, including capped and polyA-tailed oligonucleotides. Also provided are compositions comprising one or more of the capped or tailed RNA oligonucleotides provided herein, and methods of using said compositions for therapeutic applications.Equivalents

[0020] 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 advantages described 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.

[0021] 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.

[0022] 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, i.e., 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.g., “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.”

[0023] 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 (e 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.).

[0024] 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.

[0025] 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 RNAmolecule 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.).

[0026] 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.

[0027] In some embodiments of the modified mRNAs provided herein, the mRNA comprises 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.

[0028] In some embodiments of the modified mRNAs provided herein, the mRNA comprises, 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 someembodiments, the first nucleotide of the poly-A region is 3’ to (downstream of) the last nucleotide of the 3’ UTR.

[0029] 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.

[0030] In some embodiments, the mRNA is a circular mRNA. A circular mRNA is an mRNA 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 mRNA with a nucleic acid sequence comprising every nucleotide of the circular mRNA 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 mRNAs 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.

[0031] 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 the codon 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.

[0032] 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.

[0033] 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 phosphodi ester 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.

[0034] 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: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 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.

[0035] 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 sugarare 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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 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.

[0043] 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.

[0044] 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.

[0045] 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 intemucleotide 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 intemucleotide linkage. In some embodiments, a 5' cap comprises one or more nucleotides with a sugar modification, such as 2'-O-methylation.

[0046] 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 internucleotide linkage is reproduced below:

[0047] 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 , Cl", Br , I"), NCh", C1O4 , OH , H2PO4 , HCO3 , HSO4 , sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-l-sulfonic acid-5-sulfonate, ethan-l-sulfonic acid-2- sulfonate, and the like), carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, and the like), BF4 , PF 4 , PF 6 ", AsFe", SbFe", B[3,5-(CF3)2C6H3]4]“, B(C6FJ)4 , BPt , A1(OC(CF3)3)4- and carborane anions (e.g., CBi il I12 or (HCBnMesBre) ). 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.

[0048] 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 mRNAs

[0049] In some aspects, the present disclosure provides modified mRNAs comprising a 5’ cap region, wherein the 5’ cap region comprises a 5’ nucleotide cap and one or more modifiednucleotides. In some embodiments, a modified mRNA is a modified linear mRNA. In some embodiments, a modified mRNA is a modified circular mRNA.

[0050] The “5' cap region”, as used herein, refers to a region of an mRNA 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 controls mRNA 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.

[0051] In some embodiments of the modified mRNAs provided herein, the modified mRNA comprises one or more modified nucleotides in the 5' cap region of the mRNA. In some embodiments, the 5' cap region includes one or more nucleotides that are not canonical adenosine, cytidine, guanosine, or uridine nucleotides. In some embodiments, the 5' cap region comprises between 1 and 3, between 3 and 5, between 5 and 7, or between 7 and 10 5' caps. In some embodiments, the 5' cap region comprises between 10-500 nucleotides. In some embodiments, the 5' cap region comprises between 10 and 15, between 15 and 20, between 20 and 25, between 25 and 50, between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 and 500 nucleotides.Chemical synthesis of 5’ cap regions

[0052] Existing methods for preparing capped linear mRNA do not accommodate modifications that are not tolerated by RNA polymerase or capping enzymes, nor modifications that extend beyond the first two bases, creating a screening bias due to differing cap incorporation efficiencies. To overcome these challenges, the capping process was decoupled from mRNA synthesis as described herein.

[0053] In some embodiments of the methods provided herein, the method comprises first synthesizing a 5 ’-phosphorylated RNA oligonucleotide with a specific sequence and / or desired modifications. In the methods described herein, the synthesized 5 ’-phosphorylated RNA oligonucleotide defines the 5’ UTR when ligated to an RNA transcript. Thus, as used herein, the terms “5’-phosphorylated RNA oligonucleotide,” “5 ’-phosphorylated oligonucleotide,” and “5’- phosphorylated UTR” are used interchangeably. In some embodiments, the 5 ’-phosphorylated RNA oligonucleotide comprises one or more modified nucleotides which may affect RNA translation and / or stability.

[0054] In some embodiments, the 5 ’-phosphorylated oligonucleotide comprises a modified phosphate, resulting in a modified intemucleotide 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, hydroxy phosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate. In someembodiments, more than one modified phosphate is used. In some embodiments, the 5’- phosphorylated oligonucleotide 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 some embodiments, the 5 ’-phosphorylated oligonucleotide 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 5 ’-phosphorylated oligonucleotide 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 5 ’-phosphorylated oligonucleotide.

[0055] In some embodiments, the 5 ’-phosphorylated oligonucleotide comprises 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'-deoxyadenosine (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 D-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 5 ’-phosphorylated oligonucleotide 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 5 ’ -phosphorylated oligonucleotide 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 5 ’-phosphorylated oligonucleotide 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 5 ’-phosphorylated oligonucleotide.

[0056] In some embodiments, the 5 ’-phosphorylated oligonucleotide comprises 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 -carboxy cytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5 -formyluracil, 5 -hydroxy cytosine, 5-hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5 -methoxy cytosine, 5- methoxyuracil, 5-methylcytosine, 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, N1 -ethylpseudouracil, Nl- methoxymethylpseudouracil, N 1 -methyladenine, N1 -methylpseudouracil, Nl- 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 5 ’-phosphorylated oligonucleotide 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 5 ’-phosphorylated oligonucleotide 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 5 ’-phosphorylated oligonucleotide 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 5’- phosphorylated oligonucleotide.

[0057] In some embodiments, the 5 ’-phosphorylated oligonucleotide is synthesized on a solidphase support. In some embodiments, the solid support is controlled-pore glass (CPG) or polystyrene (PS). In some embodiments, the 5 ’-phosphorylated oligonucleotide is synthesized via phosphorami di te oligonucleotide synthesis. In some embodiments, the 5 ’-phosphorylated oligonucleotide is synthesized in a solvent system comprising a nonpolar counterion. In some embodiments, the nonpolar counterion used in oligonucleotide synthesis is ammonium. In some embodiments, the nonpolar counterion used in oligonucleotide synthesis is ammonium.

[0058] In some embodiments, a 5’ cap is added to the 5 ’-phosphorylated oligonucleotide to produce a 5’-capped oligonucleotide (i.e., a 5’-capped UTR). A 5’ cap can be added to an RNA oligonucleotide via enzymatic or chemical reactions. In some embodiments, the cap is added to the 5 ’-phosphorylated oligonucleotide through chemical capping methods. Chemical capping may be performed by any method known in the art. Preferably, the chemical capping reaction is performed through an anhydrous reaction between the 5 ’-phosphorylated RNA oligonucleotide and a capping nucleotide conjugated to imidazole in the presence of 1 -methylimidazole (see Abe et al., “Complete Chemical Synthesis of Minimal Messenger RNA by Efficient Chemical Capping Reaction” ACS Chem. Biol. 2022, 17: 1308-1314). In this method, the cap of interest is first conjugated to imidazole. A chemical reaction is then performed between the imidazole- conjugated capping oligonucleotide and a 5’-phoshporylated oligonucleotide under anhydrous conditions and in the presence of 1 -methylimidazole. In some embodiments, the capping reaction is performed in dimethyl sulfoxide (DMSO). The desired product of this reaction is an oligonucleotide capped on its 5’ end with the cap of interest.

[0059] In some embodiments, the 5’ cap used in the present invention may be, but is not limited to, 7-methy guanosine (m7G), N7,3’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7G- 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], phenylethyl) guanosine [7-(l-PhEt)G], m7GpppBH3G (DI and D2 stereoisomers), (DI and D2 stereoisomers), m7GpBH3G (DI and D2 stereoisomers), m7GppBH3pm°GpppBH3G (DI and D2 stereoisomers), m27’2°GppBH3pG (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, ARC A, modified ARC A, inosine, Nl- methylguanosine, LNA-guanosine, 2-azido-guanosine, and a bridging or non-bridging methylphosphonate moiety.

[0060] Thus, in some embodiments, a 5’ cap region provided herein comprises a 5’-capped oligonucleotide (i.e., a 5’-capped UTR) synthesized as described above. In some embodiments, the 5’ cap region comprises a modified 5’ cap, one or more modified phosphates, one or more modified sugars, and / or one or more modified nucleobases. The 5’ cap region may comprise any combination of modifications. In some embodiments, the 5’ cap region is between 5 and 50, between 10 and 45, between 15 and 40, between 20 and 35, between 25 and 30, or more than 30 nucleotides in length. In some embodiments, the 5’ cap region 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 nucleotides. In some embodiments, the 5’ cap region 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 and100, or between 100 and 200 modified nucleotides. In some embodiments, the modified nucleotides of the 5’ cap region 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 nucleotides in the 5’ cap region.

[0061] In some embodiments, a capped RNA transcript as provided herein comprises more than one 5’ cap or 5’ UTR region. In some embodiments, a capped RNA transcript as provided herein comprises more than one poly-A tail. Methods of producing multi-capped RNA strands have been described in U.S. Patent Application Number 63 / 300,602, the contents of which are incorporated herein in their entirety. These methods comprise incorporating azide handles into an RNA molecule such that it is compatible with an alkyne-containing nucleotide to undergo a click chemistry reaction. The present application builds upon these techniques. In some embodiments, an azide handle is introduced into the RNA molecule through tRNA guanine transglycosylase (TGT) in combination with a pre-queuosine 1 (preQi) substrate (Ehret et al. “Site-specific covalent conjugation of modified mRNA by tRNA guanine transglycosylase.” Mol. Pharm. 15, 737-742 (2018)). In some embodiments, an azide handle is incorporated into an RNA molecule (e.g., 5 ’-phosphorylated RNA oligonucleotide or a capped RNA transcript) through during transcription, providing an azide-linked nucleotide as substrate for incorporation into a growing RNA strand (e.g., 5-Azido-PEG4-CTP). In some embodiments, more than one azide handle is introduced into an RNA molecule. In some embodiments, more than one azide handle is introduced into an RNA molecule using more than one introduction technique (e.g., both TGT and IVT).Producing RNA transcripts with a modified 5’ cap region

[0062] In some aspects, the methods provided herein produce a capped RNA transcript with a modified 5’ cap region. In some embodiments, the methods comprise attaching a 5’ cap region as described herein to an RNA precursor, thereby producing a capped RNA transcript comprising a modified 5’ cap and UTR.

[0063] In some aspects, the present disclosure provides methods of producing modified RNAs comprising ligating an RNA (e.g., an RNA precursor) to a 5’ cap region comprising a 5’ cap and a 5’ UTR in the presence of a ligase, whereby the ligase forms a covalent bond between the 3’ nucleotide of the 5’ cap region and the 5’ nucleotide of the RNA (e.g., the RNA precursor) toproduce capped RNA transcript, (e.g., a modified capped RNA transcript). In some embodiments, a 5’ cap region is produced as described herein. When a ligase forms a covalent bond between two linear nucleic acids, a new nucleic acid is produced, with the produced nucleic acid comprising the nucleic acid sequences of both nucleic acids. Ligation of the 3’ terminal nucleotide of a first nucleic acid to the 5’ terminal nucleotide of a second nucleic acid produces a third nucleic acid, with the third nucleic acid comprising the sequence of the first nucleic acid and the second nucleic acid, and the second nucleic acid being 3’ to (downstream of) the first nucleic acid sequence. 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)).

[0064] In some embodiments, the RNA precursor 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. For example, IPEX syndrome in humans is caused by a mutation in the F0XP3 gene, which hinders development of FOXP3+ regulatory T cells and results in increased susceptibility to autoimmune and inflammatory disorders. Expression of an essential enzyme, clotting factor, transcription factor, growth factor, cytokine, chemokine, antibody (or antibodyfragment 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 (Cast, 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 colony-stimulating 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.

[0065] In some embodiments, the ORF encodes an antigen. As used herein, “antigen” refers to a molecule (e.g., 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.

[0066] In another aspect, the methods disclosed herein provide an RNA precursor and / or a capped RNA transcript comprising one or more noncoding genes. In some embodiments, the noncoding heterologous genes are therapeutic nucleic acids. As used herein, a therapeutic nucleic acid is a nucleic acid or related compound that alters gene expression to prevent or treat diseases or disorders. In some embodiments, the therapeutic nucleic acid is an antisense oligonucleotide (ASO), N-acetylgalactosamine (GalNAc) ligand-modified short interfering RNA (siRNA) conjugate, DNA aptamer, RNA aptamer, ribozyme, RNA decoy, siRNA, shRNA, miRNA, gRNA, or CRISPRi molecule.

[0067] In some embodiments, a composition provided herein (e.g, a pharmaceutical composition) further comprises one or more additional agents. In some embodiments, the additional agent is 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 is an agent which has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is an agent that is capable of modulating expression of a gene and / or protein is a subject, such as a short hairpin RNA (shRNA), a small interfering RNA (siRNA), or an antisense oligonucleotide (ASO). In some embodiments, the additional agent is a small molecular inhibitor. In some embodiments, the additional agent is an agent that is capable of eliciting or enhancing an immune response in a subject. In some embodiments, the additional agent is an antigen (e.g, a viral antigen, a bacterial antigen). In some embodiments, the additional agent is an adjuvant, which is defined as an agent that is sufficient for enhancing an immune response in a subject when administered at an effective amount, but does not elicit an immune response in a subject when administered alone. In some embodiments, the additional agent is an enzyme, such as an enzyme that is capable of catalyzing one or more chemical reactions in a subject or in cells of a subject.

[0068] In some embodiments of the methods provided herein, an RNA precursor for which 5’ capping is desired must first be chemically prepared for capping. In vitro transcription (IVT) of RNA results in uncapped 5 ’-triphosphorylated RNA. The 5 ’ -triphosphate of the in vitro transcribed RNA is incompatible with enzymatic ligation on its 5’ end, as enzymatic ligation requires 5 ’-monophosphorylated RNA. For example, T4 RNA Ligase 1 catalyzes the ligation of a 5’-phosphoryl-terminated nucleic acid donor (e.g., the RNA precursor described herein) to a 3’ hydroxyl -terminated nucleic acid acceptor (e g., the 5’ cap region described herein) through theformation of a 3’D 5’ phosphodiester bond with hydrolysis of adenosine triphosphate (ATP) to adenosine monophosphate (AMP) and pyrophosphate (PPi).Thus, in some embodiments, an RNA precursor is prepared for capping by removing pyrophosphate from the 5’ end of the triphosphorylated RNA, leaving a 5 ’-monophosphorylated RNA to be used in a ligation reaction. In some embodiments of the methods provided herein, RNA 5’ Pyrophosphohydrolase (RppH) is used to produce 5 ’-monophosphorylated RNA from 5 ’-triphosphorylated RNA.

[0069] Ligation of the 5’ cap region to the RNA precursor using conventional enzymatic ligation methods demonstrated the requirement of a high ratio (>200) of 5’ cap region oligo:RNA precursor (oligo:mRNA) to achieve complete labeling of the RNA precursor. The requirement for such high levels of 5’ cap region oligos is inconsistent with scalability of such methods. Thus, to address this issue, in some embodiments a short unstructured spacer region was introduced into the 5’ end of the RNA precursor such that the 5’ end was exposed, allowing enzymes (e.g., RNA ligase) better access to the 5’ end.

[0070] In some embodiments, the spacer region comprises a plurality of identical consecutive nucleotides. In some embodiments, the spacer region comprises between 5 and 10, between 10 and 20, between 15 and 30, between 20 and 50, between 50 and 100, or between 100 and 200 consecutive adenosine, cytosine, guanine, or thymine nucleotides. In some embodiments, the spacer region comprises about 15 consecutive adenosine nucleotides. In some embodiments, the spacer region comprises at least one non-canonical nucleotide (e.g., inosine).

[0071] In some embodiments, the RNA precursor to which the 5’ cap region is ligated comprises one or more exonuclease-resistant nucleotide modifications in its 3’ end. Modified nucleotides containing one or more structural modifications to the nucleobase, sugar, or phosphate linkage of the RNA can interfere with 3’ and 5’ exonuclease activity, rendering the RNA more stable. Nucleotide modifications conferring exonuclease resistance are known in the art. In some embodiments, the RNA precursor comprises one or more modified phosphates, sugars, and / or nucleobases to confer exonuclease resistance. In some embodiments, the RNA precursor comprises one or more 2’-O-Methyl (2’0Me) modifications. In some embodiments, the RNA precursor comprises one or more 2’-fluoro bases. In some embodiments, the RNA precursor comprises one or more phosphorothioate (PS) or thiophosphate (SP) linkages. In some embodiments, the 3’ end of the RNA precursor comprises a phosphate group. In some embodiments, the RNA precursor comprises a C3 spacer incorporated internally or at its 3’ end.A C3 spacer modification adds a 3-carbon spacer to the 3’ terminus of an oligonucleotide. In some embodiments, the RNA precursor comprises a 2’-O-methoxy-ethyl base (2’-M0E), a G- quadruplex, or a 2’-3’-dideoxy nucleotide (ddN). In some embodiments, the RNA precursor comprises one or a combination of any of the modifications known in the art to confer exonuclease resistance (see, e.g., Clave et al., “Modified internucleoside linkages for nuclease- resistant oligonucleotides.” RSC Chem. Biol. (2021) 2:94-150)Capped-circular mRNA (QRNA)

[0072] Despite advances in circular RNA (circRNA) engineering, current constructs rely on IRES (Internal Ribosome Entry Site) or TEE (Translation Enhancing Element)-mediated translation, which are embodiments that enable cap-independent translation. Linear mRNAs are capable of undergoing cap-dependent translation through interaction with eIF4E and other eukaryotic translation initiation factors, which is the predominant form of translation in cells (Sonenberg and Hinnebusch, 2009, Cell 136: 731-745) and is generally more efficient than capindependent translation (Koch etal., 2020, Nat. Struct. Mol. Biol. 27:1095-1104).

[0073] As described herein, a “capped-circular mRNA” is a circular mRNA characterized by one or more covalent linkages to one or more cap structures (or a derivative thereof). The circular mRNA can contain all the canonical elements of a linear mRNA: (1) Cap, (2) 5’ UTR (untranslated region), (3) protein-coding regions (CDS), (4) 3’ UTR, and (5) poly(A) tail. By circularizing these features into a capped-circular RNA, it is intended to enhance half-life (increased nuclease resistance) of a canonical circular mRNA, while retaining the benefits of efficient cap-dependent translation, such as in linear mRNA.

[0074] The RNA embodiments and methods disclosed herein take advantage of the exonucleaseresistant feature of circRNA while utilizing the strong m7G-cap dependent translation initiation machinery. Such features can be achieved via chemical conjugation of a capped oligonucleotide with a circRNA through click chemistries such as copper catalyzed azide-alkyne cycloaddition (CuAAC) or tetrazine-trans cyclooctene inverse electron demand Diels- Alder reaction (IEDDA). There are at least two generic structures of capped circular messenger RNAs (QRNAs): Type 1 QRNA and Type 2 QRNA. In Type 1 QRNA, a circular poly-phosphodi ester backbone is present while capping is achieved via chemical ligation of a short, capped oligonucleotide to an internal handle on the circular mRNA through click chemistry. The 5’ cap may comprise of a 7-methylguanylate that enables efficient translation of an mRNA or alternative common mRNA cap structures, as shown, for example, in Mccaffreyanton, 2019, Genetic Engineering & Biotechnology News. 39. In Type 2 QRNA, a continuous mRNA poly-phosphodiester backbone is present; circularization is achieved via chemical conjugation between the 3’-end and 5’-UTR of the mRNA through click chemistry.

[0075] The 5’ capping and 3’ poly(A) tailing steps are useful in producing active synthetic mRNA; these modifications prevent mRNA degradation and facilitate translation initiation in eukaryotic cells. As used herein, “capping” means modification at the 5’ end of an mRNA by an addition of a “cap” molecule such as a 7-methylguanosine (m7G) cap. Other cap structures and modifications of the cap as described below can be used to optimize the translation efficiency.

[0076] Enzymes capable of catalyzing the reaction of linking a cap molecule to the mRNA include, but are not limited to, Vaccinia capping system including 2’-O-Methyl Transferase, tRNA guanine transglycosylase (TGT), Faustovirus capping enzyme, and T4-RNA ligase. Capping can also occur during the synthesis of mRNA called co-transcriptional capping.

[0077] As used herein, the term “molecular handle” or “handle” refers to a chemical group that is attached to a nucleotide on mRNA and can form a covalent bond to another molecule that is separate from the mRNA to link this other molecule to the mRNA. The covalent bond can be formed via various appropriate functional crosslinking reactions. In some embodiments described herein, the crosslinking reaction is click chemistry. As used herein, the term “click handle” refers to a molecule on mRNA that can covalently bind to another molecule via click chemistry reaction. Examples of a handle include, but are not limited to, alkyne or azide (when CuAAC is used in click chemistry), or trans-cyclotene or tetrazine (when IEDDA is used in click chemistry), or hydrozone or oxime, or any equivalent structures thereof. Other crosslinking chemistries including thio-ene and tiol-yne reactions (Escorihuela et al., 2014, Bioconjug. Chem. 25:618-627), a phosphate-amine based reaction (El-Sagheer and Brown, 2017, Chem. Commun. 53: 10700-10702; Kalinowski et al., 2016, Chembiochem. T. 1150-1155), thiol-yne, amino-yne, and hydroxyl-yne reactions (Worch et al., 2021, Chem Rev. 121(12): 6744-6776), and other bioconjugation reactions (Gassensmith, chem.libretexts.org / Bookshelves / Organic_Chemistry / Supplemental_Modules_(Organic_Chemist ry) / Reactions / Introduction_to_Bioconjugation, accessed June 23, 2023) have also been contemplated.

[0078] As used herein, the term “hairpin” or “hairpin oligonucleotide” refers to a single-stranded oligonucleotide that has a sequence of complementary base pairs at both ends capable of forming a “stem-and-loop” structure.

[0079] As used herein and understood in the art, the term “click chemistry” is intended to encompass chemical methods for linking chemical components together, including but not limited to nucleotides into polynucleotides and amino acids into peptides and polypeptides, that are “simple to perform, have high yields, require no or minimal purification, and are versatile in joining diverse structures without the prerequisite of protection steps” (see, for example, Hein et al., 2006, Pharm. Res. 10: 2216-2230). In current chemical synthetic practice four primary reactions are employed: 1) cycloadditions (including for example monovalent copper-catalyzed Huisgen 1,3-dipolar cycloadditions of azides and alkynes, the most widely used); 2) nucleophilic ring openings (including ring systems comprising strained heterocyclic electrophiles); 3) non- Aldol carbonyl chemistry (including for example hydrazone / oxime ether formation); and 4) carbon multiple bond additions (including for example certain Michael additions and formation of various three-membered rings by inter alia epoxidations). Click chemistry has been found to be particularly useful for polymeric substances such as proteins and nucleic acids as illustrated herein.

[0080] As used herein, the term “equivalent structure” means any molecule that are sufficiently structurally similar and perform the same function in a chemical reaction.

[0081] As used herein, the terms “derivatized” or “functionalized” means modification of a nucleotide that leads to some functional consequences in its chemical properties or reactivity or both. Both terms shall be understood to be equivalent to the extent that particular embodiments of the capped, circular RNA molecules have by benefit of derivatization thereof a function, particularly with regard to crosslink-dependent circularization embodiments provided herein. In some embodiments, a derivatized nucleotide is a nucleotide that is modified to comprise a chemical group / handle can participate in a cross-linking reaction.

[0082] As used herein, the term “QRNA” is intended as a generic term meaning capped circular messenger RNAs. Particularly encompassed by this term are the various species of circularized RNA molecules and in particular circularized mRNA molecules disclosed herein, but these examples are not intended to be limiting.

[0083] In some embodiments, the synthesis pathway of Type 1 and Type 3 QRNA enables multiple oligonucleotides containing 5’ cap binding to the circular RNA. For example, circular RNA can include multiple derivatized nucleotides that can covalently bind to multiple oligonucleotides containing 5’ cap. Alternatively, a single circular RNA backbone can encode multiple TGT sites to enable binding of multiple oligonucleotides containing 5’ cap onto the circular RNA simultaneously.

[0084] In some embodiments, the capped, circular RNA molecule comprises an mRNA region encoding one or a plurality of peptides or polypeptides.

[0085] As provided herein, the cap used in the capped, circularized RNA molecules of the invention can include 7-methylguanine (m7G) but in addition cap analogues as set forth, inter alia, in U.S. patent application No. 2020 / 0055891 to Walczak et al.; Holstein et al., 2016, Agnew Chem. Int. Ed. Engl. 55: 10899-10903; Walczak et al., 2017, Chem. Sci. 8: 260-267; Muttach et al., 2017, J. Org. Chem. 13: 2819-2832) can be incorporated into the circular RNA molecule precursors to create the capped, circularized RNA molecules provided herein.Cap modifications for QRNA

[0086] Several variations of the cap structure have been contemplated here to optimize translation efficiency of QRNA. These variations include: including multiple cap structures (cap 0, 1, and 2; Shanmugasundaram Qt al., 2022, Chem Rec. 22(8): e202200005); including N6, 2’-O- dimethyladenosine (m6Am) as a terminal modification adjacent to the mRNA cap (Sun et al., 2021, Nat Commun. 12(1): 4778); using cap structures with modified triphosphate bridges (Sun et al., 2021, Nat Commun. 12(1): 4778; Wojtczak et al., 2018, J Am Chem Soc. 140(18): 5987- 5999); incorporating Locked Nucleic Acid (LNA)-modified cap analogs (Kore et al., 2009, J Am Chem Soc. 131(18): 6364-5); introducing cap analogs with alternative functionalities such as light reactivity and click groups (Klocker et al., 2022, Nat Chem. 14(8): 905-913; Nowakowska et al., 2014, Org. biomol. Chem. 12: 4841-4847); hydrophobic cap analogs (WO 2017066782 Al); and others (Wojcik et al, 2021, Pharmaceutics 13(11): 1941; Grudzien et al., RNA 10(9): 1479-1487; Grzela et al., 2023, RNA 29(2): 200-216).

[0087] In some embodiments, the methyl group in 7-methylguanosine (m7G) cap structure can be modified to produce 7-benzylguanosine (Bn7G), 7-chlorobenzylguanosine (ClBn7G), and chlorobenzyl-O-ethoxyguanosine (ClBnOEt7G). Introduction of one or more Locked NucleicAcid (LNA), 2’-methoxy (20Me), and 2-methoxy ethoxy (2M0E) into m7G structure significantly increase mRNA translation. In some embodiments, the cap structures include, but are not limited to, m7G-LNA, LNAm7G-LNA, LNAm7G-LNAx6, LNAm7G-2OMex6. In some embodiments, the cap structure is m7G diphosphate imidazolide (m7GDP-Im).Nucleotide modifications

[0088] In some embodiments, as disclosed and recognized herein it is beneficial to alter the type of nucleotide / nucleotide identity, specifically incorporation of adenosine (A), guanosine (G), 6- methyladenosine (m6A), or the non-canonical inosine (I) in the mRNA, preferably, at the +1 position, increases translation efficiency. In some embodiments, substitution of some or all uridine residues to ^-methylpseudouridinein the mRNA also boosts the translation. The nucleotides are numbered according to their position immediately downstream of the cap structure. For example, the cap structure found at the 5' end of eukaryotic mRNAs consists of a 7-methylguanosine (m7G) moiety linked to the first nucleotide (+1 position) of the transcript via a 5 —5' triphosphate bridge.

[0089] Other modified nucleotides include, but are not limited to, pseudouridine, 5- methylcytidine, 2-thiouridine, 5 -methoxy uridine, 4-acetylcytidine, xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenine, 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 -formyl cytosine, 5-formyluracil, 5- hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5- iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 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, 06-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-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A) have also been contemplated at +1 and other positions.

[0090] In some embodiments, the modified phosphate backbone can be 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, or guanidinopropyl phosphoramidate.

[0091] In some embodiments, introduction of locked nucleic acid (LNA), 2’-methoxyribose (2- OMe), and 2-methoxyethoxy (2 -MOE) into the ribose sugar backbone increases mRNA translation. Addition of multiple 2-OMe and 2-MOE modified bases increases translation further. LNA specifically increased expression at the +1 position.

[0092] In some embodiments, the modified sugar can be 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 ’-dideoxyribose, 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, or 2’-O,4’-C-thio-linked ribose.

[0093] In these backbone modifications, stereoisomer structures are also considered since they have been shown to impact the RNA’s nuclease-resistance properties (Iwamoto etal., 2017, Nat. Biotech. 35: 845-851; Jahns etal., 2022, Nucleic Acids Res. 50(3): 1221-1240).

[0094] Modification of nucleotides on traditional circRNA is limited because not all of them are compatible with the internal ribosome entry site (IRES). QRNA translation does not require an IRES; thus, is tolerable to more modified nucleotides in a wide range of percentage. These modifications could be spiked into the circular backbone in varying percentages (m6A is typically spiked in at 5%). And the “stem” oligo containing the cap, or the 573’ UTR and tails could likely tolerate a higher percentage of modifications. Alternatively, these modifications can be present in different percentages along different regions of the circular RNA backbone (e.g. in the 5’ UTR, or 3’ UTR, or CDS, or close to the cap structure, or combinations thereof).Furthermore, the “stem” oligo of a Type 1 QRNA (the oligonucleotide containing the cap) is chemically synthesized and could potentially tolerate more complex structures that are difficult to enzymatically incorporate, such as locked nucleic acids (LNAs), 2’ O-methyl nucleotides, peptide nucleic acids (PNAs), morpholinos, and various internal chemical linkers as provided herein.Peptides and polypeptides encoded by QRNA

[0095] Polypeptides encoded by the capped, circularized RNA molecules provided by the invention include any therapeutically useful polypeptide for treatment or intervention of any disease process associated with or dependent on polymorphic or mutant polypeptide species, heritable or acquired as a result of environmental insult or injury. QRNA can encode multiple polypeptides, for example, self-amplifying mRNA cassettes, or multiple therapeutic peptides or polypeptides. In some embodiments, the capped, circular RNA molecule comprises an mRNA region encoding one or a plurality of peptides or polypeptides. A plurality of polypeptides include multiple copies of the same polypeptide or multiple copies of different polypeptides.

[0096] An IRES, or self-cleaving peptide such as T2A sequence, can exist between the multiple polypeptide coding sequences on the QRNA. Alternatively, an RNA oligonucleotide containing cap residue site is located before each polypeptide coding sequence, which ultimately will resultin a QRNA with multiple cap residue-containing RNA oligonucleotides and ensure that all coding sequences are translated efficiently.

[0097] Peptides encoded by capped, circularized RNA molecules of the invention can include but are not limited to therapeutic peptides or antigenic peptides, particularly antigenic peptides suitable for presentation by antigen-presenting cells to humoral (B cells) or cellular (T cells) immune system cells. In certain embodiments these antigenic peptides are adapted to and effective for use as vaccines. In other embodiments the antigenic peptides are adapted to or effective in suppressing immune responses, for example in autoimmune diseases or transplant patients. In additional embodiments the antigenic peptides are adapted to and effective for eliciting specific antitumor immune responses in tumor cells or in attracting cytotoxic native (natural killer cells) or engineered (e.g., CAR-T) cells. Therapeutic peptides encoded by capped, circularized RNA molecules of the invention can include but are not limited to human parathyroid hormone, filgrastim, oxytocin, somatostatin, calcitonin, glucagon, insulin, liraglutide, vasopressin, and the like (see, Fosgerau & Hoffman, 2015, Drug Discovery Today 20: 122-128; al Musaimi et al., 2021, Pharmaceuticals (Basil) 14: 145; Wang etal., 2022, Signal Transduct, and Targeted Therap. 7: 1-27).

[0098] In some embodiments, peptides encoded by the capped, circular RNA molecules of the invention can include, but are not limited, to Cas9 or derivatives (Rothgangl et al., 2021, Nat. Biotechnol. 39: 949-957) and adenine base editors or other base editors (Gaudelli et al., 2017, Nature 551: 464-471), or RNA base editors for delivery of genome or epigenome editing therapies.

[0099] In some embodiments, peptides encoded by the capped, circular RNA molecules of the invention can be selected from any of several target categories including, but not limited to, biologies, antibodies, vaccines , therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, or targeting moieties.Synthesis of QRNA

[0100] Type 2: The invention also provides methods for producing a type 2 capped, circularized RNA molecules of this aspect of the invention, the methods comprising: synthesizing an RNA oligonucleotide comprising a 5’ end containing a cap structure, an mRNA encoding a peptide or polypeptide, a derivatized nucleotide located between the cap structure and the mRNA region encoding the polypeptide, and a 3’ end containing moiety; and reacting thederivatized nucleotide with the 3’ end moiety to form the covalently linked capped circular RNA molecule.

[0101] Type 1: The invention also provides methods for producing a type 1 capped, circularized RNA molecules of this aspect of the invention, the methods comprising the steps of: producing a circularized RNA molecule comprising an mRNA region encoding a peptide or polypeptide, and a derivatized nucleotide outside the mRNA region; synthesizing an RNA oligonucleotide comprising a 5’ end containing cap structure and a 3’ end containing a moiety reactive with the derivatized nucleotide; and reacting the derivatized nucleotide with the 3’ end moiety of the RNA oligonucleotide form a covalently link between the RNA oligonucleotide and the circular RNA. In certain embodiments, the derivatized nucleotide comprises a moiety that can react with the 3’ end moiety by bioconjugation chemistry, wherein in the bioconjugation chemistry is click chemistry. In addition, the circularized RNA is produced by ribozyme- mediated splicing, enzymatic ligation, or click chemistry-mediated circularization.

[0102] Type 3: The invention also provides methods for producing a type 3 capped, circularized RNA molecules of this aspect of the invention, the methods comprising the steps of: producing a circularized RNA molecule comprising an mRNA region encoding a peptide or polypeptide, and a derivatized nucleotide outside the mRNA region; synthesizing an RNA oligonucleotide comprising a 5’ end containing cap structure and a 3’ end containing a moiety reactive with the derivatized nucleotide; and reacting the derivatized nucleotide with the 3’ end moiety of the RNA oligonucleotide form a covalently link between the RNA oligonucleotide and the circular RNA, wherein the synthesis of the circular RNA oligonucleotide, further comprises the steps of: synthesizing an RNA oligonucleotide comprising the mRNA region encoding a peptide or polypeptide, a hairpin structure containing an enzyme-recognition site, and a twister ribozyme sequence on both 5’ and 3’ ends; reacting the RNA oligonucleotide with the enzyme to produce the derivatized nucleotide within the hairpin structure; circularizing the RNA oligonucleotide using the twister ribozyme sequence.

[0103] The derivatized nucleotide in these 3 types of QRNA can be generated using different strategies. Namely, the derivatized nucleotide can be specifically targeted by having a hairpin structure containing a specific enzyme-recognition site. The enzyme described in some examples in tRNA guanine transferases (TGT). In other examples, the derivatized nucleotide is generated by replacement of a single cytidine with azide-cytidine.

[0104] Circularization of RNA molecule in type 1 and type 3 can be achieved by ligation with T4 ligase, RtcB ligase, or ribozyme-mediated splicing. In these embodiments, the 5’ end and 3’ end of the linear oligonucleotide comprise the appropriate moiety to participate in the enzymatic reaction to form the circular RNA. Alternatively, the click chemistry moiety has also been contemplated for the circularization. In some embodiments, additional splint probe containing complementary sequences to the 5’ and 3’ ends of the linear oligonucleotide can be used to bring the two ends in proximity and facilitate circularization.Purification of capped RNA

[0105] In some embodiments, the nucleic acids described herein are purified by any method known in the art to remove undesired components from IVT or associated reactions (including unincorporated rNTPs, protein enzymes, salts, metal ions, etc.). Techniques for the isolation of RNA molecules are well known in the art. Well-known procedures include phenol / chloroform extraction and or precipitation with alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional non-limiting examples of purification procedures which can be used include size exclusion chromatography (Lukavsky, P.J. and Puglisi, J.D., 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, et al. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012). Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).Techniques to remove contaminants, such as dsDNA, have been developed and are known in the art including but not limited to scalable HPLC purification (see, e.g., Kariko, et al., 2011, Nucl Acid Res, v. 39 el42; Weissman, et al., 2012, Synthetic Messenger RNA and Cell Metabolism Modulation v.969 (Rabinovich, P.H. Ed)). In a preferred embodiment, the capped RNA described herein is purified through HPLC, as HPLC-purified RNA has been reported to be translated at much greater levels compared to other purification methods, particularly in primary cells and in vivo.

[0106] In some aspects, the capped RNA oligonucleotides provided herein (e.g., comprising one or more modified oligonucleotides) are purified by high-performance liquid chromatography (HPLC). In some embodiments, the capped RNA oligonucleotide is purified by reverse-phase HPLC (RP-HPLC). The addition of a counterion to the mobile phase of a HPLC setup can improve separation of the desired product from unwanted products. In a preferred embodiment, HPLC gradients used to isolate the capped RNA oligonucleotides comprise hydrophobic hexylammonium ions. In some embodiments, the gradient is chosen from ethyl ammonium, diethyl ammonium, triethyl ammonium, propyl ammonium, dipropyl ammonium, hexyl ammonium, dihexyl ammonium, octyl ammonium, dioctyl ammonium, etc. The number and lengths of carbon chains may be altered based on the lengths of the oligonucleotide to be captured and the desired feature for separation. In some embodiments, the concentration of hydrophobic ions (e.g., hexylammonium ions) used for HPLC purification of RNA oligonucleotides is between 10 mM to 200 mM. In some embodiments, the concentration of hydrophobic ions is between 10 mM and 20 mM, between 20 mM and 30 mM, between 30 mM and 40 mM, between 40 mM and 50 mM, between 50 mM and 60 mM, between 60 mM and 70 mM, between 70 mM and 80 mM, between 80 mM and 90 mM, between 90 mM and 100 mM, between 100 mM and 110 mM, between 110 mM and 120 mM, between 120 mM and 130 mM, between 130 mM and 140 mM, between 140 mM and 150 mM, between 150 mM and 160 mM, between 160 mM and 170 mM, between 170 mM and 180 mM, between 180 mM and 190 mM, or between 190 mM and 200 mM. In some embodiments, the concentration of hydrophobic ions is greater than 200 mM.Compositions comprising capped and / or tailed RNAs

[0107] In some aspects, the present disclosure provides a delivery reagent comprising any of the capped or tailed RNAs provided herein. In some embodiments, any of the capped or tailed RNAs provided herein are conjugated to a delivery agent. Any of the capped or tailed RNAs provided 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 modified mRNAs are generally known in the art, and include, for example, cholesterol. Peptides, proteins, antibodies, and carbohydrates used in the conjugation and delivery of modified mRNAs 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., aprotein) on the surface of a target cell type. Methods for conjugating a lipid, peptide, protein, antibody, or carbohydrate to a capped or tailed RNA include, for example, methods of conjugating a lipid, peptide, protein, antibody, or carbohydrate to a capped or tailed RNA at a 5’ or 3’ terminus, and are generally known in the art.

[0108] In some embodiments, any of the capped or tailed RNAs 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, non-cationic lipids, sterols, and polyethylene glycol-modified lipids. See, e.g., Buschmann et al. Vaccines. 2021. 9(1):65. In some embodiments, the capped or tailed 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 capped or tailed RNA is dispersed throughout 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 asphospholipids, 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 capped or tailed RNAs to tissues within the brain. Exosomes comprising capped or tailed 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 capped or tailed RNA, or mixing exosomes with a lipid-conjugated capped or tailed RNA, such as, for example, a capped or tailed RNA that has been conjugated to cholesterol. See, e.g., Roberts et al. Nat Rev Drug Discov. 2020. 19(10):673-694.

[0109] 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, polyethylenimine (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 capped or tailed 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 capped or tailed RNA is dispersed throughout the polymers of the polymeric nanoparticle or polymeric microparticle.

[0110] 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, andinclude 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 capped or tailed 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 capped or tailed 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 capped or tailed 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 capped or tailed RNA and protein each to a protein or peptide of a protein / peptide pair known to react to form a covalent linkage.

[0111] 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 some embodiments, a capped or tailed RNA is conjugated to the external surface of a solid nanoparticle or solid microparticle. Solid nanoparticles and solid microparticles comprising capped or tailed RNAs may be produced by any means generally known in the art, such as, for example, by linking the capped or tailed RNAs 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 capped or tailed RNAs are present. See, e.g., Roberts et al. Nat Rev Drug Discov. 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.

[0112] In some aspects, the present disclosure provides cells comprising any of the capped or tailed RNAs provided herein. In some embodiments, the cell is a human cell comprising any one of the capped or tailed RNAs 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 capped or tailed RNA in the cell is 15-900 minutes. In some embodiments, the half-life of the capped or tailed RNA in the cell is 30-600 minutes. In some embodiments, the half-life of the capped or tailed RNA in the cell is 60-300 minutes. In some embodiments, the half-life of the capped or tailed 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 capped or tailed 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 cells provided 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 capped or tailed RNAs,delivery agents, or cells provided herein, and a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients, carriers, buffers, stabilizers, 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.

[0113] In some aspects, the present disclosure provides a method of administering to a subject any of the capped or tailed RNAs, delivery agents, cells, compositions, or pharmaceutical compositions provided herein. In some embodiments, the subject 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 capped or tailed 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, e g., Somiari et al. Molecular Therapy., 2000. 2(3): 178-187. The capped or tailed 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 capped or tailed 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 capped or tailed RNA. In some embodiments, the capped or tailed RNA is administered with other elements, such as buffers and / or excipients, that increase the efficiency of electroporation.

[0114] In some aspects, the present disclosure provides a kit comprising any of the capped or tailed RNA oligonucleotides, RNA precursors, or capped or tailed RNAs provided herein. The capped RNA oligonucleotide and RNA precursor can be combined in the presence of an RNA ligase to produce a capped or tailed RNA, such as one of the capped or tailed RNAsprovided herein. 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. In some embodiments, the kit comprises an RtcB RNA ligase. In some embodiments, the kit further comprises a buffer for carrying out the ligation. 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.

[0115] 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 further comprises instructions for administering any of the pharmaceutical compositions provided herein to a subject.Pharmaceutical compositions for delivery and methods therefore

[0116] This invention provides pharmaceutical compositions comprising capped or tailed RNA molecules of the disclosure, particularly linear and circularized mRNA molecules. In certain embodiments pharmaceutical compositions of the invention further comprise pharmaceutically acceptable excipients and in certain other embodiments comprise one or more additional therapeutics agents.

[0117] In some embodiments, the compositions are suitable to be administered to a human subject in need thereof. In the context of the present disclosure, “active ingredient” refers generally to the capped or tailed RNA molecules described herein, particularly linear and circularized mRNA molecules as well as any additional therapeutic agents provided therewith.

[0118] It is generally understood by a person of ordinary skill in the art that the compositions described herein are also suitable for administration to any non-human subjects aswell. A person of ordinary skill in the veterinary arts will understand that pharmaceutical compositions described herein can be suitable for administration to mammals including but not limited to primates, cattle, pigs, horses, sheep, goats, cats, dogs, mice, rats, whales, and other mammals. A person of ordinary skill in the veterinary arts also will understand that pharmaceutical compositions described herein can be suitable for administration to birds including by not limited to chickens, ducks, geese, turkey, and other domesticated birds, as well as wild birds particularly endangered species of such birds. Additionally, a person of ordinary skill in the veterinary arts will understand that pharmaceutical compositions described herein can be suitable for administration to a wide variety of fish including commercial or wild salmon, tuna, cod, sardine, zebra fish, shark, or the like.

[0119] Pharmacological compositions described herein can be prepared by any method known or developed in the art of pharmacology, immunology, virology, or in biotechnology in general.

[0120] In some embodiments, the formulations of a pharmacological composition described herein can comprise a unit dose of at least one RNA, in addition to at least one other pharmaceutically acceptable excipient. Such excipients can include but are not limited to, solvents, dispersions, buffers, diluents, surfactants, emulsifiers, isotonic agents, preservatives, thickeners, lubricating agents, oils, or the like.

[0121] In some embodiments, the pharmacological composition can comprise a delivery mechanism further comprising a lipid nanoparticle. The size of the lipid nanoparticle can be altered to counteract immunogenic response from the subject, or to allow for increased potency and pharmacological activity.

[0122] In other embodiments, the pharmacological composition can comprise a delivery mechanism further comprising a lipidoid as previously described in the art. See Akinc etal, 2008, Nat Biotechnol. 26:561-596; Frank-Kamenetsky etal., Proc Natl Acad Sci USA. 2008 105: 11915- 11920; Akinc et al., 2009, Mol Ther. 17:872-879; Love et al., 2010. Proc Natl Acad Sci USA 107: 1864- 1869; Leuschner et al., 2011 , Nat Biotechnol. 29 : 1005-1010, all of which is incorporated herein in their entirety. Lipidoids refers broadly to lipid nanoparticles, liposomes, lipid emulsions, lipid micelles and the like. Lipidoids containing the pharmacological composition comprising the derivatized RNA can be administered parenterally by means including but not limited to,intravenous injection, intramuscular injection, subcutaneous injection, via dialysate, intrathecal injection, or intracranial injection.

[0123] A person of ordinary skill in the art would also recognize that other nucleotide delivery mechanisms exist such as the use of viral like, or viral derived particles. See Rohovie et al., 2016, Bioengineering & Translational Med. 2(1): 43-57. Virus like particles can include coat proteins or viral capsids of a virus. Such particles can be PEGylated or further annealed to compounds that avoid phagocytotic clearance. Additionally, the surface of the virus like particle can be further functionalized to provide cellular specific targeting, facilitate extravasation, facilitate radio labeling, improve permeability across cellular boundaries, or to transcytose the blood-brain barrier. The virus like particles can be derived for animal viruses, bacteriophages, or plant viruses. Examples of suitable virus for derivation of a virus like particle delivery mechanism include but are not limited to cowpea chlorotic mottle virus, cowpea mosaic virus, hepatitis B virus (core), enterobacteria phage MS2, Salmonella typhimurium P22, enterobacteria phage Q0 amongst other suitable viruses. Derivatized RNA payloads can be loaded into the virus like particles by electrostatic adsorption or any other suitable method known to a person of ordinary skill in the art.

[0124] Various exemplary embodiments of compositions and methods according to this invention are now described in the following non-limiting Examples. The Examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.Methods for RNA sensing and translation induction

[0125] Programmable approaches to sense, respond to and regulate specific RNA species in a biological system is of great value for basic research, diagnostics, and therapeutics. As shown in Example 1, the proximity of the m7G cap structure to an appropriate region of mRNA molecule through either covalent or non-covalent linkage contributes to the activation of translation (See also U.S. Application No. 63 / 640,571). In view of this phenomenon, Applicantdesigned mRNA “cap-snatching” and “tail-snatching” technologies, which take advantage of proximal cap / tail induced translation as a synthetic biology tool for RNA sensing.Cap-snatching

[0126] A “cap-snatching” scheme as described herein can include a two-component system, a three-component system, or a system comprising more than 3 components. In some embodiments, and as shown in FIG. 1A, such a system can include: (1) an untranslatable reporter mRNA (with or without nucleotide modifications) that does not comprise a m7G cap or other translation-initiation elements, (2) a donor RNA species (with or without nucleotide modifications) that includes an m7G cap or other translation-initiation elements, and / or (3) one or more RNA binding proteins. By designing the homology sequences between cap donor RNA and acceptor RNA, or other sequences and modifications that bridge the donor-acceptor RNA pairs (e g. G-qudraplex, triplex, molecular glues), or covalently linking the donor RNA and acceptor, the expression of the reporter mRNA can be activated through cap snatching (proximal)-induced translation.

[0127] Each component of the system is modular. Regarding the cap donor RNA, endogenous RNA, exogenous RNA, or any oligonucleotide having a m7G cap can be utilized. In addition, a cap donor oligonucleotide (e.g., cap donor RNA) can also be generated through a nucleolytic reaction, such as by a CRISPR system, siRNA mediated RNA cleavage, or miRNA mediated cleavage (See FIG. IB). In some embodiments, regarding the reporter mRNA (cap acceptor RNA), endogenous RNA or exogenous mRNA / circRNA that does not comprise a m7G cap can be used. The reporter RNA species are translationally inactive before the interaction with cap donor RNA species. The reporter RNA can also be encoded by DNA vectors which transcribe the corresponding RNA species in cellulo (See FIG. 1C).

[0128] The cap donor and cap acceptor RNA species can be introduced to each other in various ways. For example, in some embodiments, the cap donor RNA and the cap acceptor RNA are introduced by direct hybridization in a homology-dependent manner. In some embodiments, the cap donor RNA and the cap acceptor RNA are introduced by RNA-binding proteins assisted binding (e.g., dCas!3, CRISPR-Csm complex, Argonaute system, etc.). In some embodiments, for capped RNA species (e.g., cap donor RNA species) generated through nucleolytic reactions, endogenous or exogenously introduced RNA repairing mechanisms (e.g.RtcB-based RNA ligation or ribozyme ligase) can be employed to form covalent linkages between a cap donor and cap acceptor RNA (See FIG. ID).

[0129] In one aspect, provided herein is a method for producing a translatable RNA. In some embodiments, the method comprises contacting a cap acceptor RNA with a cap donor RNA under conditions sufficient to permit binding of the cap acceptor RNA and the cap donor RNA, wherein before binding of the cap acceptor RNA and the cap donor RNA, the cap acceptor RNA does not comprise a 5’cap and is untranslatable. In some embodiments, the method further comprises contacting the cap acceptor RNA and the cap donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme. In some embodiments, the cap donor RNA is an endogenous mRNA comprising an m7G cap. In some embodiments, the cap donor RNA is an exogenous mRNA comprising an m7G cap. In some embodiments, the cap donor RNA is a capped oligonucleotide generated through a nucleolytic reaction. In some embodiments, the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage. In some embodiments, the cap acceptor RNA is an endogenous mRNA that does not comprise an m7G cap. In some embodiments, the cap acceptor RNA is an exogenous mRNA that does not comprise an m7G cap. In some embodiments, the exogenous mRNA is encoded by a DNA vector. In some embodiments, the cap acceptor RNA is an endogenous uncapped RNA. In some embodiments, the cap acceptor RNA is an endogenous circRNA. In some embodiments, the cap acceptor RNA is an exogenous circRNA. In some embodiments, the exogenous circRNA is encoded by a DNA vector. In some embodiments, binding of the cap acceptor RNA and the cap donor RNA comprises one or more non-covalent interactions. In some embodiments, the cap acceptor RNA and the cap donor RNA comprises one or more covalent interactions. In some embodiments, binding of the cap acceptor RNA and the cap donor RNA comprises direct hybridization. In some embodiments, the one or more RBPs comprises a CRISPR protein. In some embodiments, the one or more RBPs comprises an Ago complex protein. In some embodiments, the one or more RBPs comprises an Rtcb enzyme. In some embodiments, the cap donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA. In some embodiments, the cap donor RNA is generated through a nucleolytic reaction, and the ribozymecatalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA.

[0130] In one aspect, provided herein is a method for inducing translation of a target cap acceptor RNA, comprising contacting the cap acceptor RNA with a cap donor RNA under conditions sufficient to permit binding of the cap acceptor RNA and the cap donor RNA, wherein before binding of the cap acceptor RNA and the cap donor RNA, the cap acceptor RNA does not comprise a 5’cap and is untranslatable. In some embodiments, the method further comprises contacting the cap acceptor RNA and the cap donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme. In some embodiments, the cap donor RNA is an endogenous mRNA comprising an m7G cap. In some embodiments, the cap donor RNA is an exogenous mRNA comprising an m7G cap. In some embodiments, the cap donor RNA is a capped oligonucleotide generated through a nucleolytic reaction. In some embodiments, the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage. In some embodiments, the cap acceptor RNA is an endogenous mRNA that does not comprise an m7G cap. In some embodiments, the cap acceptor RNA is an exogenous mRNA that does not comprise an m7G cap. In some embodiments, the exogenous mRNA is encoded by a DNA vector. In some embodiments, the cap acceptor RNA is an endogenous uncapped RNA. In some embodiments, the cap acceptor RNA is an endogenous circRNA. In some embodiments, the cap acceptor RNA is an exogenous circRNA. In some embodiments, the exogenous circRNA is encoded by a DNA vector. In some embodiments, binding of the cap acceptor RNA and the cap donor RNA comprises one or more non-covalent interactions. In some embodiments, the cap acceptor RNA and the cap donor RNA comprises one or more covalent interactions. In some embodiments, binding of the cap acceptor RNA and the cap donor RNA comprises direct hybridization. In some embodiments, the one or more RBPs comprises a CRISPR protein. In some embodiments, the one or more RBPs comprises an Ago complex protein. In some embodiments, the one or more RBPs comprises an Rtcb enzyme. In some embodiments, the cap donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA. In some embodiments, the cap donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing acovalent linkage between the cap donor RNA and the cap acceptor RNA. In some embodiments, translation occurs in cellulo.

[0131] In one aspect, provided herein is an RNA produced by any of the methods described above.

[0132] In one aspect, provided herein is a delivery agent comprising the RNA described above, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle. In some embodiments, the nanoparticle or microparticle is a lipid nanoparticle or microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle.

[0133] In one aspect, provided herein is a cell comprising the RNA described above. In some embodiments, the cell is a mammalian cell.

[0134] In one aspect, provided herein is a composition comprising the RNA described above, the delivery agent described above, or the cell described above. In some embodiments, the composition further comprises an additional agent. In some embodiments, the additional agent is an agent which has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is chosen from the list consisting of 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 is a shRNA, a siRNA, or an antisense oligonucleotide (ASO). In some embodiments, the additional agent is an antigen or adjuvant. In some embodiments, the composition is a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0135] In one aspect, provided herein is the RNA described above, the delivery agent described above, the cell described above, or the composition described above for use in preventing or treating a disease in a subject.

[0136] In one aspect, provided herein is a kit comprising one or more of the RNA described above, the delivery agent described above, the cell described above, or the composition described above. In some embodiments, the kit further comprises one or more RNA binding proteins (RBPs) or polynucleotides encoding the one or more RBPs.

[0137] In one aspect, provided is a kit comprising the composition described above, a device for administering the composition to a subject, and instructions for administering the composition to the subject.Tail-snatching

[0138] A “tail-snatching” scheme as described herein can include a two-component system, a three-component system, or a system comprising more than 3 components. In some embodiments, and as shown in FIG. 2A, such a system can include: (1) a reporter mRNA (without a polyA tail and thus likely having low translatability), (2) a tail donor RNA (which includes a polyA tail), and (3) one or more RNA binding proteins. By designing the homology sequences between tail donor RNA and acceptor RNA, or other sequences and modifications that bridge the donor-acceptor RNA pairs (e.g. G-qudraplex, triplex, molecular glues), or covalently linking the donor RNA and acceptor, the expression of the reporter mRNA can be activated through tail snatching (proximal)-induced translation.

[0139] Each component of the system is modular. Regarding the tail donor RNA, endogenous RNA, exogenous RNA, or any oligonucleotide having a polyA tail can be utilized. In addition, a tail donor oligonucleotide (e.g., tail donor RNA) can also be generated through a nucleolytic reaction, such as by a CRISPR system, siRNA mediated RNA cleavage, or miRNA mediated cleavage (See FIG. 2B). In some embodiments, regarding the reporter mRNA (tail acceptor RNA), endogenous RNA or exogenous mRNA / circRNA that does not comprise a polyA tail can be used. The reporter RNA species are translationally inactive or have a low level of translatability before the interaction with the tail donor RNA species. The reporter RNA can also be encoded by DNA vectors which transcribe the corresponding RNA species in cellulo (See FIG. 2C)

[0140] The tail donor and tail acceptor RNA species can be introduced to each other in various ways. For example, in some embodiments, the tail donor RNA and the tail acceptor RNA are introduced by direct hybridization in a homology-dependent manner. In some embodiments, the tail donor RNA and the tail acceptor RNA are introduced by RNA-binding proteins assisted binding (e.g., dCasl3, CRISPR-Csm complex, Argonaute system, etc.) In some embodiments, for tailed RNA species (e.g., tail donor RNA species) generated through nucleolytic reactions, endogenous or exogenously introduced RNA repairing mechanisms (e.g. RtcB-based RNAligation or ribozyme ligase) can be employed to form covalent linkages between a tail donor and tail acceptor RNA (See FIG. 2D).

[0141] In one aspect, the present disclosure provides a method for producing a translatable RNA, comprising contacting a tail acceptor RNA with a tail donor RNA under conditions sufficient to permit binding of the tail acceptor RNA and the tail donor RNA, wherein before binding of the tail acceptor RNA and the tail donor RNA, the tail acceptor RNA does not comprise a polyA tail and is untranslatable or has low translatability. In some embodiments, the method further comprises contacting the tail acceptor RNA and the tail donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme. In some embodiments, the tail donor RNA is an endogenous mRNA comprising polyA tail. In some embodiments, the tail donor RNA is an exogenous mRNA comprising a polyA tail. In some embodiments, the tail donor RNA is a polyA-tailed oligonucleotide generated through a nucleolytic reaction. In some embodiments, the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage. In some embodiments, the tail acceptor RNA is an endogenous mRNA that does not comprise polyA tail. In some embodiments, the tail acceptor RNA is an exogenous mRNA that does not comprise a polyA tail. In some embodiments, the exogenous mRNA is encoded by a DNA vector. In some embodiments, the tail acceptor RNA is an endogenous un-tailed RNA. In some embodiments, the tail acceptor RNA is an endogenous circRNA. In some embodiments, the tail acceptor RNA is an exogenous circRNA. In some embodiments, the exogenous circRNA is encoded by a DNA vector. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises one or more non-covalent interactions. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises one or more covalent interactions. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises direct hybridization. In some embodiments, the one or more RBPs comprises a CRISPR protein. In some embodiments, the one or more RBPs comprises an Ago complex protein. In some embodiments, the one or more RBPs comprises an Rtcb enzyme. In some embodiments, the tail donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA. In some embodiments, the tail donor RNA is generated through a nucleolytic reaction, andthe ribozyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA.

[0142] In one aspect, the present disclosure provides a method for inducing translation of a target tail acceptor RNA, comprising contacting the tail acceptor RNA with a tail donor RNA under conditions sufficient to permit binding of the tail acceptor RNA and the tail donor RNA, wherein before binding of the tail acceptor RNA and the tail donor RNA, the tail acceptor RNA does not comprise a polyA tail and is untranslatable or has low translatability. In some embodiments, the method further comprises contacting the tail acceptor RNA and the tail donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme. In some embodiments, the tail donor RNA is an endogenous mRNA comprising polyA tail. In some embodiments, the tail donor RNA is an exogenous mRNA comprising a polyA tail. In some embodiments, the tail donor RNA is a polyA-tailed oligonucleotide generated through a nucleolytic reaction. In some embodiments, the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage. In some embodiments, the tail acceptor RNA is an endogenous mRNA that does not comprise polyA tail. In some embodiments, the tail acceptor RNA is an exogenous mRNA that does not comprise a polyA tail. In some embodiments, the exogenous mRNA is encoded by a DNA vector. In some embodiments, the tail acceptor RNA is an endogenous un-tailed RNA. In some embodiments, the tail acceptor RNA is an endogenous circRNA. In some embodiments, the tail acceptor RNA is an exogenous circRNA. In some embodiments, the exogenous circRNA is encoded by a DNA vector. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises one or more non-covalent interactions. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises one or more covalent interactions. In some embodiments, binding of the tail acceptor RNA and the tail donor RNA comprises direct hybridization. In some embodiments, the one or more RBPs comprises a CRISPR protein. In some embodiments, the one or more RBPs comprises an Ago complex protein. In some embodiments, the one or more RBPs comprises an Rtcb enzyme. In some embodiments, the tail donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA. In some embodiments, the tail donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, therebyproducing a covalent linkage between the tail donor RNA and the tail acceptor RNA. In some embodiments, translation occurs in cellulo.

[0143] In one aspect, provided herein is an RNA produced by any of the methods described above.

[0144] In one aspect, provided herein is a delivery agent comprising the RNA described above, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle. In some embodiments, the nanoparticle or microparticle is a lipid nanoparticle or microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle.

[0145] In one aspect, provided herein is a cell comprising the RNA described above. In some embodiments, the cell is a mammalian cell.

[0146] In one aspect, provided herein is a composition comprising the RNA described above, the delivery agent described above, or the cell described above. In some embodiments, the composition further comprises an additional agent. In some embodiments, the additional agent is an agent which has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is chosen from the list consisting of 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 is a shRNA, a siRNA, or an antisense oligonucleotide (ASO). In some embodiments, the additional agent is an antigen or adjuvant. In some embodiments, the composition is a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0147] In one aspect, provided herein is the RNA described above, the delivery agent described above, the cell described above, or the composition described above for use in preventing or treating a disease in a subject.

[0148] In one aspect, provided herein is a kit comprising one or more of the RNA described above, the delivery agent described above, the cell described above, or the composition described above. In some embodiments, the kit further comprises one or more RNA binding proteins (RBPs) or polynucleotides encoding the one or more RBPs.

[0149] In one aspect, provided is a kit comprising the composition described above, a device for administering the composition to a subject, and instructions for administering the composition to the subject.EXAMPLES OF EMBODIMENTS

[0150] The following examples are given to illustrate the present disclosure. It should be understood, however, that the disclosure is not to be limited to the specific conditions or details described in these examples.

[0151] Embodiment 1 : A method for producing a translatable RNA, comprising contacting a cap acceptor RNA with a cap donor RNA under conditions sufficient to permit binding of the cap acceptor RNA and the cap donor RNA, wherein before binding of the cap acceptor RNA and the cap donor RNA, the cap acceptor RNA does not comprise a 5 ’cap and is untranslatable.

[0152] Embodiment 2: The method of embodiment 1, further comprising contacting the cap acceptor RNA and the cap donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme.

[0153] Embodiment 3: The method of embodiment 1 or 2, wherein the cap donor RNA is an endogenous mRNA comprising an m7G cap.

[0154] Embodiment 4: The method of embodiment 1 or 2, wherein the cap donor RNA is an exogenous mRNA comprising an m7G cap.

[0155] Embodiment 5: The method of embodiment 1 or 2, wherein the cap donor RNA is a capped oligonucleotide generated through a nucleolytic reaction.

[0156] Embodiment 6: The method of embodiment 5, wherein the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage.

[0157] Embodiment 7: The method of any one of embodiments 1-6, wherein the cap acceptor RNA is an endogenous mRNA that does not comprise an m7G cap.

[0158] Embodiment 8: The method of any one of embodiments 1-6, wherein the cap acceptor RNA is an exogenous mRNA that does not comprise an m7G cap.

[0159] Embodiment 9: The method of embodiment 8, wherein the exogenous mRNA is encoded by a DNA vector.

[0160] Embodiment 10: The method of any one of embodiments 1-6, wherein the cap acceptor RNA is an endogenous uncapped RNA.

[0161] Embodiment 11 : The method of any one of embodiments 1-6, wherein the cap acceptor RNA is an endogenous circRNA.

[0162] Embodiment 12: The method of any one of embodiments 1-6, wherein the cap acceptor RNA is an exogenous circRNA.

[0163] Embodiment 13: The method of embodiment 12, wherein the exogenous circRNA is encoded by a DNA vector.

[0164] Embodiment 14: The method of any one of embodiments 1-13, wherein binding of the cap acceptor RNA and the cap donor RNA comprises one or more non-covalent interactions.

[0165] Embodiment 15: The method of any one of embodiments 1-13, wherein binding of the cap acceptor RNA and the cap donor RNA comprises one or more covalent interactions.

[0166] Embodiment 16: The method of any one of embodiments 1-13, wherein binding of the cap acceptor RNA and the cap donor RNA comprises direct hybridization.

[0167] Embodiment 17: The method of any one of embodiments 2-16, wherein the one or more RBPs comprises a CRISPR protein.

[0168] Embodiment 18: The method of any one of embodiments 2-17, wherein the one or more RBPs comprises an Ago complex protein.

[0169] Embodiment 19: The method of any one of embodiments 2-18, wherein the one or more RBPs comprises an Rtcb enzyme.

[0170] Embodiment 20: The method of embodiment 19, wherein the cap donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA.

[0171] Embodiment 21 : The method of any one of embodiments 2-18, wherein the cap donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA.

[0172] Embodiment 22: A method for producing a translatable RNA, comprising contacting a tail acceptor RNA with a tail donor RNA under conditions sufficient to permitbinding of the tail acceptor RNA and the tail donor RNA, wherein before binding of the tail acceptor RNA and the tail donor RNA, the tail acceptor RNA does not comprise a polyA tail and is untranslatable or has low translatability.

[0173] Embodiment 23: The method of embodiment 22, further comprising contacting the tail acceptor RNA and the tail donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme.

[0174] Embodiment 24: The method of embodiment 22 or 23, wherein the tail donor RNA is an endogenous mRNA comprising polyA tail.

[0175] Embodiment 25: The method of embodiment 22 or 23, wherein the tail donor RNA is an exogenous mRNA comprising a polyA tail.

[0176] Embodiment 26: The method of embodiment 22 or 23, wherein the tail donor RNA is a polyA-tailed oligonucleotide generated through a nucleolytic reaction.

[0177] Embodiment 27: The method of embodiment 26, wherein the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage.

[0178] Embodiment 28: The method of any one of embodiments 22-27, wherein the tail acceptor RNA is an endogenous mRNA that does not comprise polyA tail.

[0179] Embodiment 29: The method of any one of embodiments 22-27, wherein the tail acceptor RNA is an exogenous mRNA that does not comprise a polyA tail.

[0180] Embodiment 30: The method of embodiment 29, wherein the exogenous mRNA is encoded by a DNA vector.

[0181] Embodiment 31 : The method of any one of embodiments 22-27, wherein the tail acceptor RNA is an endogenous un-tailed RNA.

[0182] Embodiment 32: The method of any one of embodiments 22-27, wherein the tail acceptor RNA is an endogenous circRNA.

[0183] Embodiment 33: The method of any one of embodiments 22-27, wherein the tail acceptor RNA is an exogenous circRNA.

[0184] Embodiment 34: The method of embodiment 33, wherein the exogenous circRNA is encoded by a DNA vector.

[0185] Embodiment 35: The method of any one of embodiments 22-34, wherein binding of the tail acceptor RNA and the tail donor RNA comprises one or more non-covalent interactions.

[0186] Embodiment 36: The method of any one of embodiments 22-34, wherein binding of the tail acceptor RNA and the tail donor RNA comprises one or more covalent interactions.

[0187] Embodiment 37: The method of any one of embodiments 22-34, wherein binding of the tail acceptor RNA and the tail donor RNA comprises direct hybridization.

[0188] Embodiment 38: The method of any one of embodiments 23-37, wherein the one or more RBPs comprises a CRISPR protein.

[0189] Embodiment 39: The method of any one of embodiments 23-38, wherein the one or more RBPs comprises an Ago complex protein.

[0190] Embodiment 40: The method of any one of embodiments 23-39, wherein the one or more RBPs comprises an Rtcb enzyme.

[0191] Embodiment 41 : The method of embodiment 40, wherein the tail donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA.

[0192] Embodiment 42: The method of any one of embodiments 22-39, wherein the tail donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA.

[0193] Embodiment 43: An RNA produced by the method of any one of embodiments 1- 42.

[0194] Embodiment 44: A delivery agent comprising the RNA of embodiment 43, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.

[0195] Embodiment 45: The delivery agent of embodiment 44, wherein the nanoparticle or microparticle is a lipid nanoparticle or microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle.

[0196] Embodiment 46: A cell comprising the RNA of embodiment 43.

[0197] Embodiment 47: The cell of embodiment 46, wherein the cell is a mammalian cell.

[0198] Embodiment 48: A composition comprising the RNA of embodiment 43, the delivery agent of embodiment 44 or 45, or the cell of embodiment 46 or 47.

[0199] Embodiment 49: The composition of embodiment 48 further comprising an additional agent.

[0200] Embodiment 50: The composition of embodiment 49, wherein the additional agent is an agent which has a therapeutic effect when administered to a subject.

[0201] Embodiment 51 : The composition of embodiment 49 or 50, wherein the additional agent is chosen from the list consisting of a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a small molecule, an aptamer, a lipid, or a carbohydrate.

[0202] Embodiment 52: The composition of embodiment 51, wherein the additional agent is a shRNA, a siRNA, or an antisense oligonucleotide (ASO).

[0203] Embodiment 53: The composition of any one of embodiments 50-52, wherein the additional agent is an antigen or adjuvant.

[0204] Embodiment 54: The composition of any one of embodiments 50-53, wherein the composition is a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0205] Embodiment 55: The RNA of embodiment 43, the delivery agent of embodiment 44 or 45, the cell of embodiment 46 or 47, or the composition of any one of embodiments 48-54 for use in preventing or treating a disease in a subject.

[0206] Embodiment 56: A kit comprising one or more of the RNA of embodiment 43, the delivery agent of embodiment 44 or 45, the cell of embodiment 46 or 47, or the composition of any one of embodiments 48-54.

[0207] Embodiment 57: The kit of embodiment 56, further comprising one or more RNA binding proteins (RBPs) or polynucleotides encoding the one or more RBPs.

[0208] Embodiment 58: A kit comprising the composition of any one of embodiments 48-54, a device for administering the composition to a subject, and instructions for administering the composition to the subject.

[0209] Embodiment 59: A method for inducing translation of a target cap acceptor RNA, comprising contacting the cap acceptor RNA with a cap donor RNA under conditions sufficientto permit binding of the cap acceptor RNA and the cap donor RNA, wherein before binding of the cap acceptor RNA and the cap donor RNA, the cap acceptor RNA does not comprise a 5 ’cap and is untranslatable.

[0210] Embodiment 60: The method of embodiment 59, further comprising contacting the cap acceptor RNA and the cap donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme.

[0211] Embodiment 61 : The method of embodiment 59 or 60, wherein the cap donor RNA is an endogenous mRNA comprising an m7G cap.

[0212] Embodiment 62: The method of embodiment 59 or 60, wherein the cap donor RNA is an exogenous mRNA comprising an m7G cap.

[0213] Embodiment 63: The method of embodiment 59 or 60, wherein the cap donor RNA is a capped oligonucleotide generated through a nucleolytic reaction.

[0214] Embodiment 64: The method of embodiment 63, wherein the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage.

[0215] Embodiment 65: The method of any one of embodiments 59-64, wherein the cap acceptor RNA is an endogenous mRNA that does not comprise an m7G cap.

[0216] Embodiment 66: The method of any one of embodiments 59-64, wherein the cap acceptor RNA is an exogenous mRNA that does not comprise an m7G cap.

[0217] Embodiment 67: The method of embodiment 66, wherein the exogenous mRNA is encoded by a DNA vector.

[0218] Embodiment 68: The method of any one of embodiments 59-64, wherein the cap acceptor RNA is an endogenous uncapped RNA.

[0219] Embodiment 69: The method of any one of embodiments 59-64, wherein the cap acceptor RNA is an endogenous circRNA.

[0220] Embodiment 70: The method of any one of embodiments 59-64, wherein the cap acceptor RNA is an exogenous circRNA.

[0221] Embodiment 71 : The method of embodiment 70, wherein the exogenous circRNA is encoded by a DNA vector.

[0222] Embodiment 72: The method of any one of embodiments 59-71, wherein binding of the cap acceptor RNA and the cap donor RNA comprises one or more non-covalent interactions.

[0223] Embodiment 73: The method of any one of embodiments 59-71, wherein binding of the cap acceptor RNA and the cap donor RNA comprises one or more covalent interactions.

[0224] Embodiment 74: The method of any one of embodiments 59-71, wherein binding of the cap acceptor RNA and the cap donor RNA comprises direct hybridization.

[0225] Embodiment 75: The method of any one of embodiments 60-74, wherein the one or more RBPs comprises a CRISPR protein.

[0226] Embodiment 76: The method of any one of embodiments 60-75, wherein the one or more RBPs comprises an Ago complex protein.

[0227] Embodiment 77: The method of any one of embodiments 60-76, wherein the one or more RBPs comprises an Rtcb enzyme.

[0228] Embodiment 78: The method of embodiment 77, wherein the cap donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA.

[0229] Embodiment 79: The method of any one of embodiments 60-76, wherein the cap donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA.

[0230] Embodiment 80: The method of any one of embodiments 59-79, wherein translation occurs in cellulo.

[0231] Embodiment 81 : A method for inducing translation of a target tail acceptor RNA, comprising contacting the tail acceptor RNA with a tail donor RNA under conditions sufficient to permit binding of the tail acceptor RNA and the tail donor RNA, wherein before binding of the tail acceptor RNA and the tail donor RNA, the tail acceptor RNA does not comprise a polyA tail and is untranslatable or has low translatability.

[0232] Embodiment 82: The method of embodiment 81, further comprising contacting the tail acceptor RNA and the tail donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme.

[0233] Embodiment 83: The method of embodiment 81 or 82, wherein the tail donor RNA is an endogenous mRNA comprising polyA tail.

[0234] Embodiment 84: The method of embodiment 81 or 82, wherein the tail donor RNA is an exogenous mRNA comprising a polyA tail.

[0235] Embodiment 85: The method of embodiment 81 or 82, wherein the tail donor RNA is a polyA-tailed oligonucleotide generated through a nucleolytic reaction.

[0236] Embodiment 86: The method of embodiment 85, wherein the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage.

[0237] Embodiment 87: The method of any one of embodiments 81-86, wherein the tail acceptor RNA is an endogenous mRNA that does not comprise polyA tail.

[0238] Embodiment 88: The method of any one of embodiments 81-86, wherein the tail acceptor RNA is an exogenous mRNA that does not comprise a polyA tail.

[0239] Embodiment 89: The method of embodiment 88, wherein the exogenous mRNA is encoded by a DNA vector.

[0240] Embodiment 90: The method of any one of embodiments 81-86, wherein the tail acceptor RNA is an endogenous un-tailed RNA.

[0241] Embodiment 91 : The method of any one of embodiments 81-86, wherein the tail acceptor RNA is an endogenous circRNA.

[0242] Embodiment 92: The method of any one of embodiments 81-86, wherein the tail acceptor RNA is an exogenous circRNA.

[0243] Embodiment 93 : The method of embodiment 89, wherein the exogenous circRNA is encoded by a DNA vector.

[0244] Embodiment 94: The method of any one of embodiments 81-93, wherein binding of the tail acceptor RNA and the tail donor RNA comprises one or more non-covalent interactions.

[0245] Embodiment 95: The method of any one of embodiments 81-93, wherein binding of the tail acceptor RNA and the tail donor RNA comprises one or more covalent interactions.

[0246] Embodiment 96: The method of any one of embodiments 81-93, wherein binding of the tail acceptor RNA and the tail donor RNA comprises direct hybridization.

[0247] Embodiment 97: The method of any one of embodiments 82-96, wherein the one or more RBPs comprises a CRISPR protein.

[0248] Embodiment 98: The method of any one of embodiments 82-97, wherein the one or more RBPs comprises an Ago complex protein.

[0249] Embodiment 99: The method of any one of embodiments 82-98, wherein the one or more RBPs comprises an Rtcb enzyme.

[0250] Embodiment 100: The method of embodiment 99, wherein the tail donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA.

[0251] Embodiment 101 : The method of any one of embodiments 82-98, wherein the tail donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA.

[0252] Embodiment 102: The method of any one of embodiments 81-101, wherein translation occurs in cellulo.EXAMPLESExample 1: Induction of translation via a trans-acting intermolecular cap

[0253] Given that 3 '-cap can efficiently initiate translation via the proximity between the 573'-UTRs by homology base-pairing, Applicant hypothesized that a “trans-acting intermolecular cap” that hybridizes to the circRNA 5'-UTR without covalent linkage might also drive translation. Applicant thus inserted a 20-nt sequence in m^-NLuc-circRNA (without IRES) for the hybridization with a complementary capped oligonucleotide at varying molar ratios (FIG. 3A). Interestingly, the non-covalent trans-acting caps also enhanced protein production by up to 5-fold at 1 : 80 mRNA to trans-acting cap ratio, while still significantly lower than the QRNA, potentially due to RNA helicase activity of elFs or ribosome scanning to unwind the hybridized RNA duplex.

[0254] To test whether the branched cap could drive the translation for upstream AUG via either proximity looping or bidirectional ribosome scanning to opt for upstream open reading frames (ORFs), Applicant generated a two-ORF reporter mRNA that contained a 5' full-length FLuc ORF and a 3' 3*HiBiT ORF with an out-of-frame start codon from the FLuc ORF (construct 3). The two-ORF mRNA was circularized (construct 4) and contained a branched cap (construct5) where the LNAm7G cap was located 77 nucleotides upstream of the FLuc ORF and 240 nucleotides downstream of the 3*HiBiT (34 amino acids) ORF (FIG. 3B). Compared to the circRNA (uncapped QRNA control), QRNA exhibited higher expression of both the FLuc ORF by 166-fold and the HiBiT ORF by 18.7-fold. These results indicated the ability of the internal branched cap to induce translation of an upstream ORF through either direct 3D proximity of the branched cap with upstream UTR and start codon or through ID ribosome 3' to 5' back scanning, providing potential avenues for replacing IRES for encoding two proteins of interest onto a single mRNA transcript.

[0255] Collectively, these observations suggest a general “cap-proximal” mechanism, permitting cap and 5' UTR sequences connected to mRNA via natural phosphodi ester backbone, unnatural covalent linkages, or noncovalent hybridization, to effectively initiate translation (FIG. 3C). Such a model is first supported by dual-capped mRNA and QRNA, and further supported by successful translation initiation driven by proximal but noncovalent cap structures mediated by either intramolecular proximity (a distal cis-acting “3 '-cap” base pairing with the 5' end of mRNA, or intermolecular proximity (a trans-acting, non-covalent 5'-cap, FIG. 3B). Such a mechanism may provide inspiration for future therapeutic mRNA architectures.SEQUENCES

[0256] The following tables contain sequences of oligonucleotides used in the experiments shown in each referenced figure. Nucleotide modifications are denoted as follows: r = ribose sugar m = 2OMe-modified sugar* = phosphorothioate (PS) linkage+ = locked nucleic acid (LNA) i2FA = internal 2'-deoxy fluoro iN6Me = internal N6-methyladenosine ibetaL = internal Z-adenosine i5OCTdU = internal3AzideN = 3' 6-diazynyl-A-heptylhexanamideTable 1. oligo Sequences used in FIG. 3 to ligate in between 5’ & 3’ of the mRNA sequence to circularize the RNA

Claims

CLAIMSWhat is claimed is:

1. A method for producing a translatable RNA, comprising contacting a cap acceptor RNA with a cap donor RNA under conditions sufficient to permit binding of the cap acceptor RNA and the cap donor RNA, wherein before binding of the cap acceptor RNA and the cap donor RNA, the cap acceptor RNA does not comprise a 5 ’cap and is untranslatable.

2. The method of claim 1, further comprising contacting the cap acceptor RNA and the cap donor RNA with one or more RNA binding proteins (RBPs) and / or a riboyzyme.

3. The method of claim 1 or 2, wherein the cap donor RNA is an endogenous mRNA comprising an m7G cap.

4. The method of claim 1 or 2, wherein the cap donor RNA is an exogenous mRNA comprising an m7G cap.

5. The method of claim 1 or 2, wherein the cap donor RNA is a capped oligonucleotide generated through a nucleolytic reaction.

6. The method of claim 5, wherein the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage.

7. The method of any one of claims 1-6, wherein the cap acceptor RNA is an endogenous mRNA that does not comprise an m7G cap.

8. The method of any one of claims 1-6, wherein the cap acceptor RNA is an exogenous mRNA that does not comprise an m7G cap.

9. The method of claim 8, wherein the exogenous mRNA is encoded by a DNA vector.

10. The method of any one of claims 1-6, wherein the cap acceptor RNA is an endogenous uncapped RNA.11 . The method of any one of claims 1 -6, wherein the cap acceptor RNA is an endogenous circRNA.

12. The method of any one of claims 1-6, wherein the cap acceptor RNA is an exogenous circRNA.

13. The method of claim 12, wherein the exogenous circRNA is encoded by a DNA vector.

14. The method of any one of claims 1-13, wherein binding of the cap acceptor RNA and the cap donor RNA comprises one or more non-covalent interactions.

15. The method of any one of claims 1-13, wherein binding of the cap acceptor RNA and the cap donor RNA comprises one or more covalent interactions.

16. The method of any one of claims 1-13, wherein binding of the cap acceptor RNA and the cap donor RNA comprises direct hybridization.

17. The method of any one of claims 2-16, wherein the one or more RBPs comprises a CRISPR protein.

18. The method of any one of claims 2-17, wherein the one or more RBPs comprises an Ago complex protein.

19. The method of any one of claims 2-18, wherein the one or more RBPs comprises an Rtcb enzyme.

20. The method of claim 19, wherein the cap donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA.

21. The method of any one of claims 2-18, wherein the cap donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA.

22. A method for producing a translatable RNA, comprising contacting a tail acceptor RNA with a tail donor RNA under conditions sufficient to permit binding of the tail acceptor RNA and the tail donor RNA, wherein before binding of the tail acceptor RNA and the tail donor RNA, the tail acceptor RNA does not comprise a polyA tail and is untranslatable or has low translatability.

23. The method of claim 22, further comprising contacting the tail acceptor RNA and the tail donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme.

24. The method of claim 22 or 23, wherein the tail donor RNA is an endogenous mRNA comprising polyA tail.

25. The method of claim 22 or 23, wherein the tail donor RNA is an exogenous mRNA comprising a polyA tail.

26. The method of claim 22 or 23, wherein the tail donor RNA is a polyA-tailed oligonucleotide generated through a nucleolytic reaction.

27. The method of claim 26, wherein the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage.

28. The method of any one of claims 22-27, wherein the tail acceptor RNA is an endogenous mRNA that does not comprise polyA tail.

29. The method of any one of claims 22-27, wherein the tail acceptor RNA is an exogenous mRNA that does not comprise a polyA tail.

30. The method of claim 29, wherein the exogenous mRNA is encoded by a DNA vector.

31. The method of any one of claims 22-27, wherein the tail acceptor RNA is an endogenous un-tailed RNA.

32. The method of any one of claims 22-27, wherein the tail acceptor RNA is an endogenous circRNA.

33. The method of any one of claims 22-27, wherein the tail acceptor RNA is an exogenous circRNA.

34. The method of claim 33, wherein the exogenous circRNA is encoded by a DNA vector.

35. The method of any one of claims 22-34, wherein binding of the tail acceptor RNA and the tail donor RNA comprises one or more non-covalent interactions.

36. The method of any one of claims 22-34, wherein binding of the tail acceptor RNA and the tail donor RNA comprises one or more covalent interactions.

37. The method of any one of claims 22-34, wherein binding of the tail acceptor RNA and the tail donor RNA comprises direct hybridization.

38. The method of any one of claims 23-37, wherein the one or more RBPs comprises a CRISPR protein.

39. The method of any one of claims 23-38, wherein the one or more RBPs comprises an Ago complex protein.

40. The method of any one of claims 23-39, wherein the one or more RBPs comprises an Rtcb enzyme.

41. The method of claim 40, wherein the tail donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA.

42. The method of any one of claims 22-39, wherein the tail donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA.

43. An RNA produced by the method of any one of claims 1-42.

44. A delivery agent comprising the RNA of claim 43, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.

45. The delivery agent of claim 44, wherein the nanoparticle or microparticle is a lipid nanoparticle or microparticle, a polymer nanoparticle or a polymer microparticle, a protein nanoparticle or a protein microparticle, or a solid nanoparticle or a solid microparticle.

46. A cell comprising the RNA of claim 43.

47. The cell of claim 46, wherein the cell is a mammalian cell.

48. A composition comprising the RNA of claim 43, the delivery agent of claim 44 or 45, or the cell of claim 46 or 47.

49. The composition of claim 48 further comprising an additional agent.

50. The composition of claim 49, wherein the additional agent is an agent which has a therapeutic effect when administered to a subject.

51. The composition of claim 49 or 50, wherein the additional agent is chosen from the list consisting of a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a small molecule, an aptamer, a lipid, or a carbohydrate.

52. The composition of claim 51, wherein the additional agent is a shRNA, a siRNA, or an antisense oligonucleotide (ASO).

53. The composition of any one of claims 50-52, wherein the additional agent is an antigen or adjuvant.

54. The composition of any one of claims 50-53, wherein the composition is a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

55. The RNA of claim 43, the delivery agent of claim 44 or 45, the cell of claim 46 or 47, or the composition of any one of claims 48-54 for use in preventing or treating a disease in a subject.

56. A kit comprising one or more of the RNA of claim 43, the delivery agent of claim 44 or 45, the cell of claim 46 or 47, or the composition of any one of claims 48-54.

57. The kit of claim 56, further comprising one or more RNA binding proteins (RBPs) or polynucleotides encoding the one or more RBPs.

58. A kit comprising the composition of any one of claims 48-54, a device for administering the composition to a subject, and instructions for administering the composition to the subject.

59. A method for inducing translation of a target cap acceptor RNA, comprising contacting the cap acceptor RNA with a cap donor RNA under conditions sufficient to permit binding of the cap acceptor RNA and the cap donor RNA, wherein before binding of the cap acceptor RNA and the cap donor RNA, the cap acceptor RNA does not comprise a 5 ’cap and is untranslatable.

60. The method of claim 59, further comprising contacting the cap acceptor RNA and the cap donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme.

61. The method of claim 59 or 60, wherein the cap donor RNA is an endogenous mRNA comprising an m7G cap.

62. The method of claim 59 or 60, wherein the cap donor RNA is an exogenous mRNA comprising an m7G cap.

63. The method of claim 59 or 60, wherein the cap donor RNA is a capped oligonucleotide generated through a nucleolytic reaction.

64. The method of claim 63, wherein the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage.

65. The method of any one of claims 59-64, wherein the cap acceptor RNA is an endogenous mRNA that does not comprise an m7G cap.

66. The method of any one of claims 59-64, wherein the cap acceptor RNA is an exogenous mRNA that does not comprise an m7G cap.

67. The method of claim 66, wherein the exogenous mRNA is encoded by a DNA vector.

68. The method of any one of claims 59-64, wherein the cap acceptor RNA is an endogenous uncapped RNA.

69. The method of any one of claims 59-64, wherein the cap acceptor RNA is an endogenous circRNA.

70. The method of any one of claims 59-64, wherein the cap acceptor RNA is an exogenous circRNA.

71. The method of claim 70, wherein the exogenous circRNA is encoded by a DNA vector.

72. The method of any one of claims 59-71, wherein binding of the cap acceptor RNA and the cap donor RNA comprises one or more non-covalent interactions.

73. The method of any one of claims 59-71, wherein binding of the cap acceptor RNA and the cap donor RNA comprises one or more covalent interactions.

74. The method of any one of claims 59-71, wherein binding of the cap acceptor RNA and the cap donor RNA comprises direct hybridization.

75. The method of any one of claims 60-74, wherein the one or more RBPs comprises a CRISPR protein.

76. The method of any one of claims 60-75, wherein the one or more RBPs comprises an Ago complex protein.

77. The method of any one of claims 60-76, wherein the one or more RBPs comprises an Rtcb enzyme.

78. The method of claim 77, wherein the cap donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA.

79. The method of any one of claims 60-76, wherein the cap donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the cap donor RNA and the cap acceptor RNA, thereby producing a covalent linkage between the cap donor RNA and the cap acceptor RNA.

80. The method of any one of claims 59-79, wherein translation occurs in celhilo.

81. A method for inducing translation of a target tail acceptor RNA, comprising contacting the tail acceptor RNA with a tail donor RNA under conditions sufficient to permit binding of the tail acceptor RNA and the tail donor RNA, wherein before binding of the tail acceptor RNA and the tail donor RNA, the tail acceptor RNA does not comprise a polyA tail and is untranslatable or has low translatability.

82. The method of claim 81, further comprising contacting the tail acceptor RNA and the tail donor RNA with one or more RNA binding proteins (RBPs) and / or a ribozyme.

83. The method of claim 81 or 82, wherein the tail donor RNA is an endogenous mRNA comprising polyA tail.

84. The method of claim 81 or 82, wherein the tail donor RNA is an exogenous mRNA comprising a polyA tail.

85. The method of claim 81 or 82, wherein the tail donor RNA is a polyA-tailed oligonucleotide generated through a nucleolytic reaction.

86. The method of claim 85, wherein the nucleolytic reaction comprises CRISPR-mediated cleavage, siRNA-mediated cleavage, or miRNA-mediated cleavage.

87. The method of any one of claims 81-86, wherein the tail acceptor RNA is an endogenous mRNA that does not comprise polyA tail.

88. The method of any one of claims 81-86, wherein the tail acceptor RNA is an exogenous mRNA that does not comprise a polyA tail.

89. The method of claim 88, wherein the exogenous mRNA is encoded by a DNA vector.

90. The method of any one of claims 81-86, wherein the tail acceptor RNA is an endogenous un-tailed RNA.

91. The method of any one of claims 81-86, wherein the tail acceptor RNA is an endogenous circRNA.

92. The method of any one of claims 81-86, wherein the tail acceptor RNA is an exogenous circRNA.

93. The method of claim 92, wherein the exogenous circRNA is encoded by a DNA vector.

94. The method of any one of claims 81-93, wherein binding of the tail acceptor RNA and the tail donor RNA comprises one or more non-covalent interactions.

95. The method of any one of claims 81-93, wherein binding of the tail acceptor RNA and the tail donor RNA comprises one or more covalent interactions.

96. The method of any one of claims 81-93, wherein binding of the tail acceptor RNA and the tail donor RNA comprises direct hybridization.

97. The method of any one of claims 82-96, wherein the one or more RBPs comprises a CRISPR protein.

98. The method of any one of claims 82-97, wherein the one or more RBPs comprises an Ago complex protein.

99. The method of any one of claims 82-98, wherein the one or more RBPs comprises an Rtcb enzyme.

100. The method of claim 99, wherein the tail donor RNA is generated through a nucleolytic reaction, and the Rtcb enzyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA.

101. The method of any one of claims 82-98, wherein the tail donor RNA is generated through a nucleolytic reaction, and the ribozyme catalyzes the ligation of the tail donor RNA and the tail acceptor RNA, thereby producing a covalent linkage between the tail donor RNA and the tail acceptor RNA.

102. The method of any one of claims 81-101, wherein translation occurs in cellulo.

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