Polynucleotides, 3' chain terminating tails, ligation and uses thereof

Abstract

The present disclosure provides constructs and methods for RNA ligation of 3' chain terminating tails and uses for improving nucleic acid stability and protein expression.

Description

[0001] POLYNUCLEOTIDES, 3' CHAIN TERMINATING TAILS, LIGATION AND USES THEREOF

[0002] CROSS-REFERENCE

[0003]

[0001] This application claims the benefit of the filing date under 35 U.S.C. § 119(e) of Provisional U.S. Patent Application Ser. No. 63 / 631,742, filed April 9, 2024, U.S. Patent Application Ser. No. 63 / 769,373, filed March 10, 2025, and Provisional U.S. Patent Application Ser. No. 63 / 782,248, filed April 02, 2025, each of which is hereby incorporated by reference.

[0004] BACKGROUND

[0005]

[0002] Despite advances in messenger RNA (mRNA)-based therapies, challenges associated with stability (e.g., short half-life), immunogenicity, translation efficiency, and delivery present obstacles to effective bench to bedside translation of mRNA-based therapies. For example, mRNA is inherently unstable and prone to degradation by nucleases (RNases) present in the body, leading to a short half-life and reduced efficacy. While chemical modifications to mRNA have been explored to address the challenges of mRNA stability, chemical modifications generally have not made their way into therapeutics and the primarily focus of mRNA modifications have been on substitutions of uridine with Nl-methyl-pseudouridine and cap methylation state.

[0006] SUMMARY

[0007]

[0003] Provided in certain embodiments herein are modified poly-A tails useful for increasing the stability of an mRNA molecule and / or production of a therapeutic protein (e.g., relative to an mRNA having an unmodified poly-A tail). Moreover, in certain instances, modified poly-A tails described herein are useful because they can be effectively and / or efficiently ligated onto an mRNA molecule (e.g., using a T4 RNA ligase and / or under reduced amounts of polyethylene glycol), providing advantages in the manufacture a therapeutic mRNA. In some instances, increases in stability and protein expression can be conferred by modified poly-A tails described herein, such as through the use of one or more phosphorothioate modifications within the terminal 3' inter-nucleotide linkage(s). In some instances, the increases in ligation efficiency (e.g., under reduced polyethylene glycol) can be achieved by incorporation of a monocyclic nucleobase (e.g., a pyrimidine such as cytidine) at the 5' nucleotide position of the poly-A tail being ligated to the mRNA. In certain instances, the modified poly-A tails can be useful in formats having one or more poly-A tails linker to one another such that they for a multi-terminus poly-A (e.g., a poly- A tail having additional poly-A branches).

[0008]

[0004] In some embodiments, provided herein are messenger ribonucleic acid (mRNA) polynucleotides comprising a poly-A tail, wherein the poly-A tail comprises a terminal 3' nucleotide comprising (a) a 5' phosphorothioate linkage and (b) a nucleoside lacking a 3' hydroxyl group (in some instances, referred to herein as a free 3' hydroxyl, e.g., indicating that the 3' position is not hydroxyl and is not connected to another nucleotide). In certain embodiments, the poly-A tail further comprises (i) a 5' nucleotide comprising a monocyclic nucleobase. To be clear, in some instances, such a poly-A tail may be attached to an additional poly-A group, such as wherein the poly-A tail was attached via a ligation technique to an existing 3'-poly-A group of an mRNA. Also provided in certain embodiments herein are poly-A tails (e.g., wherein the poly-A tail is not connected to other mRNA elements, such as until after ligation) useful in the synthesis of an mRNA molecule via ligation, wherein the poly-A tails comprise (i) a terminal 5' nucleotide comprising a monocyclic nucleobase and free 5' phosphate, and (ii) a terminal 3' nucleotide comprising (a) a 5' phosphorothioate linkage and (b) a nucleoside lacking a free 3' hydroxyl.

[0009]

[0005] In certain embodiments, the last 2-10 inter-nucleotide linkages at the 3' terminus of the poly-A tail are phosphorothioate inter-nucleotide linkages. In certain embodiments, the last 2-8 inter-nucleotide linkages at the 3' terminus of the poly-A tail are phosphorothioate inter- nucleotide linkages. In certain embodiments, the last 3-6 inter-nucleotide linkages at the 3' terminus of the poly-A tail are phosphorothioate inter-nucleotide linkages. In certain embodiments, provided herein is an mRNA comprising any poly-A tail provided herein. In some embodiments, the mRNA comprises a second poly-A region. In some embodiments, the poly-A tail is connected (e.g., through the 5'-chain terminating nucleotide of the poly-A tail) to the second poly-A region. In some embodiments the poly-A tail and / or the combined poly-A tail and the second poly-A region comprise an adenine nucleobase content of 85% or greater. In certain embodiments, the combined poly-A tail and second poly-A region comprises any suitable number of adenine nucleobases. In certain embodiments, the poly-A tails comprise an adenine nucleobase content of 85% or greater. In certain embodiments, the poly-A tail comprises 10-250 nucleotides (including, e.g., 10-200, 10-150, 10-100, 10-50, 10-25, or 10-20). In some embodiments, any poly-A tail provided herein comprises a 5' (when not attached to a larger mRNA structure) nucleotide that comprises a monocyclic nucleobase. In certain embodiments, the monocyclic nucleobase is a pyrimidine nucleobase. In certain embodiments, the monocyclic nucleobase is cytidine.

[0010]

[0006] In some embodiments, any poly-A tail provided herein comprises a 5'-chain terminating (when not attached to a larger mRNA structure) abasic nucleotide (e.g., wherein the base is replaced with any suitable group, such as described herein). In some embodiments, the abasic nucleotide is substituted at the 1' position with a group that is smaller than a bicyclic base, such as a purine base. In some embodiments, the abasic nucleotide is substituted at the 1' position with H (at both 1' positions), OR0, NR°2, alkyl (saturated or unsaturated), heteroalkyl, aryl, heteroaryl, cycloaklyl, or heterocyclyl, such as wherein each R° is independently H, alkyl (saturated or unsaturated), heteroalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. In some embodiments, each alkyl (saturated or unsaturated), heteroalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl is substituted or unsubstituted. In some embodiments, such a substituent is a C1-C3 alkyl.

[0011]

[0007] Provided in certain embodiments herein are mRNA polynucleotides comprising a 3' chain terminating polynucleotide (e.g., a poly-A tail) according to Formula (I): p-Z-(p'-A)n-p"-(R-pc)m-R1-pd-N (I).

[0012]

[0008] In some embodiments, p is an internucleotide linker or a chain terminal group. In certain embodiments, Z is a natural (e.g., a nucleoside comprising a monocyclic nucleobase) or modified nucleoside (e.g., an abasic nucleoside). In some embodiments, each p is independently an internucleotide linker (e.g., a phosphate connected to another nucleotide, such as an adenosine) or a chain terminating group (e.g., a phosphate, such as being capable of being ligated to a 3'- hydroxyl of a 3' chain terminating nucleotide of an mRNA). In some embodiments, each p' is independently an internucleotide linker (e.g., a phosphate). In some embodiments, each A is independently an adenosine nucleoside, or an analog thereof (e.g., a natural analog thereof or an adenosine substituted with one or more Cl-3 alkyl group(s)). In some embodiments, p" is an internucleotide linker (e.g., phosphate or a phosphorothioate). In some embodiments, n is an integer of 0 to 30 (e.g., 5-15). In some embodiments, each R is independently a nucleoside (e.g., adenosine). In some embodiments, each pcis independently a phosphorothioate. In some embodiments, m is an integer of 2 to 8 (e.g., 2 to 6). In some embodiments, R1is a nucleoside (e.g., a natural nucleoside, such as adenosine). In some embodiments, pdis a phosphorothioate. In some embodiments, N is a chain terminating nucleoside (e.g., comprising a ribose lacking a 3' hydroxyl). In some embodiments, each nucleoside independently comprises a ribose or analog thereof. In some embodiments, the ribose analog is a locked ribose, an unlocked ribose, a spiroribose, or a deoxyribose. In some embodiments, the ribose analog is a furanose or other cyclized sugar, such as a pyranose (e.g., glucose). In some embodiments, the ribose analog is a nitrogencontaining heterocycle (e.g., wherein the ring has a nitrogen instead of or in addition to the ribose oxygen).

[0013]

[0009]

[0014]

[0010] Further provided herein are mRNA polynucleotides comprising a 3' chain terminating polynucleotide according to Formula (II): p-Z-(p'-A)n-p"-(R-pc)m-R1-pd-N (II) wherein: p is an inter-nucleotide linker or a chain terminal group;

[0015] Z is a natural or modified nucleoside; each p' is independently an inter-nucleotide linker; each A is independently an adenosine nucleoside, or an analog thereof; p" is an inter-nucleotide linker; n is an integer of 0 to 30; each R is independently a nucleoside; each pcis independently a phosphorothioate; m is an integer of 2 to 8;

[0016] R1is a nucleoside; pdis a phosphorothioate; and

[0017] N is a chain terminating nucleoside (e.g., comprising a ribose lacking a free 3' hydroxyl), wherein each nucleoside independently comprises a ribose or analog thereof. [Oil] In some embodiments, p is a chain terminal group selected from the group consisting of phosphate and diphosphate. In some embodiments, p is an inter-nucleotide linker selected from the group consisting of phosphate and diphosphate. In some embodiments, p is a chain terminal group selected from the group consisting of hydroxyl, phosphate, phosphorothioate, phosphorodithioate, thiophosphate, phosphonate, phosphanate, phosphoroselenoate, selenophosphate, and phosphoramidate.

[0018]

[0012] In some embodiments, each nucleoside independently comprises a ribose or a ribose analog that is a deoxyribose, heteroribose, a locked ribose, or an unlocked ribose.

[0019]

[0013] In some embodiments, Z is a nucleoside comprising a monocyclic base, such as a natural or modified pyrimindine base (e.g., cytosine or uracil).

[0020]

[0014] In some embodiments, Z is an abasic nucleoside.

[0021]

[0015] In some embodiments, Z is a nucleoside substituted at the 1-position with a hydrogen and B, wherein B is hydrogen, C2-C6alkynyl, C2-C8heterocycloalkyl, aryl, or heteroaryl, and wherein C2- Cealkynyl, C2-C8heterocycloalkyl, aryl, and heteroaryl are unsubstituted or substituted.

[0022]

[0016] In some embodiments, B is monocyclic C2-C8heterocycloalkyl, monocyclic aryl, or monocyclic heteroaryl. In some embodiments, B is a substituted or unsubstituted 5- or 6- membered aryl or a substituted or unsubstituted 5- or 6-membered heteroaryl. In some embodiments, B is a pyrimidine nucleobase. In some embodiments, B is cytosine.

[0023]

[0017] In some embodiments, B is:

[0019] In some embodiments, Z is a nucleoside comprising a monocyclic nucleobase. In some embodiments, Z is cytidine (C) or an analog thereof. In some embodiments, Z is cytidine (C). In some embodiments, the ribose of Z is substituted with a hydrogen and a second group R4, wherein R4is a hydrogen, a halogen, a hydroxyl, a substituted or unsubstituted alkoxy, a substituted or unsubstituted heteroalkoxy, or a substituted or unsubstituted alkoxy. In some embodiments, R4is hydroxyl.

[0024]

[0020] In some embodiments, each p' is a phosphate or analog thereof. In some embodiments, p" is phosphate or a phosphorothioate. In some embodiments, each p' is phosphate. In some embodiments, p" is a phosphorothioate (PS) or diphosphorothioate (PS2). In some embodiments, p" is a phosphorothioate.

[0025]

[0021] In some embodiments, each A is independently an adenosine. In some embodiments, n is an integer of 2 to 25. In some embodiments, n is an integer of 6 to 20.

[0026]

[0022] In some embodiments, p" is a phosphate or analog thereof. In some embodiments, p" is phosphate.

[0027]

[0023] In some embodiments, each R and R1is independently a natural or modified nucleoside. In some embodiments, each R and R1is independently adenosine or an analog thereof. In some embodiments, each R and R1is independently adenosine. In some embodiments, at least 50% of the total of R and R1are adenosine. In some embodiments, at least 80% of the total of R and R1are adenosine.

[0028]

[0024] In some embodiments, m is an integer of 3 to 7. In some embodiments, m is an integer of 4 to 6. In some embodiments, m is an integer of 5.

[0029]

[0025] In some embodiments, the ribose of N does not have a 3'-hydroxyl. In some embodiments, N is a non-hydroxyl nucleoside. In some embodiments, N is a dideoxynucleoside (e.g., ddC or ddA). In some embodiments, N is an inverted nucleoside (e.g., wherein N is attached at the 3' position, through pd, to the 3' position of R1). In some embodiments, N is an inverted 2'- deoxynucleoside (InvdN) (e.g., inverted 2'-deoxythymidine (InvdT), inverted 2'-deoxycytidine (InvdC) or inverted 2'-deoxyadenosine (InvdA)). In some embodiments, N is inverted 2'- deoxythymidine (InvdT). In some embodiments, N is a nucleoside substituted at the 2'-position with a non-hydroxyl. In some embodiments, N is a nucleoside substituted at the 2'-position with R7, wherein R7is hydrogen, a halogen, an alkoxy, a heteroalkoxy, an alkylthio, an amino, an alkyl, or a heteroalkyl, wherein the alkoxy, heteroalkoxy, alkylthio, amino, alkyl, or heteroalkyl is substituted or unsubstituted. In some embodiments, N is a nucleoside substituted at the 3'- position with a non-hydroxyl. In some embodiments, N is a nucleoside substituted at the 3'- position with R6, wherein R6is hydrogen, a halogen, an alkoxy, a heteroalkoxy, an alkylthio, an amino, an alkyl, or a heteroalkyl, wherein the alkoxy, heteroalkoxy, alkylthio, amino, alkyl, or heteroalkyl is substituted or unsubstituted. In some embodiments, the ribose of nucleoside N is a spiro-ribose, a bridged-ribose, bicyclic ribose, or a locked ribose.

[0030]

[0026] In some embodiments, a nucleoside within six upstream nucleosides of R and R1(e.g., R, N, or a nucleoside of the polynucleotide) comprises a 2' hydroxyl. In some embodiments, R or R1comprises a 2' hydroxyl.

[0031]

[0027] In some embodiments, p' is bonded to an upstream nucleoside (e.g., Z or another nucleoside of the polynucleotide) that is natural or modified, wherein the modified nucleoside is not: (i) a locked nucleic acid or (ii) substituted in the 2'-position with methoxy or methoxyethoxy (MOE).

[0032]

[0028] In some embodiments, N comprises a nucleobase. In some embodiments, the nucleobase of N is a modified or natural nucleobase. In some embodiments, the nucleobase of N is a cytosine. In some embodiments, N is 2',3'-dideoxycytosine. In some embodiments, N is a nucleoside reverse transcriptase inhibitor (NRTI), such as Lamivudine. In some embodiments, the ribose of N is substituted at the 1' position with Q, wherein: Q is hydrogen, alkyl, Cz-Cealkynyl, C2- Csheterocycloalkyl, aryl, or heteroaryl, wherein Cz-Cealkynyl, C2-Cgheterocycloalkyl, aryl, and heteroaryl are unsubstituted or substituted (e.g., a substituted heteroaryl that is N6- methyladenosine (m6A)).

[0033]

[0029] In some embodiments, m is 5, each R is adenosine, and each pcis independently a phosphorothioate. In some embodiments, m is 5, each R is adenosine, each pcis independently a phosphorothioate, R1is adenosine, and pdis a phosphorothioate. In some embodiments, m is 5, each R is adenosine, each pcis independently a phosphorothioate, R1is adenosine, pdis a phosphorothioate, and N is a dideoxynucleoside (e.g., ddC or ddA) or an inverted 2'- deoxynucleoside (InvdN).

[0030] In some embodiments, N is abasic. In some embodiments, N is unlocked.

[0034]

[0031] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 1. In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 2. In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 3. In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 4. In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 5.

[0035]

[0032] In some embodiments, the polynucleotide further comprises: at least one 5'-cap structure; a 5'-UTR; a coding region; and a 3'-UTR. In some embodiments, the polynucleotide further comprises a poly-A. In some embodiments, the poly-A is connected 3' to 5' to the 3' chain terminating polynucleotide.

[0036]

[0033] Provided herein is a composition comprising a polynucleotide described herein and a lipid nanoparticle (LNP).

[0037]

[0034] Provided herein is aa method of ligating a polynucleotide tail to an mRNA polynucleotide, the method comprising: a. providing a first (donor) polynucleotide, the first polynucleotide being a 3'-chain terminating polynucleotide described herein; b. providing a second (acceptor) polynucleotide, wherein the first polynucleotide has a 3' terminal hydroxyl group; c. contacting an RNA ligase enzyme with the first polynucleotide and the second polynucleotide.

[0038]

[0035] In some embodiments, the first polynucleotide has a 5'-terminal phosphate group. In some embodiments, the second polynucleotide comprises a poly A. In some embodiments, the second polynucleotide comprises a 5'-UTR, a coding region, or a 3'-UTR. In some embodiments, the second polynucleotide comprises a 5'-UTR, a coding region, and a 3'-UTR.

[0039]

[0036] In some embodiments, the RNA ligase enzyme is a T4 RNA ligase. In some embodiments, the contacting an RNA ligase enzyme with the first polynucleotide and the second polynucleotide occurs in a medium comprising less than 20% polyethylene glycol (PEG), less than 15% PEG, less than 10% PEG, less than 5% PEG, less than 2% PEG, or that is substantially free of PEG. In some embodiments, the contacting an RNA ligase enzyme with the first polynucleotide and the second polynucleotide occurs in a medium comprising less than 20% high molecular weight polyethylene glycol (PEG), less than 15% high molecular weight PEG, less than 10% high molecular weight PEG, less than 5% high molecular weight PEG, less than 2% high molecular weight PEG, or that is substantially free of high molecular weight PEG. In some embodiments, the contacting an RNA ligase enzyme with the first polynucleotide and the second polynucleotide is conducted in a composition comprising a total polynucleotide concentration of at least 0.5 mg / mL.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041]

[0037] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also "Figure" and "FIG." herein) of which:

[0042]

[0038] FIG. 1A is a PAGE gel depicting the amount of ligated and unligated product after an oligo containing DNA bases was hybridized with the 3'UTR of modified polynucleotides provided herein, and then cleaved with RNase H to release a part of 3'UTR with poly-A.

[0043]

[0039] FIG. IB provide charts shows levels of cellular expression of luciferase by ligated constructs comprising modified polynucleotides provided herein.

[0044]

[0040] FIG. 2 is a PAGE gel depicting the amount of ligated and unligated product after ligation reactions using modified polynucleotides provided herein were performed in reaction medium containing varying levels of PEG8000.

[0045]

[0041] FIG. 3 illustrates increased expression in mice cells (Panel A) and human cells (Panel B) for various polynucleotides having a 3' chain terminating tail described herein.

[0046]

[0042] FIG. 4 shows increased expression of mRNAs having ligated 6PS rA tails in mice and NHPs as compared to traditional, unmodified mRNA.

[0047] DETAILED DESCRIPTION

[0043] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "at least," "greater than" or "greater than or equal to" applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0048]

[0044] Whenever the term "up to," "no more than," "less than," or "less than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "no more than," "less than," or "less than or equal to" applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0049]

[0045] In some instances, a value is "about" a recited value if the value is within ±10% of the recited value. In some instances, disclosure of "about" a recited value includes disclosure of a value that is within ±5% of the recited value.

[0050]

[0046] The term "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense, that is, as "including, but not limited to." It is also to be understood that disclosure of "comprising" is also intended to include disclosures of "consisting essentially of" and "consisting of" the same recited elements. Generally, "consisting essentially of" is to construed at partially open, being directed to those elements set forth and to those other elements that do not materially affect the basic and novel characteristics of the invention described. Generally, "consisting of" means that the claim is directed only to the specified elements.

[0051]

[0047] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise.

[0052]

[0048] As used herein, the term "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each were set out individually herein.

[0049] In some embodiments, "nucleotides" or "nucleosides" are referred to herein interchangeably. If a "nucleotide" is referred to is where a "nucleoside" should be referred to, such as wherein the group called a "nucleotide" is connected to a 5' (p), it is understood that a "nucleoside" is also described, as applicable. Similarly, unless stated otherwise, "internucleoside linker" and "inter-nucleotide linker" are referred to herein interchangeably.

[0053]

[0050] A "nucleic acid," or "polynucleotide," as used herein, refers to an organic molecule comprising two or more covalently bonded nucleotides. A "nucleotide," as used herein, refers to an organic molecule comprising a 1) a nucleoside comprising a sugar covalently bonded to a nitrogenous base (nucleobase); and 2) a phosphate group that is covalently bonded to the sugar of the nucleoside. Nucleotides in a polynucleotide are typically joined by a phosphodiester bond, in which the 3' carbon of the sugar of a first nucleotide is linked to the 5' carbon of the sugar of a second nucleic acid by a bridging phosphate group. Typically, the bridging phosphate comprises two non-bridging oxygen atoms, which are bonded only to a phosphorus atom of the phosphate, and two bridging oxygen atoms, each of which connects the phosphorus atom to either the 3' carbon of the first nucleotide or the 5' carbon of the second nucleotide. In a nucleic acid sequence describing the order of nucleotides in a nucleic acid, a first nucleotide is said to be 5' to (upstream of) a second nucleotide if the 3' carbon of first nucleotide is connected to the 5' carbon of the second nucleotide. Similarly, a second nucleotide is said to be 3' to (downstream of) a first nucleotide if the 5' carbon of the second nucleotide is connected to the 3' carbon of the first nucleotide. Nucleic acid sequences are typically read in 5'->3' order, starting with the 5' nucleotide and ending with the 3' nucleotide.

[0054]

[0051] A "modified nucleotide," as used herein, refers to a nucleotide with a structure that is not the canonical structure of an adenosine nucleotide, a cytidine nucleotide, a guanosine nucleotide, a uridine nucleotide, such as a 2'-deoxyadenosine nucleotide, a 2'-deoxythymidine nucleotide, a 2'-deoxycytidine nucleotide, or a 2'-deoxyguanosine 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. As used herein, a "modified nucleotide" may also refer to a nucleotide which comprises a nucleobase or sugar (ribose or deoxyribose) that is not canonical, such as including nucleotides that result from natural or unnatural modifications. A "modified nucleotide" may also refer to a nucleotide that is covalently linked to a second nucleotide through an internucleoside linkage that is not a canonical internucleoside linkage (i.e., not a phosphodiester internucleoside linkage, e.g., a phosphorothioate internucleoside linkage). In some instances, a "modified nucleotide" may also refer to a nucleotide that does not comprise a nitrogenous base (e.g., a nucleotide where the nitrogenous base has been replaced with another moiety, e.g., an abasic nucleotide).

[0055]

[0052] A "modified nucleoside," as used herein, refers to a nucleoside with a structure that is not the canonical structure of adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'- deoxythymidine, 2'-deoxycytidine, or 2'-deoxyguanosine. As used herein, a "modified nucleoside" may also refer to a nucleoside which comprises a nucleobase or sugar (ribose or deoxyribose) that is not canonical, which can include nucleotides that result from natural or unnatural modifications.

[0056]

[0053] A "natural nucleoside," as used herein, includes natural unmodified nucleosides and naturally modified nucleosides. "Natural unmodified nucleosides," as used herein, are nucleosides with a structure that is the canonical structure of adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxythymidine, 2'-deoxycytidine, or 2'-deoxyguanosine. A "naturally modified nucleoside," as used herein, is a canonical nucleoside with modification(s) that can result from natural biological processes, and can include, for example, Nl- methyladenosine or 1-methyladenosine, N2-methyladenosine or 2-methyladenosine, 2'-O- methyladenosine, 2-Methylthio-N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)-adenosine, 2-Methylthio-N6-isopentenyladenosine, 2-Methylthio-N6-(cis- hydroxyisopentenyl)-adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-Methylthio-N6-threonylcarbamoyl-adenosine, N6-methyl-N6- threonylcarbamoyladenosine, N6-hydroxynorvalylcarbamoyladenosine, 2-Methylthio-N6- hydroxynorvalylcarbamoyladenosine, 2'-O-ribosyladenosine (phosphate), Inosine, Nl-inosine or 1-methylinosine, l,2'-O-dimethylinosine, N6,N6-dimethyladenosine, 2'-O-methylinosine, N6,2'- O-dimethyladenosine, N6,N6,2'-O-trimethyladenosine, Nl,2'-O-dimethyladenosine, N6- acetyladenosine, 8-Methyladenosine, N6-formyladenosine, CyclicN6- threonylcarbamoyladenosine, N6-hydroxymethyladenosine, 2,8-Dimethyladenosine, 2- methylthio-N6-threonylcarbamoyladenosine, N6-hydroxythreonylcarbamoyladenosine, 2- Methylthiomethylenethio-N6-isopentenyl-adenosine, , 3-Methylcytidine, 5-Methylcytidine, 2'- O-methylcytidine, 2-Thiocytidine, N4-acetylcytidine, 5-Formylcytidine, 5,2'-O-dimethylcytidine, Lysidine, N4-methylcytidine, 4,2'-O-dimethylcytidine, 5-Hydroxymethylcytidine, 5-Formyl-2'-O- methylcytidine, N4,N4,2'-O-trimethylcytidine, Agmatidine, 5-Hydroxycytidine, N4-acetyl-2'-O- methylcytidine, N4,N4-dimethylcytidine, 2-Methylthiocytidine, 2'-O-methyl-5- hydroxymethylcytidine, 1-Methylguanosine, N2-methylguanosine, 7-Methylguanosine, 2'-O- methylguanosine, N2,N2-dimethylguanosine, N2-2'-O-dimethylguanosine, N2,N2,2'-O- trimethylguanosine, 2'-O-ribosylguanosine (phosphate), Wybutosine, Peroxywybutosine, Hydroxywybutosine, Undermodified hydroxywybutosine, Wyosine, Methylwyosine, Queuosine, Epoxyqueuosine, Galactosyl-queuosine, Mannosyl-queuosine, Glutamyl-queuosine, Pre- queuosineO, Pre-queuosinel, Archaeosine, N2,7-dimethylguanosine, N2,2-7- trimethylguanosine, l,2'-O-dimethylguanosine, 4-Demethylwyosine, Isowyosine, N2,2'-O-7- trimethylguanosine, 7-Aminocarboxypropylwyosine methyl ester, 7-Aminocarboxypropyl- demethylwyosine, 7-Aminocarboxypropylwyosine, Methylated undermodified hydroxywybutosine, 2-Hydroxymethylguanosine, Pseudouridine, Dihydrouridine, 5- Methyluridine, ribosylthymine, or ribothymidine, 2'-O-methyluridine, 5,2'-O-dimethyluridine, 1- Methylpseudouridine, 2'-O-methylpseudouridine, 2-Thiouridine, 4-Thiouridine, 2-Thio-2'-O- methyluridine, 3-(3-Amino-3-carboxypropyl)uridine, 5-Hydroxyuridine, 5-Methoxyuridine, Uridine 5-oxyacetic acid, Uridine 5-oxyacetic acid methyl ester, 5-Carboxyhydroxymethyluridine, 5-Carboxyhydroxymethyluridine methyl ester, 5-Methoxycarbonylmethyluridine, 5- Methoxycarbonylmethyl-2'-O-methyluridine, 5-Aminomethyl-2-thiouridine, 5- Methylaminomethyl uridine, 5-Methylaminomethyl-2-thiouridine, 5-Methylaminomethyl-2- selenouridine, 5-Carbamoylmethyluridine, 5-Carbamoylmethyl-2'-O-methyluridine, 5- Carboxymethylaminomethyl uridine, 5-Carboxymethylaminomethyl-2'-O-methyluridine, 5- Carboxymethylaminomethyl-2-thiouridine, 3-Methyluridine, l-Methyl-3(3-amino-3- carboxypropyl) pseudouridine, 5-Carboxymethyluridine, 3,2'-O-dimethyluridine, 5- Methyldihydrouridine, 3-Methylpseudouridine, 5-Taurinomethyluridine, 5-Taurinomethyl-2- thiouridine, 5-(lsopentenylaminomethyl)uridine, 5-(lsopentenylaminomethyl)-2-thiouridine, 5- (lsopentenylaminomethyl)-2'-O-methyl uridine, 5-Cyanomethyluridine, 5-

[0057] (Carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 5-Carboxymethylaminomethyl-2- selenouridine, 5-Carboxymethylaminomethyl-2-geranylthiouridine, 5-Methylaminomethyl-2- geranylthiouridine, 5-Aminomethyl-2-geranylthiouridine, 5-Methoxycarbonylmethyl-2- thiouridine, 5-Carbamoylmethyl-2-thiouridine, 3(3-Amino-3-carboxypropyl)-5,6-dihydrouridine, 5-Aminomethyl-2-selenouridine, 5-Carbamoyl hydroxymethyl uridine, 5-Carboxymethyl-2- thiouridine, 5-Methyl-2-thiouridine, 2-Geranylthiouridine, 2-Selenouridine, 5- Aminomethyluridine, 2'-O-methyluridine 5-oxyacetic acid methyl ester, 3-(3-Amino-3- carboxypropyl)pseudouridine, and 5-cyanomethyl-2-thiouridine.

[0058]

[0054] As used herein, "inter-nucleotide linker" refers to any suitable divalent moiety that links a nucleoside to one or more adjacent nucleoside in a polynucleotide. Unless stated otherwise, "internucleoside linker" and "inter-nucleotide linker" are referred to herein interchangeably. Generally, an inter-nucleotide linker links a 3'-substitutent of a nucleoside (e.g., oxygen) to a 5'- substituent (e.g., oxygen) of an adjacent nucleoside, unless specified otherwise. In some instances, an inter-nucleotide linker is an internucleoside linker between two or more ribose groups. In some instances, an inter-nucleotide linker is a inter-nucleotide between two or more polynucleotides. In some embodiments, an inter-nucleotide linker is a phosphate, phosphorothioate, phosphorodithioate, or thiophosphate. It shall be understood in embodiments where "a phosphate, phosphorothioate, phosphorodithioate, or thiophosphate" is used to describe an inter-nucleotide linker, those terms refer to the chemical moiety formed when the 3'-substituent of a nucleoside (e.g., oxygen) and a 5'-substitutent of an adjacent nucleoside (e.g., oxygen) are taken together with the divalent group to which both said 3' and 5' substituents are bound. For example, "phosphate" refers to the group , wherein X1is the 3'-oxygen of the immediately upstream nucleoside and X2is the 5'-oxygen of the immediately downstream nucleoside.

[0059]

[0055] As used herein, "phosphate analog" refers to any suitable phosphorus-containing group for attachment to a 5'-substituent or 3'-substituent of a nucleoside. In some embodiments, a phosphate analog is a phosphate, phosphorothioate, phosphorodithioate, or thiophosphate. In some embodiments, a phosphate analog comprises -O-P(X2)2-O- or -O-P(X2)2-O-, where each X2is independently O or S.

[0060]

[0056] "Amino" refers to the -NH2 radical.

[0061]

[0057] "Cyano" refers to the CN radical.

[0062]

[0058] " Nitro" refers to the NO2 radical.

[0063]

[0059] "Oxo" refers to the =0 radical.

[0064]

[0060] "Hydroxyl" refers to the -OH radical.

[0065]

[0061] "Alkyl" generally refers to an acyclic (e.g., straight or branched) or cyclic hydrocarbon (e.g., chain) radical consisting solely of carbon and hydrogen atoms, such as having from one to fifteen carbon atoms (e.g., C1-C15 alkyl). Unless otherwise state, alkyl is saturated or unsaturated (e.g., an alkenyl, which comprises at least one carbon-carbon double bond). Disclosures provided herein of an "alkyl" are intended to include independent recitations of a saturated "alkyl," unless otherwise stated. Alkyl groups described herein are generally monovalent, but may also be divalent (which may also be described herein as "alkylene" or "alkylenyl" groups). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (e.g., C1-C13 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., Ci-C8alkyl). ). In certain embodiments, an alkyl comprises one to six carbon atoms (e.g., Ci-Ce alkyl). In other embodiments, an alkyl comprises one to five carbon atoms (e.g., C1-C5 alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (e.g., C1-C4 alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (e.g., C1-C3 alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (e.g., C1-C2 alkyl). In other embodiments, an alkyl comprises one carbon atom (e.g., Ci alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., Cs-Cs alkyl). In other embodiments, an alkyl comprises two to five carbon atoms (e.g., C2-C5alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (e.g., C3-C5 alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso propyl), 1-butyl (n butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl is attached to the rest of the molecule by a single bond. In general, alkyl groups are each independently substituted or unsubstituted. In some instances, an alkyl group provided herein is substituted or further substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, -0Ra, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -C(O)N(Ra)2, -N(Ra)C(O)ORa, -OC(O)-N(Ra)2, -N(Ra)C(O)Ra, -N(Ra)S(O)tRa(where t is 1 or 2), - S(O)tORa(where t is 1 or 2), -S(O)tRa(where t is 1 or 2) and -S(0)tN(Ra)2 (where t is 1 or 2) where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

[0066]

[0062] "Alkoxy" refers to a radical bonded through an oxygen atom of the formula — O-alkyl, where alkyl is an alkyl group as defined above.

[0067]

[0063] "Alkenyl" refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon double bond, and having from two to twelve carbon atoms. In certain embodiments, an alkyl comprises two to six carbon atoms (e.g., C2-Cealkenyl). In certain embodiments, an alkyl comprises two to eight carbon atoms (e.g., C2-Csalkenyl). In certain embodiments, an alkyl comprises two to four carbon atoms (e.g., C2-C4alkenyl). Unless stated otherwise specifically in the specification, an alkenyl group provided herein is substituted or further substituted as described for "alkyl" hereinabove.

[0064] "Alkynyl" refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and having from two to twelve carbon atoms. In certain embodiments, an alkynyl comprises two to six carbon atoms (e.g., C2-C6alkynyl). In certain embodiments, an alkynyl comprises two to eight carbon atoms (e.g., C2-Csalkynyl). In certain embodiments, an alkynyl comprises two to four carbon atoms (e.g., C2-C4alkynyl). Unless stated otherwise specifically in the specification, an alkenyl group provided herein is substituted or further substituted as described for "alkyl" hereinabove.

[0068]

[0065] "Alkylene" or "alkylene chain" generally refers to a straight or branched divalent alkyl group linking the rest of the molecule to a radical group, such as having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, / -propylene, n-butylene, and the like. Unless stated otherwise specifically in the specification, an alkylene chain is substituted or further substituted as described for "alkyl" hereinabove.

[0069]

[0066] "Aryl" refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon from five to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) 7i-electron system in accordance with the Huckel theory. The ring system from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin and naphthalene. In some instances, an aryl group provided herein is substituted or further substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-ORa, -Rb-OC(O)-Ra, -Rb-OC(O)-ORa, -Rb-OC(O)-N(Ra)2, - Rb-N(Ra)2, -Rb-C(O)Ra, -Rb-C(O)ORa, -Rb-C(O)N(Ra)2, -Rb-O-Rc-C(O)N(Ra)2, -Rb-N(Ra)C(O)ORa, -Rb- N(Ra)C(O)Ra, -Rb-N(Ra)S(O)tRa(where t is 1 or 2), -Rb-S(O)tRa(where t is 1 or 2), -Rb-S(O)tORa(where t is 1 or 2) and -Rb-S(O)tN(Ra)2(where t is 1 or 2), where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rbis independently a direct bond or a straight or branched alkylene or alkenylene chain, and Rcis a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated. In certain embodiments, an aryl comprises 4 to 10 ring members. In certain embodiments, an aryl comprises 4 to 6 ring members. In certain embodiments, an aryl 5 to 10 ring members. In certain embodiments, an aryl comprises 5 to 8 ring members. In certain embodiments, an aryl comprises 5 or 6 ring members.

[0070]

[0067] "Carbocyclyl" or "cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which includes fused or bridged ring systems, having from three to fifteen carbon atoms. In certain embodiments, a carbocyclyl comprises three to ten carbon atoms. In other embodiments, a carbocyclyl comprises five to seven carbon atoms. The carbocyclyl is attached to the rest of the molecule by a single bond. Carbocyclyl or cycloalkyl is saturated (i.e., containing single C-C bonds only) or unsaturated (i.e., containing one or more double bonds or triple bonds). Examples of saturated cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. An unsaturated carbocyclyl is also referred to as "cycloalkenyl." Examples of monocyclic cycloalkenyls include, e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Polycyclic carbocyclyl radicals include, for example, adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbornenyl, decalinyl, 7,7-dimethyl bicyclo[2.2.1]heptanyl, and the like. In some instances, a carbocyclyl group provided herein is substituted or further substituted with one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-ORa, -Rb-OC(O)-Ra, -Rb-OC(O)-ORa, -Rb-OC(O)-N(Ra)2, -Rb-N(Ra)2, -Rb-C(O)Ra, - Rb-C(O)ORa, -Rb-C(O)N(Ra)2, -Rb-O-Rc-C(O)N(Ra)2, -Rb-N(Ra)C(O)ORa, -Rb-N(Ra)C(O)Ra, -Rb- N(Ra)S(O)tRa(where t is 1 or 2), -Rb-S(O)tRa(where t is 1 or 2), -Rb-S(O)tORa(where t is 1 or 2) and -Rb-S(O)tN(Ra)2(where t is 1 or 2), where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rbis independently a direct bond or a straight or branched alkylene or alkenylene chain, and Rcis a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

[0068] "Halo" or "halogen" refers to fluoro, bromo, chloro, or iodo substituents.

[0071]

[0069] "Haloalkyl" refers to an alkyl radical, as defined above, that is substituted by one or more halogen radicals, as defined above, for example, trihalomethyl, dihalomethyl, halomethyl, and the like. In some embodiments, the haloalkyl is a fluoroalkyl, such as, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl 2-fluoroethyl, and the like. In some embodiments, the alkyl part of the fluoroalkyl radical is substituted or further substituted as defined above for an alkyl group.

[0072]

[0070] The term "heteroalkyl" refers to an alkyl group as defined above in which one or more skeletal carbon atoms of the alkyl are substituted with a heteroatom (with the appropriate number of substituents or valencies - for example, -CH2- may be replaced with -NH- or -O-). For example, each substituted carbon atom is independently substituted with a heteroatom, such as wherein the carbon is substituted with a nitrogen, oxygen, sulfur, or other suitable heteroatom. In some instances, each substituted carbon atom is independently substituted for an oxygen, nitrogen (e.g. -NH-, -N(alkyl)-, or -N(aryl)- or having another substituent contemplated herein), or sulfur (e.g. -S-, -S(=O)-, or -S(=O)z-). In some embodiments, a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. In some embodiments, a heteroalkyl is attached to the rest of the molecule at a heteroatom of the heteroalkyl. In some embodiments, a heteroalkyl is a Ci-Cis heteroalkyl. In some embodiments, a heteroalkyl is a C1-C12 heteroalkyl. In some embodiments, a heteroalkyl is a Ci-Ce heteroalkyl. In some embodiments, a heteroalkyl is a C1-C4 heteroalkyl. In some embodiments, heteroalkyl includes alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, heterocycloalkyl, heterocyclyl, and heterocycloalkylalkyl, as defined herein. Unless stated otherwise specifically in the specification, heteroalkyl does not include alkoxy as defined herein. Unless stated specifically otherwise, a heteroalkyl group is substituted or further substituted as defined above for an alkyl group.

[0073]

[0071] "Heteroalkylene" refers to a divalent heteroalkyl group defined above which links one part of the molecule to another part of the molecule. Unless stated specifically otherwise, a heteroalkylene is substituted or further substituted, as defined above for an alkyl group.

[0074]

[0072] "Heterocyclyl" refers to a stable 3 to 18 membered non-aromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which optionally includes fused or bridged ring systems. The heteroatoms in the heterocyclyl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocyclyl radical is partially or fully saturated. The heterocyclyl radical is saturated (i.e., containing single C-C bonds only) or unsaturated (e.g., containing one or more double bonds or triple bonds in the ring system). In some instances, the heterocyclyl radical is saturated. In some instances, the heterocyclyl radical is saturated and substituted. In some instances, the heterocyclyl radical is unsaturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl [l,3]dithia nyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2- oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1 oxo thiomorpholinyl, and 1,1-dioxo thiomorpholinyl. In some instances, a heterocyclyl group is substituted or further substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb- ORa, -Rb-OC(O)-Ra, -Rb-OC(O)-ORa, -Rb-OC(O)-N(Ra)2, -Rb-N(Ra)2, -Rb-C(O)Ra, -Rb-C(O)ORa, -Rb- C(O)N(Ra)2, -Rb-O-Rc-C(O)N(Ra)2, -Rb-N(Ra)C(O)ORa, -Rb-N(Ra)C(O)Ra, -Rb-N(Ra)S(O)tRa(where t is 1 or 2), -Rb-S(O)tRa(where t is 1 or 2), -Rb-S(O)tORa(where t is 1 or 2) and -Rb-S(O)tN(Ra)2(where t is 1 or 2), where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rbis independently a direct bond or a straight or branched alkylene or alkenylene chain, and Rcis a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.

[0075]

[0073] "Heteroaryl" refers to a radical derived from a 3 to 18 membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, the heteroaryl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) n-electron system in accordance with the Huckel theory. Heteroaryl includes fused or bridged ring systems. The heteroatom(s) in the heteroaryl radical is optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl is attached to the rest of the molecule through any atom of the ring(s). Examples of heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][l,4]dioxepinyl, benzo[b][l,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro 5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[3,2 c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10- hexa hydrocycloocta [d] pyridazi nyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano 5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5, 6, 6a, 7, 8, 9, 10, 10a- octahydrobenzo[h]quinazolinyl, 1-phenyl-lH-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8- tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6, 7,8,9- tetrahydro 5H-cyclohepta[4,5]thieno[2,3 d] pyrimidi nyl, 5,6,7,8-tetrahydropyrido[4,5- c] pyridazi nyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e. thienyl). In some instances, a heteroaryl group is substituted or further substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, haloalkenyl, haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-ORa, -Rb-OC(O)-Ra, - Rb-OC(O)-ORa, -Rb-OC(O)-N(Ra)2, -Rb-N(Ra)2, -Rb-C(O)Ra, -Rb-C(O)ORa, -Rb-C(O)N(Ra)2, -Rb-O-Rc- C(O)N(Ra)2, -Rb-N(Ra)C(O)ORa, -Rb-N(Ra)C(O)Ra, -Rb-N ( Ra)S(O)tRa(where t is 1 or 2), -Rb-S(O)tRa(where t is 1 or 2), -Rb-S(O)tORa(where t is 1 or 2) and -Rb-S(O)tN(Ra)2(where t is 1 or 2), where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rbis independently a direct bond or a straight or branched alkylene or alkenylene chain, and Rcis a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated. In some embodiments, a heteroaryl comprises 4 to 10 ring members. In some embodiments, a heteroaryl comprises 4 to 6 ring members. In some embodiments, a heteroaryl comprises 5 to 10 ring members. In some embodiments, a heteroaryl comprises 5 to 8 ring members. In some embodiments, a heteroaryl comprises 5 or 6 ring members.

[0076]

[0074] "Heterocycloalkyl" refers to a cycloalkyl containing at least one annular carbon and at least one annular heteroatom selected from the group consisting of N, O and S, wherein the ring is not aromatic but can contain unsaturations. The nitrogen and sulfur atoms in a heterocyclic group can be oxidized and the nitrogen atom(s) may optionally be quaternized. The heterocyclic group can be fused to an additional carbocyclic or heterocyclic ring. A heterocyclic group can be attached to the remainder of the molecule at an annular carbon or annular heteroatom. In certain embodiments, a heterocycloalkyl comprises 3 to 18 ring members with two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. In certain embodiments, a heterocycloalkyl comprises two to eight carbon atoms (C2- Csheterocycloalkyl). In certain embodiments, a heterocycloalkyl comprises two to six carbon atoms (C2-C6heterocycloalkyl).

[0075] In general, optionally substituted groups are each independently substituted or unsubstituted. Each recitation of an optionally substituted group provided herein, unless otherwise stated, includes an independent and explicit recitation of both an unsubstituted group and a substituted group (e.g., substituted in certain embodiments, and unsubstituted in certain other embodiments). In some instances, a substituted group provided herein (e.g., substituted alkyl) is substituted by one or more substituent, each substituent being independently selected from the group consisting of halo, cyano, nitro, oxo, thioxo, imino, oximo, tri methylsi lanyl, -ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -C(O)N(Ra)2, -N(Ra)C(O)ORa, -OC(O)-N(Ra)2, - N(Ra)C(O)Ra, -N(Ra)S(O)tRa(where t is 1 or 2), -S(O)tORa(where t is 1 or 2), -S(O)tRa(where t is 1 or 2) and -S(O)tN(Ra)2(where t is 1 or 2), where each Rais independently hydrogen, alkyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

[0077]

[0076] A "poly-A tail" or "poly-A polynucleotide" or "poly-A region" can be used interchangeably and referto a polynucleotide (e.g., polyribonucleotide) having a nucleobase sequence comprising 75% or greater adenine (A) nucleobases or modified adenine nucleobases. In certain embodiments, the poly-A tail nucleobase sequence comprises 80% or greater adenine (A) nucleobases. In certain embodiments, the poly-A tail nucleobase sequence comprises 85% or greater adenine (A) nucleobases. In certain embodiments, the poly-A tail nucleobase sequence comprises 90% or greater adenine (A) nucleobases. In certain embodiments, the poly-A tail nucleobase sequence comprises 95% or greater adenine (A) nucleobases. In certain embodiments, the poly-A tail nucleobase sequence comprises 96%, 97%, 98, 99% or greater adenine (A) nucleobases. In some embodiments, a poly-A tail comprises a structure represented by the formula: (pN)n wherein each (p) is independently an inter-nucleotide linker, each (N) is independently a nucleoside, (n) is an integer great than 1 (e.g., 2, 5, 10, 15, 25, 50, 100, etc.), and wherein the nucleobase content of the poly-A tail is consistent with the nucleobase contents described herein (e.g., 80% adenine or greater than 80% adenine). In certain embodiments, a poly-A tail comprises a structure represented by the formula: (pA)n wherein (pA) is a nucleotide having an adenine or modified adenine nucleobase, and (n) is an integer great than 1 (e.g., 2, 5, 10, 15, 25, 50, 100, etc.). In certain embodiments, a poly-A tail comprises about 10-15 nucleotides, about 10-20 nucleotides, about 10-25 nucleotides, about 10-50 nucleotides, about 10-100 nucleotides, about 10-150 nucleotides, about 10-200 nucleotides, about 10-250 nucleotides, or about 10-400. In certain embodiments, the 5' nucleotide or beginning nucleotide of the poly-A tail comprises a monocyclic or abasic nucleobase such as those described herein (e.g., cytidine or a modified cytidine). As used herein, a 3' chain terminating tail generally refers to a poly-A tail having a 3' chain terminating nucleotide. In certain embodiments, the chain terminating nucleoside is a nucleoside that having a modification that blocks 3' ligation (e.g., by a T4 RNA ligase). In certain embodiments, non-limiting examples of chain terminating nucleotides include dideoxynucleotides, deoxynucleotides (e.g., lacking a 3' hydroxyl), inverted nucleotides, and 3' spacer nucleotides (e.g., 3'-Spacer C3 nucleotides). In certain embodiments, blocking 3' ligation (e.g., reducing ligation by 50% or greater) can be determined by the methods described herein and in the examples. In certain embodiments, the chain terminating nucleotide is a 3' terminal nucleotide that lacks a 3' hydroxyl. The chain terminating nucleotide can also be a dideoxy nucleotide. In certain instances, the chain terminating nucleotide is a 3' terminal nucleotide that lacks both a 2' hydroxyl and 3' hydroxyl. In certain instances, the chain terminating nucleotide is an inverted nucleotide.

[0078]

[0077] Provided in certain embodiments herein are modified polynucleotides useful for increasing the stability of an mRNA molecule and / or production of a therapeutic protein. In some instances, when ligated to a second mRNA molecule (such as a mRNA that encodes an antigen or therapeutic protein), the modified polynucleotide confers to the resulting ligated polynucleotide increased longevity of expression relative to polynucleotides that lack the modified polynucleotides disclosed herein. In some embodiments, the modified polynucleotides exhibit high ligation efficiency and / or improved reaction parameters.

[0079]

[0078] Provided herein, in some embodiments, are messenger ribonucleic acid (mRNA) polynucleotides comprising a poly-A tail, wherein the poly-A tail comprises a terminal 3' nucleotide comprising (a) a 5' phosphorothioate linkage and (b) a nucleoside lacking a free 3' hydroxyl. In certain embodiments, the poly-A tail further comprises (i) a 5' nucleotide comprising a monocyclic nucleobase. Also provided are poly-A tails comprising (i) a terminal 5' nucleotide comprising a monocyclic nucleobase and free 5' phosphate, and (ii) a terminal 3' nucleotide comprising (a) a 5' phosphorothioate linkage and (b) a nucleoside lacking a 3' hydroxyl.

[0080]

[0079] In certain embodiments, the last 2-8 inter-nucleotide linkages at the 3' terminus of the poly-A tail are phosphorothioate inter-nucleotide linkages. In certain embodiments, the last 2-8 inter-nucleotide linkages at the 3' terminus of the poly-A tail are phosphorothioate inter- nucleotide linkages. In certain embodiments, the last 6 inter-nucleotide linkages at the 3' terminus of the poly-A tail are phosphorothioate inter-nucleotide linkages. In certain embodiments, the poly-A tails comprise an adenine nucleobase content of 85% or greater. In certain embodiments, the poly-A tails comprise an adenine nucleobase content of 90% or greater. In certain embodiments, the poly-A tails comprise an adenine nucleobase content of 90% or greater. In certain embodiments, the poly-A tail comprises about 10-400 nucleotides (including, e.g., 10-200, 10-150, 10-100, 10-50, 10-25, or 10-20 nucleotides). In certain embodiments, the monocyclic nucleobase is a pyrimidine nucleobase. In certain embodiments, the monocyclic nucleobase is cytidine or a modified cytidine (e.g., m5C, hm5C, f5C, etc.). In certain embodiments, the terminal 3' nucleotide also lacks a 2' hydroxyl (is a dideoxy nucleotide).

[0080] Also, in some embodiments, provided herein are polyribonucleotides comprising a structure represented by the formula: pZ - (p'N)n - p"N* wherein :

[0081] (p) is a free 5' terminal phosphate;

[0082] (Z) is a nucleoside comprising a monocyclic nucleobase; each (p') is independently an inter-nucleotide linkage (e.g., phosphorothioate linkage or phosphate linkage);

[0083] (p") is a phosphorothioate inter-nucleotide linkage; each (N) is independently a nucleoside;

[0084] (N*) is a nucleoside lacking a 3' hydroxyl;

[0085] (n) is an integer equal to or greater than 1; and the nucleobase content of the polyribonucleotide is 80% adenine or greater than 80% adenine.

[0086]

[0081] In some embodiments, provided herein are mRNA molecules comprising a poly-A tail having a structure represented by the formula: pZ - (p'N)n - p"N* wherein :

[0087] (p) is a phosphate inter-nucleotide linkage;

[0088] (Z) is a nucleoside comprising a monocyclic nucleobase; each (p') is independently an inter-nucleotide linkage (e.g., phosphorothioate linkage or phosphate linkage);

[0089] (p") is a phosphorothioate inter-nucleotide linkage; each (N) is independently a nucleoside;

[0090] (N*) is a nucleoside lacking a 3' hydroxyl;

[0091] (n) is an integer equal to or greater than 1; and the nucleobase content of the polyribonucleotide is 80% adenine or greater than 80% adenine.

[0092]

[0082] In certain embodiments, (n) is 10-400, 10-200, 10-150, 10-100, 10-50, 10-25, 10-20, or 10- 15. In certain embodiments, (Z) comprises a pyrimidine nucleobase. In certain embodiments, (Z) is a nucleoside comprising a cytidine nucleobase or modified cytidine nucleobase (e.g., m5C, hm5C, f5C, etc.). In certain embodiments, the nucleobase content of the polyribonucleotide is 85% adenine or greater than 85% adenine. In certain embodiments, the nucleobase content of the polyribonucleotide is 90% adenine or greaterthan 90% adenine. In certain embodiments, the nucleobase content of the polyribonucleotide is 95% adenine or greater than 95% adenine. In certain embodiments, (N) is a nucleoside comprising an adenine nucleobase or modified adenine nucleobase (e.g., m6A, mlA, m2A). In certain embodiments, the mRNA molecule comprises, from 5' to 3', a 5' cap structure, a 5' UTR, a protein coding sequence, a 3' UTR, and the poly-A tail.

[0093]

[0083] Further provided, in some embodiments, are polyribonucleotides comprising a structure represented by the formula: pZ - (p'N)n1- (p"A)n2- p"'N* wherein :

[0094] (p) is a free 5' terminal phosphate;

[0095] (Z) is a nucleoside comprising a monocyclic nucleobase (e.g., cytosine); each (p') is independently a phosphate inter-nucleotide linkage; each (N) is independently a nucleoside; each (p") is independently a phosphorothioate inter-nucleotide linkage; each (A) is independently a nucleoside comprising an adenine nucleobase or modified adenine nucleobase;

[0096] (p'") is a phosphorothioate inter-nucleotide linkage;

[0097] (N*) is a nucleoside lacking a 3' hydroxyl;

[0098] (n1) is an integer equal to or greater than 1;

[0099] (n2) is 0, 1, 2, 3, 4, 5, 6, or 7; and the nucleobase content of the polyribonucleotide is 80% adenine or greater than 80% adenine.

[0100]

[0084] In some embodiments, provided herein are mRNA molecules comprising a poly-A tail having a structure represented by the formula: pZ - (p'N)n1- (p"A)n2- p"'N* wherein :

[0101] (p) is a phosphate inter-nucleotide linkage;

[0102] (Z) is a nucleoside comprising a monocyclic nucleobase (e.g., cytosine); each (p') is independently a phosphate inter-nucleotide linkage; each (N) is independently a nucleoside; each (p") is independently a phosphorothioate inter-nucleotide linkage; each (A) is independently a nucleoside comprising an adenine nucleobase or modified adenine nucleobase;

[0103] (p'") is a phosphorothioate inter-nucleotide linkage;

[0104] (N*) is a nucleoside lacking a 3' hydroxyl;

[0105] (n1) is an integer equal to or greater than 1;

[0106] (n2) is 0, 1, 2, 3, 4, 5, 6, or 7; and the nucleobase content of the polyribonucleotide is 80% adenine or greater than 80% adenine.

[0107]

[0085] In certain embodiments, (n2) is 2-8. In certain embodiments (n2) is 4-6. In certain embodiments (n2) is 6. In certain embodiments, (n1) is 5-400, 5-200, 5-150, 5-100, 5-50, 5-25, 10- 20, 5-15, or 5-10. In certain embodiments, (Z) comprises a pyrimidine nucleobase. In certain embodiments, (Z) is a nucleoside comprising a cytidine nucleobase or modified cytidine nucleobase (e.g., m5C, hm5C, f5C, etc.). In certain embodiments, the nucleobase content of the polyribonucleotide is 85% adenine or greater than 85% adenine. In certain embodiments, the nucleobase content of the polyribonucleotide is 90% adenine or greater than 90% adenine. In certain embodiments, the nucleobase content of the polyribonucleotide is 95% adenine or greater than 95% adenine. In certain embodiments, (N) is a nucleoside comprising an adenine nucleobase or modified adenine nucleobase (e.g., m6A, mlA, m2A). In certain embodiments, the mRNA molecule comprises, from 5' to 3', a 5' cap structure, a 5' UTR, a protein coding sequence, a 3' UTR, and the poly-A tail.

[0108]

[0086] In some embodiments, provided herein are mRNA molecules comprising a poly-A tail having a structure represented by the formula: pZ - (p'N)n1- (p"A)6- p"'N* wherein :

[0109] (p) is a phosphate inter-nucleotide linkage;

[0110] (Z) is a nucleoside comprising a monocyclic nucleobase (e.g., cytosine); each (p') is independently a phosphate inter-nucleotide linkage; each (N) is independently a nucleoside; each (p") is independently a phosphorothioate inter-nucleotide linkage; each (A) is independently a nucleoside comprising an adenine nucleobase or modified adenine nucleobase;

[0111] (p'") is a phosphorothioate inter-nucleotide linkage;

[0112] (N*) is a nucleoside lacking a 3' hydroxyl;

[0113] (n1) is an integer of 1 to 150; the nucleobase content of the polyribonucleotide is 80% adenine or greater than 80% adenine.

[0114]

[0087] In certain embodiments, (n2) is 2-8. In certain embodiments (n2) is 4-6. In certain embodiments (n2) is 6. In certain embodiments, (n1) is 5-400, 5-200, 5-150, 5-100, 5-50, 5-25, 10- 20, 5-15, or 5-10. In certain embodiments, (Z) comprises a pyrimidine nucleobase. In certain embodiments, (Z) is a nucleoside comprising a cytidine nucleobase or modified cytidine nucleobase (e.g., m5C, hm5C, f5C, etc.). In certain embodiments, the nucleobase content of the polyribonucleotide is 85% adenine or greater than 85% adenine. In certain embodiments, the nucleobase content of the polyribonucleotide is 90% adenine or greater than 90% adenine. In certain embodiments, the nucleobase content of the polyribonucleotide is 95% adenine or greater than 95% adenine. In certain embodiments, (N) is a nucleoside comprising an adenine nucleobase or modified adenine nucleobase (e.g., m6A, mlA, m2A). In certain embodiments, the mRNA molecule comprises, from 5' to 3', a 5' cap structure, a 5' UTR, a protein coding sequence, a 3' UTR, and the poly-A tail.

[0115]

[0088] In some embodiments, provided herein are In some embodiments, provided herein is amessenger ribonucleic acid (mRNA) polynucleotide comprising a poly-A tail, wherein the poly-A tail comprises a structure represented by the formula:

[0116] -(pA)nl-(p'A)n2- wherein : each (p) is independently a phosphate linkage; each (p') is independently a phosphorothioate linkage; each (A) is independently an adenine nucleotide or a modified adenine nucleotide;

[0117] (nl) is an integer equal to or greater than 1; and

[0118] (n2) is an integer equal to or greater than 1.

[0089] In some embodiments, (nl) is an integer of 3 to 15. In some embodiments, (nl) is an integer of 7 to 12. (n2) is an integer of 1 to 10. In some embodiments, (n2) is an integer of 1 to 6.

[0090] In some embodiments, the polyA tail comprises a dideoxynucleoside (e.g., ddC or ddA) located at the terminal 3 ' position (e.g., wherein the dideoxynucleoside is attached to the penultimate A, such as via a phosphothioate linkage).

[0119]

[0091] T In some embodiments, the polyA tail comprises a cytosine (C) attached to (e.g., the 5' terminal) p.

[0120]

[0092] Provided in certain embodiments herein are systems, compositions, and methods, such as methods and compositions useful for making RNA and products thereof. In some embodiments, compositions provided herein comprise a mRNA polynucleotide comprising a 5' terminal nucleoside bearing a nucleobase or nucleobase analog, wherein the nucleobase or nucleobase analog is monocyclic C2-C8heterocycloalkyl, monocyclic aryl, or monocyclic heteroaryl. In some embodiments, the mRNA polynucleotide comprises 3 to 9 consecutive phosphorothioate nucleotides that are bound to a chain terminating nucleoside (e.g., comprising a ribose lacking a free 3' hydroxyl). In some embodiments, the mRNA polynucleotide comprises 4 to 6 consecutive phosphorothioate nucleotides that are bound to a chain terminating nucleoside (e.g., comprising a ribose lacking a free 3' hydroxyl). In some embodiments, the mRNA polynucleotide comprises 4 consecutive phosphorothioate nucleotides that are bound to a chain terminating nucleoside (e.g., comprising a ribose lacking a 3' hydroxyl). In some embodiments, the mRNA polynucleotide comprises 5 consecutive phosphorothioate nucleotides that are bound to a chain terminating nucleoside (e.g., comprising a ribose lacking a free 3' hydroxyl). In some embodiments, the mRNA polynucleotide comprises 6 consecutive phosphorothioate nucleotides that are bound to a chain terminating nucleoside (e.g., comprising a ribose lacking a 3' hydroxyl).

[0093] Provided in some embodiments herein is an mRNA polynucleotide. In some embodiments, the mRNA polynucleotide described herein comprises a 3' chain terminating dinucleotide. In some embodiments, the mRNA polynucleotide described herein comprises a 3' chain terminating dinucleotide has a structure represented by Formula (I): p-Z-(p'-A)n-p"-(R-pc)m- R1-pd-N. In some embodiments, p is an inter-nucleotide linker or a chain terminal group. In some embodiments, Z is a natural or modified nucleoside. In some embodiments, each p' is independently an inter-nucleotide linker. In some embodiments, each A is independently an adenosine nucleoside, or an analog thereof. In some embodiments, p" is an inter-nucleotide linker. In some embodiments, n is an integer of 0 to 30. In some embodiments, each R is independently a nucleoside. In some embodiments, each pcis independently a phosphorothioate. In some embodiments, m is an integer of 2 to 8. In some embodiments, R1is a nucleoside. In some embodiments, pdis a phosphorothioate. In some embodiments, N is a chain terminating nucleoside. In some embodiments, N is a chain terminating nucleoside comprising a ribose lacking a free 3' hydroxyl. In some embodiments, each nucleoside independently comprises a ribose or analog thereof.

[0121]

[0094] In some embodiments, p is an inter-nucleotide linker. In some embodiments, p is an inter- nucleotide linker that is a phosphate. In some embodiments, p is an inter-nucleotide linker that is a diphosphate.

[0122]

[0095] Unless stated otherwise herein, the inter-nucleoside and inter-nucleotide are used interchangeably for the purposes of the structures described herein.

[0123]

[0096] In some embodiments, p is a 5' terminal group. In some embodiments, p is a 5' terminal group that is a phosphate. In some embodiments, p is a 5' terminal group that is a diphosphate. In some embodiments, p is a 5' terminal group selected from the group consisting of hydroxyl, phosphate, phosphorothioate, phosphorodithioate, thiophosphate, phosphonate, phosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, diphosphate, and triphosphate. In some embodiments, p is a chain terminal group selected from the group consisting of phosphate, diphosphate, phosphorothioate, and diphosphorothioate.

[0124]

[0097] In some embodiments, p is a phosphate. In some embodiments, p is phosphate.

[0125]

[0098] In some embodiments, p is a phosphate analog. In some embodiments, p is a phosphate or a phosphorothioate. In some embodiments, p is a phosphorothioate (PS) or diphosphorothioate (PS2). In some embodiments, p is a phosphorothioate.

[0126]

[0099] In some embodiments, p' is a phosphate. In some embodiments, p' is phosphate.

[0127]

[0100] In some embodiments, p' is a phosphate analog. In some embodiments, p' is a phosphate or a phosphorothioate. In some embodiments, p' is a phosphorothioate (PS) or diphosphorothioate (PS2). In some embodiments, p' is a phosphorothioate. In some embodiments, p' is a phosphate analog selected from phosphorothioate, phosphorodithioate, thiophosphate, phosphonate, phosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, diphosphate, or triphosphate. In some embodiments, p' is a phosphate selected from phosphate, diphosphate, phosphorothioate, and diphosphorothioate.

[0128]

[0101] In some embodiments, p" is a chain terminal group. In some embodiments, p" is a chain terminal group that is a phosphate. In some embodiments, p" is a chain terminal group that is a diphosphate. In some embodiments, p" is a chain terminal group selected from the group consisting of hydroxyl, phosphate, phosphorothioate, phosphorodithioate, thiophosphate, phosphonate, phosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, diphosphate, and triphosphate. In some embodiments, p" is a chain terminal group selected from the group consisting of phosphate, diphosphate, phosphorothioate, and diphosphorothioate.

[0129]

[0102] In some embodiments, p" is a phosphate. In some embodiments, p" is phosphate.

[0130]

[0103] I n some embodiments, p" is a phosphate analog. In some embodiments, p" is a phosphate or a phosphorothioate. In some embodiments, p" is a phosphorothioate (PS) or diphosphorothioate (PS2). In some embodiments, p" is a phosphorothioate. In some embodiments, p" is a phosphate analog selected from phosphorothioate, phosphorodithioate, thiophosphate, phosphonate, phosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, diphosphate, or triphosphate. In some embodiments, p" is a phosphate selected from phosphate, diphosphate, phosphorothioate, and diphosphorothioate.

[0131]

[0104] In some embodiments, a p described herein (e.g., p, p', or p") is a phosphorothioate or a diphosphorothiorate linker. In some embodiments, a p described herein (e.g., p, p', or p") is a phosphorothioate linker. In some embodiments, a p described herein (e.g., p, p', or p") is a diphosphorothiorate linker.

[0132]

[0105] In some embodiments, each nucleoside (e.g., of any formula described herein) independently comprises a ribose or a ribose analog.

[0133]

[0106] In some embodiments, a nucleoside (e.g., of any formula described herein) comprises a ribose. In some embodiments, each nucleoside (e.g., of any formula described herein) comprises a ribose.

[0107] In some embodiments, any nucleoside provided herein (e.g., of any formula described herein), such as a 3' chain terminating nucleoside of a poly-A tail or N of formula (I), comprises a ribose analog, such as any suitable ribose analog (e.g., for the purposes provided herein). In some embodiments, the ribose analog is a locked ribose, an unlocked ribose, a spiro-ribose, or a deoxyribose (e.g, 3' deoxy, 2' deoxy). In some embodiments, the ribose analog is deoxyribose. In certain embodiments, the deoxy ribose is a 2' deoxyribose. In certain embodiments (e.g., in certain chain terminating nucleosides), the deoxyribose is a 3' deoxyribose, n certain embodiments (e.g., in certain chain terminating nucleosides), the ribose analog is dideoxyribose. In some embodiments, the ribose analog is a furanose or other cyclized sugar, such as a pyranose (e.g., glucose). In some embodiments, the ribose analog is a nitrogen-containing heterocycle (e.g., wherein the ring has a nitrogen instead of or in addition to the ribose oxygen). In some embodiments, the ribose analog a heteroribose (e.g., wherein the furanose of the ribose has the oxygen replaced with a nitrogen (e.g., NR°) group, and / or has a carbon replaced with a nitrogen (e.g., NR°) group), a locked ribose (e.g., wherein the 2' position of the ribose is bridged with the 4' position of the ribose), or an unlocked ribose (e.g., wherein the ribose ring is opened). In some embodiments, each nucleoside (e.g., of any formula described herein) independently comprises a heteroribose, a locked ribose, or an unlocked ribose. In some instances, an unlocked ribose comprises an acyclic structure group, rather than a cyclized group (e.g., ribose). In some instances, a ribose analog comprises a furanose structure and a methylene, irrespective of further hydroxyl substitution (but is not ribose).

[0134]

[0108] In some embodiments, any nucleoside provided herein, unless otherwise limited, may have any suitable structure. In some embodiments, a nucleoside provided herein is natural or modified. In some embodiments, a nucleoside provided here in comprises a nucleobase or may be abasic, such as having an abasic structure described herein.

[0135]

[0109] In some embodiments, a ribose analog described herein, unless otherwise limited, may include structures where a ribose ring methylene is added (e.g., glucose) or removed (e.g., and replaced or substituted with a heteroatom).

[0110] In some embodiments, each nucleoside (e.g., of any formula described herein) independently comprises a ribose or a ribose analog. In specific embodiments, all nucleosides of an mRNA or poly-A tail or other structure provided herein comprises a ribose.

[0136]

[0111] In some embodiments, each nucleoside (e.g., of any formula described herein) independently comprises a ribose, a heteroribose, a locked ribose, or an unlocked ribose.

[0137]

[0112] In some embodiments, Z is a natural nucleoside.

[0138]

[0113] In some embodiments, Z is a modified nucleoside.

[0139]

[0114] In some embodiments, Z is a nucleoside comprising a monocyclic nucleobase. In some embodiments, Z is cytidine (C) or an analog thereof. In some embodiments, Z is cytidine (C). In some embodiments, Z is a cytidine (C) analog.

[0140]

[0115] In some embodiments, the ribose of Z is substituted with a hydrogen and a second group R4, wherein R4is a hydrogen, a halogen, a hydroxyl, a substituted or unsubstituted alkoxy, a substituted or unsubstituted heteroalkoxy, or a substituted or unsubstituted alkoxy. In some embodiments, R4is hydroxyl.

[0141]

[0116] I n some embodiments, Z is a nucleoside substituted at the 1-position with a hydrogen and B, wherein B is hydrogen, Cz-Cealkynyl, C2-Csheterocycloalkyl, aryl, or heteroaryl, and wherein C2- C6alkynyl, C2-C8heterocycloalkyl, aryl, and heteroaryl are unsubstituted or substituted.

[0142]

[0117] In some embodiments, B is monocyclic C2-C8heterocycloalkyl, monocyclic aryl, or monocyclic heteroaryl. In some embodiments, B is a monocyclic C2-C8heterocycloalkyl. In some embodiments, B is monocyclic monocyclic aryl. In some embodiments, B is a monocyclic heteroaryl.

[0143]

[0118] In some embodiments, B is a substituted or unsubstituted 5- or 6-membered aryl or a substituted or unsubstituted 5- or 6-membered heteroaryl. In some embodiments, B is a substituted or unsubstituted 5- or 6-membered aryl. In some embodiments, B is a substituted or unsubstituted 5- or 6-membered heteroaryl.

[0144]

[0119] In some embodiments, B is a pyrimidine nucleobase. In some embodiments, B is cytosine.

[0145]

[0120] In some embodiments, B is: ; wherein each Rxis independently hydrogen, halogen, -CN, -NO2, -

[0146] NH-OH, -OH, -ORa, -NRcRd, -NRbC(=O)Ra, -C(=O)Ra, -C(=O)NRcRd, substituted or unsubstituted

[0147] Ci-Cealkyl, or substituted or unsubstituted Ci-Ceheteroalkyl.

[0148]

[0121] In some embodiments, (B) is

[0123] In some embodiments, each Rxis independently. In some embodiments, each Rxis independently hydrogen, -NH2-NO2, -NH-OH, -OH, -OCH3, -CH3, -CH2OH, -N(H)CH3, -NH-OH, or - N(H)C(=O)CH3. In some embodiments, each Rxis independently hydrogen, -NO2, -OH, -ORa, - NRcRd, -NRbC(=O)Ra, substituted or unsubstituted Ci-Cealkyl, or substituted or unsubstituted Ci-Csheteroalkyl. In some embodiments, each R1is independently hydrogen, -NH2-NO2, -NH-OH, -OH, -OCH3, -CH3, -CH2OH, -N(H)CH3, or -N(H)C(=O)CH3. In some embodiments, two Rxon the same atom are taken together to form an oxo.

[0149]

[0124] In some embodiments, each Rais independently substituted or unsubstituted Ci-CgalkyL In some embodiments, each Rbis independently hydrogen or substituted or unsubstituted Ci-Cealkyl.

[0150]

[0125] In some embodiments, each Rcand Rdare independently hydrogen or substituted or unsubstituted Ci-Cealkyl. In some embodiments, Rcand Rdare taken together with the atom to which they are attached to form a substituted or unsubstituted heterocycloalkyl.

[0151]

[0126] In some embodiments, each A is independently an adenosine.

[0152]

[0127] In some embodiments, n is an integer of 2 to 25. In some embodiments, n is an integer of 6 to 20. In some embodiments, n is an integer of 6. In some embodiments, n is an integer of 7. In some embodiments, n is an integer of 8. In some embodiments, n is an integer of 9. In some embodiments, n is an integer of 10.

[0153]

[0128] In some embodiments, each R is independently a natural or modified nucleoside. In some embodiments, each R is independently a natural nucleoside. In some embodiments, each R is independently a modified nucleoside. In some embodiments, each R is independently adenosine or an analog thereof. In some embodiments, each R is independently adenosine. In some embodiments, each R is independently an adenosine analog. In some embodiments, at least 50% of the total of R are adenosine. In some embodiments, at least 60% of the total of R are adenosine. In some embodiments, at least 70% of the total of R are adenosine. In some embodiments, at least 80% of the total of R are adenosine. In some embodiments, at least 90% of the total of R are adenosine.

[0154]

[0129] In some embodiments, R1is independently a natural or modified nucleoside. In some embodiments, R1is independently a natural nucleoside. In some embodiments, R1is independently a modified nucleoside. In some embodiments, R1is independently adenosine or an analog thereof. In some embodiments, R1is independently adenosine. In some embodiments, R1is independently an adenosine analog. In some embodiments, at least 50% of the total of R1are adenosine. In some embodiments, at least 60% of the total of R1are adenosine. In some embodiments, at least 70% of the total of R1are adenosine. In some embodiments, at least 80% of the total of R1are adenosine. In some embodiments, at least 90% of the total of R1are adenosine.

[0155]

[0130] In some embodiments, each R and R1is independently a natural or modified nucleoside. In some embodiments, each R and R1is independently a natural nucleoside. In some embodiments, each R and R1is independently a modified nucleoside. In some embodiments, each R and R1is independently adenosine or an analog thereof. In some embodiments, each R and R1is independently adenosine. In some embodiments, each R and R1is independently an adenosine analog. In some embodiments, at least 50% of the total of R and R1are adenosine. In some embodiments, at least 60% of the total of R and R1are adenosine. In some embodiments, at least 70% of the total of R and R1are adenosine. In some embodiments, at least 80% of the total of R and R1are adenosine. In some embodiments, at least 90% of the total of R and R1are adenosine.

[0156]

[0131] In some embodiments, m is an integer of 3 to 7. In some embodiments, m is an integer of 4 to 6. In some embodiments, m is an integer of 3. In some embodiments, m is an integer of 4. In some embodiments, m is an integer of 5. In some embodiments, m is an integer of 6. In some embodiments, m is an integer of 7.

[0157]

[0132] In some embodiments, the ribose of N does not have a 3'-hydroxyL

[0158]

[0133] In some embodiments, N is a nucleoside substituted at the 2'-position with a nonhydroxyl. In some embodiments, N is a nucleoside substituted at the 2'-position with R7, wherein R7is hydrogen, a halogen, an alkoxy, a heteroalkoxy, an alkylthio, an amino, an alkyl, or a heteroalkyl, wherein the alkoxy, heteroalkoxy, alkylthio, amino, alkyl, or heteroalkyl is substituted or unsubstituted. In some embodiments, N is a nucleoside substituted at the 2'- position with R7, wherein R7is a halogen, an alkoxy, a heteroalkoxy, an alkylthio, an amino, an alkyl, or a heteroalkyl, wherein the alkoxy, heteroalkoxy, alkylthio, amino, alkyl, or heteroalkyl is substituted or unsubstituted.

[0159]

[0134] In some embodiments, N is a nucleoside substituted at the 3'-position with a nonhydroxyl. In some embodiments, N is a nucleoside substituted at the 3'-position with R6, wherein R6is hydrogen, a halogen, an alkoxy, a heteroalkoxy, an alkylthio, an amino, an alkyl, or a heteroalkyl, wherein the alkoxy, heteroalkoxy, alkylthio, amino, alkyl, or heteroalkyl is substituted or unsubstituted.

[0160]

[0135] In some embodiments, N is a non-hydroxyl nucleoside. In some embodiments, N is a dideoxynucleoside. In some embodiments, N is a ddC or ddA. In some embodiments, N is a ddC. In some embodiments, N is a ddA.

[0161]

[0136] In some embodiments, N is an inverted nucleoside. In some embodiments, N is an inverted nucleoside bounded via its 3' substituent (e.g., oxygen) to the immediately upstream internucleoside linker. In some embodiments, N is an inverted nucleoside bounded via its 3' substituent (e.g., oxygen) to the pd.

[0162]

[0137] In some embodiments, N is an inverted 2'-deoxynucleoside (InvdN). In some embodiments, N is an inverted 2'-deoxythymidine (InvdT), inverted 2'-deoxycytidine (InvdC) or inverted 2'-deoxyadenosine (InvdA). In some embodiments, N is inverted 2'-deoxythymidine (InvdT). In some embodiments, N is an inverted 2'-deoxycytidine (InvdC). In some embodiments, N is an inverted 2'-deoxyadenosine (InvdA).

[0163]

[0138] In some embodiments, the ribose of N is substituted at the 1' position with Q, wherein: Q is hydrogen, alkyl, C2-Cealkynyl, Cz-Cgheterocycloalkyl, aryl, or heteroaryl, wherein Cz-Cealkynyl, C2-C8heterocycloalkyl, aryl, and heteroaryl are unsubstituted or substituted (e.g., a substituted heteroaryl that is N6-methyladenosine (m6A)).

[0164]

[0139] In some embodiments, N comprises a nucleobase. In some embodiments, the nucleobase of N is a modified nucleobase. In some embodiments, the nucleobase of N is a natural nucleobase. In some embodiments, the nucleobase of N is adenine. In some embodiments, the nucleobase of N is a guanine. In some embodiments, the nucleobase of N is a cytosine. In some embodiments, the nucleobase of N is a uracil. In some embodiments, N is 2',3'-dideoxyadenine. In some embodiments, N is 2',3'-dideoxyguanine. In some embodiments, N is 2', 3'- dideoxycytosine. In some embodiments, N is 2',3'-dideoxyuracil. In some embodiments, N is a nucleoside reverse transcriptase inhibitor (NRTI), such as Lamivudine.

[0165]

[0140] In some embodiments, N is a non-3'-hydroxyl adenosine, uridine, or guanosine. In some embodiments, N is a non-3'-hydroxyl adenosine. In some embodiments, N is a non-3'-hydroxyl uridine. In some embodiments, N is a non-3'-hydroxyl guanosine.

[0166]

[0141] In some embodiments, N is 2',3'-dideoxycytosine.

[0167]

[0142] In some embodiments, m is 5, each R is adenosine, and each pcis independently a phosphorothioate.

[0168]

[0143] In some embodiments, m is 5, each R is adenosine, each pcis independently a phosphorothioate, R1is adenosine, and pdis a phosphorothioate.

[0169]

[0144] In some embodiments, m is 5, each R is adenosine, each pcis independently a phosphorothioate, R1is adenosine, pdis a phosphorothioate, and N is a dideoxynucleoside. In some embodiments, m is 5, each R is adenosine, each pcis independently a phosphorothioate, R1is adenosine, pdis a phosphorothioate, and N is a ddC or ddA. In some embodiments, m is 5, each R is adenosine, each pcis independently a phosphorothioate, R1is adenosine, pdis a phosphorothioate, and N is a ddC. In some embodiments, m is 5, each R is adenosine, each pcis independently a phosphorothioate, R1is adenosine, pdis a phosphorothioate, and N is a ddA. In some embodiments, m is 5, each R is adenosine, each pcis independently a phosphorothioate, R1is adenosine, pdis a phosphorothioate, and N is an inverted 2'-deoxynucleoside (InvdN).

[0170]

[0145] In some embodiments, N is abasic (e.g., lacks a nucleobase).

[0171]

[0146] In some embodiments, N is unlocked (e.g., comprises a ribose that lacks the C2'-C3'-bond of the ribose ring).

[0172]

[0147] In some embodiments, the ribose of nucleoside N is a spiro-ribose, a bridged-ribose, or a locked ribose. In some embodiments, the ribose of nucleoside N is a spiro-ribose. In some embodiments, the ribose of nucleoside N is a 2'-spiro-ribose. In some embodiments, the ribose of nucleoside N is a bridged-ribose. In some embodiments, N is a 2',4'-bridged-ribose. In some embodiments, the ribose of nucleoside N is a locked ribose.

[0173]

[0148] In some embodiments, N is unlocked (e.g., comprises a ribose that lacks the C2'-C3'-bond of the ribose ring).

[0149] In some embodiments, a nucleoside within six nucleosides of N comprises a 2' hydroxyl.

[0174]

[0150] In some embodiments, a nucleoside of the polynucleotide or oligonucleotide described herein is within six nucleosides of N and comprises a 2' hydroxyl. In some embodiments, the nucleoside within six nucleosides of N is on the polynucleotide or oligonucleotide described herein.

[0175]

[0151] In some embodiments, a nucleoside on a coding region of the polynucleotide or oligonucleotide described herein is within six nucleosides of N and comprises a 2' hydroxyl. In some embodiments, the nucleoside within six nucleosides of N is on the coding region of the polynucleotide or oligonucleotide described herein.

[0176]

[0152] In some embodiments, a nucleoside within six nucleosides upstream of R comprises a 2' hydroxyl.

[0177]

[0153] In some embodiments, a nucleoside of the polynucleotide or oligonucleotide described herein is within six nucleosides upstream of R and comprises a 2' hydroxyl. In some embodiments, the nucleoside within six nucleosides upstream of R is on the polynucleotide or oligonucleotide described herein.

[0178]

[0154] In some embodiments, a nucleoside on a coding region of the polynucleotide or oligonucleotide described herein is within six nucleosides upstream of R and comprises a 2' hydroxyl. In some embodiments, the nucleoside within six nucleosides upstream of R is on the coding region of the polynucleotide or oligonucleotide described herein.

[0179]

[0155] In some embodiments, a nucleoside within six nucleosides upstream of R1comprises a 2' hydroxyl.

[0180]

[0156] In some embodiments, a nucleoside of the polynucleotide or oligonucleotide described herein is within six nucleosides upstream of R1and comprises a 2' hydroxyl. In some embodiments, the nucleoside within six nucleosides upstream of R1is on the polynucleotide or oligonucleotide described herein.

[0181]

[0157] In some embodiments, a nucleoside on a coding region of the polynucleotide or oligonucleotide described herein is within six nucleosides upstream of R1and comprises a 2' hydroxyl. In some embodiments, the nucleoside within six nucleosides upstream of R1is on the coding region of the polynucleotide or oligonucleotide described herein.

[0158] In some embodiments, a nucleoside within six nucleosides upstream of R and R1comprises a 2' hydroxyl.

[0182]

[0159] In some embodiments, a nucleoside of the polynucleotide or oligonucleotide described herein is within six nucleosides upstream of R and R1and comprises a 2' hydroxyl. In some embodiments, the nucleoside within six nucleosides upstream of R and R1is on the polynucleotide or oligonucleotide described herein.

[0183]

[0160] In some embodiments, a nucleoside on a coding region of the polynucleotide or oligonucleotide described herein is within six nucleosides upstream of R and R1and comprises a 2' hydroxyl. In some embodiments, the nucleoside within six nucleosides upstream of R and R1is on the coding region of the polynucleotide or oligonucleotide described herein.

[0184]

[0161] In some embodiments, Z comprises a 2' hydroxyl.

[0185]

[0162] In some embodiments, an R, such as an R within six nucleosides of N, comprises a 2' hydroxyl.

[0186]

[0163] In some embodiments, R1comprises a 2' hydroxyl.

[0187]

[0164] In some embodiments, p' is bonded to a nucleoside that is natural or modified. In some embodiments, p' is bonded to an upstream nucleoside (e.g., Z or another nucleoside of the polynucleotide) that is natural or modified. In some embodiments, the modified nucleoside is not a locked nucleic acid. In some embodiments, the modified nucleoside is not substituted in the 2'- position with methoxy or methoxyethoxy (MOE).

[0188]

[0165] In some embodiments, a 3' chain terminating tail (e.g., a poly-A tail having a 3' chain terminating nucleotide) described herein comprises fifty nucleotides (50mer) or less. In some embodiments, a 3' chain terminating tail described herein comprises forty nucleotides (40mer) or less. In some embodiments, a 3' chain terminating tail described herein comprises thirty nucleotides (30mer) or less. In some embodiments, a 3' chain terminating tail described herein comprises twenty nucleotides (20mer) or less. In some embodiments, a 3' chain terminating tail described herein comprises fifteen nucleotides (15mer) or less. In some embodiments, a 3' chain terminating tail described herein comprises ten nucleotides (lOmer) or less.

[0189]

[0166] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence provided in Table 1, Table 2, or Table 3.

[0167] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 1.

[0190]

[0168] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 2.

[0191]

[0169] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 3.

[0192]

[0170] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 4.

[0193]

[0171] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 5.

[0194]

[0172] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 6.

[0195]

[0173] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 7.

[0196]

[0174] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 8.

[0197]

[0175] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 9.

[0198]

[0176] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 10.

[0199]

[0177] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 11.

[0200]

[0178] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 12.

[0201]

[0179] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 13.

[0202]

[0180] In some embodiments, the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 14.

[0181] In some embodiments, a polynucleotide described herein further comprises a 5'-cap structure. In some embodiments, a polynucleotide described herein further comprises a 5'-UTR. In some embodiments, a polynucleotide described herein further comprises a coding region. In some embodiments, a polynucleotide described herein further comprises a 3'-UTR. In some embodiments, a polynucleotide described herein further comprises a coding region and a 5'-cap structure. In some embodiments, a polynucleotide described herein further comprises a coding region and a 5'-UTR. In some embodiments, a polynucleotide described herein further comprises a coding region and a 3'-UTR. In some embodiments, a polynucleotide described herein further comprises at least one 5'-cap structure, a 5'-UTR, a coding region, and a 3'-UTR.

[0203]

[0182] In some embodiments, a polynucleotide described herein further comprises a poly-A. In some embodiments, the poly-A is connected to a 3' chain terminating nucleotide (e.g., dinucleotide) described herein. In some embodiments, the poly-A is connected 3' to 5' to a 3' chain terminating nucleotide described herein.

[0204]

[0183] Provided in some embodiments herein is a composition comprising a polynucleotide described herein and a lipid nanoparticle (LNP).

[0205]

[0184] Provided in certain embodiments herein are systems, compositions, and methods, such as methods and compositions useful for making RNA and products thereof. In specific embodiments, methods provided herein are methods of making RNA from an acceptor RNA polynucleotide and a donor RNA polynucleotide.

[0206]

[0185] Provide in some embodiments herein are methods of RNA ligation. In certain embodiments, the method of RNA ligation comprises contacting an RNA ligase enzyme with (i) an acceptor RNA polynucleotide and (ii) a donor RNA polynucleotide.

[0207]

[0186] In some embodiments, donor RNA polynucleotide provided herein comprises a modified 3' nucleotide (located at the terminal 3' position). In some embodiments, a donor RNA polynucleotide provided herein comprises three or more modified nucleotides (e.g., located at 3' terminal nucleotide positions). In some embodiments, the modified 3' nucleotide comprises a modification that prevents self-ligation of the donor RNA polynucleotide. In some embodiments, the modified 3' nucleotide lacks a 3' hydroxyl group (e.g., an inverted nucleotide or dideoxy nucleotide).

[0187] In some embodiments, the donor RNA polynucleotide comprises one or one more modified nucleotides (e.g., additionally located at non-terminal nucleotide positions).

[0208]

[0188] In some embodiments, the donor polynucleotide (e.g., RNA) comprises a poly-A polynucleotide, such as provided herein. In specific embodiments, the poly-A polynucleotide comprises a nucleobase sequence having 75% or greater adenine.

[0209]

[0189] In some embodiments, provided herein is a method comprising ligating an acceptor RNA polynucleotide provided herein and a donor RNA polynucleotide provided herein. In specific embodiments, contacting the acceptor RNA polynucleotide, the donor RNA polynucleotide and the ligation enzyme results in the ligation of the acceptor RNA polynucleotide with the donor RNA polynucleotide, such as when subject to suitable conditions for ligation.

[0210]

[0190] In some embodiments, acceptor RNA polynucleotide provided herein comprises a hydroxyl group at the 3' nucleotide. In some embodiments, contacting the acceptor RNA polynucleotide, the donor RNA polynucleotide and the ligation enzyme results in the ligation of the hydroxyl group of the 3' terminal nucleotide of the acceptor RNA polynucleotide with the p (e.g., phosphate) group of the 5' terminal nucleotide (Z) of the donor RNA polynucleotide, such as when subject to suitable conditions for ligation. In some instances, use of a (Z), such as using a single ring system or other small group, facilitates high ligation efficiency and / or improved reaction parameters and / or reduced amounts of PEG to allow post-ligation processing techniques.

[0211]

[0191] In certain embodiments, the acceptor RNA polynucleotide comprises a structure represented by the formula (pN)n. In some embodiments, (N) of the acceptor is NA.

[0212]

[0192] In some embodiments, each (NA) is independently a nucleotide (or nucleoside, as applicable) or a modified nucleotide (or nucleoside, as applicable). In some embodiments, any suitable modified nucleotide is used. In some embodiments, the (p) of the 5' terminal pN (pNA) is blocked, such as with a 5' CAP.

[0213]

[0193] In some embodiments, the acceptor RNA polynucleotide comprises a modified 5' nucleotide (located at the terminal 5' position). In some embodiments, the modified 5' nucleotide lacks a 5' phosphate group. In some embodiments, the 5' terminal nucleotide comprises a CAP structure.

[0194] In certain embodiments, the acceptor RNA polynucleotide comprises a modified 5' nucleotide, such as a modification that prevents self-ligation of the donor RNA polynucleotide.

[0214]

[0195] In some embodiments, the acceptor RNA polynucleotide comprises one or one more modified nucleotides (e.g., additionally located at non-terminal nucleotide positions).

[0215]

[0196] In some embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' cap structure, a 5' UTR, a protein coding sequence, or a 3' UTR sequence element. In specific embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' UTR. In specific embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' UTR and a protein coding sequence. In specific embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' UTR, a 3' UTR, and a protein coding sequence. In further embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' CAP.

[0216]

[0197] In certain embodiments, ligating the acceptor RNA polynucleotide and the donor RNA polynucleotide forms a structure represented by the formula (pN)n Z (pN)n.

[0217]

[0198] In some embodiments, provided herein is a method of making a (e.g., mRNA) polynucleotide. In some embodiments, the method provided herein comprises ligating any acceptor polynucleotide provided herein and any donor polynucleotide (e.g., RNA) provided herein. In some embodiments, the donor polynucleotide (e.g., RNA) comprises a poly-A polynucleotide. In specific embodiments, the poly-A polynucleotide comprises a nucleobase sequence having 75% or greater adenine. In some embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' cap structure, a 5' UTR, a protein coding sequence, or a 3' UTR sequence element. In certain embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' cap structure, a 5' UTR, a protein coding sequence, and a 3' UTR sequence elements.

[0218]

[0199] In some embodiments, a donor polynucleotide (e.g., RNA) provided herein comprises a multi-terminus (e.g., branched) polynucleotide. In some certain embodiments, a donor polynucleotide (e.g., RNA) provided herein comprise a first one or more (e.g., 1-8) poly-A polynucleotides (e.g., branches) linked to a second the donor poly-A polynucleotide (e.g., backbone polynucleotide). For example, a branch poly-A polynucleotide and the donor polynucleotide can each contain a functionalized nucleotide, wherein the functionalized nucleotides comprise a modification that permits linkage (e.g., conjugation). Suitable functionalized nucleotides include those known in the art, for example and not limited to, a first functionalized nucleotide having an azide modification and a second functionalized nucleotide having an alkyne modification, facilitating a linkage to the linker through a triazole linkage. Such functionalized nucleotides can comprise the functionalizing modification within the base, sugar- backbone (e.g., 2' or 3' position), or nucleotide phosphate (e.g., 5' or 3' modifications). In certain embodiments, a branch can have one or more poly-A polynucleotides linked to the branch polynucleotide, yielding a donor poly-A polynucleotide linked to one or more (e.g., 1-8) branch poly-A polynucleotides, wherein the branch poly-A polynucleotides are further linked to one or more (e.g., 1-8) poly-A polynucleotides.

[0219]

[0200] In some embodiments, a ligation method provided herein performed in a composition comprising a vehicle, such as described herein. In some embodiments, the vehicle is an aqueous vehicle, such as comprising a buffer. In specific embodiments, a vehicle comprises a PEG, such as in any concentration described herein.

[0220]

[0201] In some embodiments, a ligation method provided herein is performed in a reaction vehicle comprising less than 20% polyethylene glycol (PEG). In some embodiments, a ligation method provided herein is performed in a reaction vehicle comprising less than 15% polyethylene glycol (PEG). In some embodiments, a ligation method provided herein is performed in a reaction vehicle comprising less than 10% polyethylene glycol (PEG). In some embodiments, a ligation method provided herein is performed in a reaction vehicle comprising less than 5% polyethylene glycol (PEG). In some embodiments, a ligation method provided herein is performed in a reaction vehicle comprising less than 2% polyethylene glycol (PEG). In some embodiments, a ligation method provided herein performed in a reaction vehicle is substantially free of PEG.

[0221]

[0202] In some embodiments, any suitable polyethylene glycol (PEG) is used in any methods or compositions provided herein. In some embodiments, the polyethylene glycol (PEG) is a high molecular weight PEG (e.g., having an average molecular mass > 1000). In specific embodiments, the polyethylene glycol (PEG) is PEG8000.

[0222]

[0203] In some embodiments, a ligation method provided herein is performed at a total polynucleotide concentration of at least 0.2 mg / mL (e.g., in the composition comprising the vehicle). In some embodiments, a ligation method provided herein is performed at a total

[0223] -M- polynucleotide concentration of at least 0.5 mg / mL (e.g., in the composition comprising the vehicle). In some embodiments, a ligation method provided herein is performed at a total polynucleotide concentration of at least 1 mg / mL (e.g., in the composition comprising the vehicle).

[0224]

[0204] In some embodiments, provided herein is a composition comprising any acceptor polynucleotide provided herein or donor polynucleotide provided herein. In specific embodiments, the composition comprises any acceptor polynucleotide provided herein and donor polynucleotide provided herein. In some embodiments, provided herein is a composition comprising a polynucleotide provided herein (e.g., a polynucleotide described herein producible by ligating a donor and an acceptor described herein). In certain embodiments, provided herein is a composition comprising a polynucleotide provided herein (e.g., a polynucleotide described herein producible by ligating a donorand an acceptor described herein), a donor described herein and an acceptor described herein. In certain embodiments, a composition provided herein comprises an enzyme.

[0225]

[0205] In some embodiments, a composition provided herein is performed in a reaction vehicle comprising less than 20% polyethylene glycol (PEG). In some embodiments, a composition provided herein is performed in a reaction vehicle comprising less than 15% polyethylene glycol (PEG). In some embodiments, a composition provided herein is performed in a reaction vehicle comprising less than 10% polyethylene glycol (PEG). In some embodiments, a composition provided herein is performed in a reaction vehicle comprising less than 5% polyethylene glycol (PEG). In some embodiments, a composition provided herein is performed in a reaction vehicle comprising less than 2% polyethylene glycol (PEG). In some embodiments, a composition provided herein performed in a reaction vehicle is substantially free of PEG.

[0226]

[0206] In some embodiments, any suitable RNA ligase enzyme is used in any method provided herein. In specific embodiments, an RNA ligase enzyme is a T4 RNA ligase. In specific embodiments, the T4 RNA ligase is a T4 RNA ligase 1.

[0227]

[0207] In some embodiments, each modified nucleotide provided herein independently comprises any suitable modified nucleotide, such as wherein each modified nucleotide may independently comprise a modified nucleobase, modified sugar (e.g., ribose), modified phosphate, or a combination thereof. In some embodiments, a modified nucleotide provided herein comprises a modified nucleobase.

[0228]

[0208] In some embodiments, a modified nucleobase provided herein is any suitable modified nucleobase, such as modified nucleobase selected from the group consisting of 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- lndolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5- bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-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, N1 -ethylpseudouracil, Nl- methoxy methylpseudouracil, Nl -methyladenine, Nl -methylpseudouracil, Nl- 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).

[0229]

[0209] In some embodiments, a modified nucleotide provided herein comprises a modified sugar. In some embodiments, a modified sugar provided herein is any suitable modified sugar, such as a modified sugar selected from the group consisting of 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- methylnbose, 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.

[0230]

[0210] In some embodiments, a modified nucleotide provided herein comprises a 2' modification. In some embodiments, a 2' modification provided herein is any suitable 2' modification, such as a 2' modification selected from the group consisting of a locked- nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F) , 2'-O-methoxy-ethyl (2'-MOE), and 2'- O- methylation (2'-0Me).

[0231]

[0211] In some embodiments, at least one modified nucleotide comprises a modified phosphate. In some embodiments, a modified phosphate provided herein is any suitable modified phosphate, such as a modified phosphate from the group consisting of phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O-methylphosphonate, 3'-O- methylphosphonate, 5'- hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0232]

[0212] In some embodiments, the 3' terminal nucleotide of the polynucleotide (e.g., RNA) does not comprise hydroxy at the 3' position of the 3' terminal nucleotide.

[0213] In some embodiments, the 3' terminal nucleotide of the donor polynucleotide does not comprise hydroxy at the 3' position of the 3' terminal nucleotide.

[0233]

[0214] In some embodiments, the 3' terminal nucleotide of the polynucleotide (e.g., RNA) comprises an inverted nucleotide.

[0234]

[0215] In some embodiments, the 3' terminal nucleotide of the donor polynucleotide comprises an inverted nucleotide.

[0235]

[0216] In some embodiments, the 3' terminal nucleotide of the polynucleotide (e.g., RNA) comprises a dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted-deoxythymidine. In some embodiments, the 3' terminal nucleotide of the polynucleotide (e.g., RNA) comprises a dideoxycytidine.

[0236]

[0217] In some embodiments, the 3' terminal nucleotide of the donor polynucleotide comprises a dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted-deoxythymidine. In some embodiments, the 3' terminal nucleotide of the donor polynucleotide comprises a dideoxycytidine.

[0237]

[0218] In some embodiments, a polynucleotide (e.g., RNA) provided herein comprises a 5' CAP. In some embodiments, the acceptor polynucleotide comprises a 5' CAP. In some embodiments, a 5' CAP provided herein comprises a 7-methylguanosine.

[0238]

[0219] In some embodiments, a 5' CAP provide herein further comprises one or more phosphates connecting the 7- methylguanosine to an adjacent nucleotide of the modified mRNA. In some embodiments, the 5' CAP comprises a 3'-O-Me-m7G(5')ppp(5')G.

[0239]

[0220] In some embodiments, one or more phosphates of the 5' cap provided herein is a modified phosphate selected from the group consisting of phosphorothioate, triazole ring, dihalogenmethylenebisphosphonate, imidodiphosphate, and methylenebis (phosphonate).

[0240]

[0221] In some embodiments, provided herein is a polynucleotide (e.g., RNA, such as a messenger ribonucleic acid (mRNA)) comprising a poly-A tail (e.g., a poly-A region). In some embodiments, a poly-A tail provided herein comprises a structure represented by the formula:

[0241]

[0222] (pA)n1Z (pA)n1.

[0242]

[0223] In some embodiments, each (A) is independently an adenine nucleotide or a modified adenine nucleotide. In some embodiments, each (n1) is an integer equal to or greater than 1.

[0224] I n some embodiments, a poly-A region (e.g., of a branch) or poly-A region provided herein comprises about 25 to about 500 nucleotides. In some embodiments, the poly-A region comprises about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to 300 nucleotides, about 300 to about 400 nucleotides, or about 400 to about 500 nucleotides. In some embodiments, the poly-A region comprises about 10 or more adenosine nucleotides. In some embodiments, about 25% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100% of nucleotides of the poly-A region are adenosine nucleotides. In some embodiments, the first poly-A tail comprises about 10 to about 50 adenosine ribonucleotides, about 1 to about 10 modified uridine deoxyribonucleotides comprising an attachment point, and a 3' terminal dideoxyribonucleotide or inverted deoxyribonucleotide; and the second poly-A tails comprises about 10 to about 50 adenosine ribonucleotides.

[0243]

[0225] In some embodiments, a modified nucleotide of an acceptor polynucleotide, a donor polynucleotide, or a polynucleotide (e.g., having a ligated acceptor and donor) provided herein comprises a linker attached to a poly-A or is modified with a linker capable of being coupled to a poly-A, such as described herein (e.g., thereby producing a poly-A branch of the nucleotide or polynucleotide). In some embodiments, at least 2 modified nucleotides (N) comprise a linker attached to a poly-A or is modified with a linker capable of being coupled to a poly-A, such as described herein. In some embodiments, the poly-A region is attached or capable of being attached to the linker at the 3' nucleotide of the poly-A region.

[0244]

[0226] In some embodiments, any suitable modified nucleotides, linkers, poly-A modifications, and click chemistries can be used herein, such as those described in WO2023141474, which is incorporated herein by reference for such disclosures.

[0245] EXAMPLES EXAMPLE 1: RNA ligation using the methods and modified nucleotides described herein exhibit good efficiency even when conducted in mediums with low PEG8000 concentrations.

[0246]

[0227] RNA ligation typically requires PEG8000 to achieve a high efficiency. However, PEG8000 is a highly viscous reagent which can interfere with the provision of consistent reaction conditions and cleaning of the reaction system. In addition, a high volume of PEG8000 dilutes the reaction system, limiting the scale of production. A comparison of ligation efficiencies was determined for oligos 5' terminal adenosine vs. cytidine (FIG. 1) at different PEG8000 concentrations.

[0247]

[0228] The study was conducted by testing the chemically modified oligos described in TABLE 1 below.

[0248]

[0229] TABLE 1

[0249] RNA nucleotides: rN; RNA phosphorothioate nucleotide: rN*; 5' Phosphate modification: / 5Phos / ; 2'-3'-dideoxycytidine (ddC) nucleotide: / 3ddC /

[0250]

[0230] The ligation reaction was evaluated by RNase H assay, and the results are provided in FIG. 1, panel A. In this assay, an oligo containing DNA bases was hybridized with 3'UTR of the mRNA polynucleotides of TABLE 1, and then cleaved with RNase H to release a part of 3'UTR with poly- A for further analysis with PAGE gel. The ligation of an oligo to the 3' end of the poly-A tail was expected to lead to a shift of the cleaved tail to a higher molecular weight. The ligations were conducted at PEG8000 concentrations of 15%, 10% and 5%.

[0251]

[0231] Reduction of PEG8000 reduced the ligation efficiency for oligos with 5' rA (adenosine), whereas the ligation efficiency remained constant for oligos with 5' rC (cytidine) (FIG. 1A). This result showed that replacing the 5' nucleotide from the larger rA to the less sterica lly hindered rC substantially and advantageously improved ligation efficiency, allowing the ligation reactions take place using lower PEG concentrations. As can be seen from left to the right in the lanes, as PEG concentration decreased, the oligo bearing 5' rA exhibited decreasing ligation efficiency, whereas the ligation efficiency of the oligo bearing 5' rC ligation was preserved.

[0252]

[0232] Cellular expressions of luciferase by different constructs comprising the modified polynucleotides of TABLE 1 were measured in HepG2 cells at days 1-4. The results are shown in FIG. IB. In the top chart of FIG. IB, the expression is normalized to a mock ligation control for each day. In the bottom chart of FIG. IB, the expression is normalized to day 1 for each construct.

[0253]

[0233] No difference in expression level and stability was observed between the mRNAs ligated with 5' rA and 5' rC oligos, confirming that the 5' rC of the donor oligo did not interfere with the biological performance of the mRNA ligated with the modified oligos.

[0254]

[0234] Also provided in certain embodiments herein are systems, compositions, and methods, such as methods and compositions useful for making RNA and products thereof. In specific embodiments, methods provided herein are methods of making RNA from an acceptor RNA polynucleotide and a donor RNA polynucleotide.

[0255]

[0235] In some embodiments, provided herein are messenger ribonucleic acid (mRNA) polynucleotides comprising a poly-A tail, wherein the poly-A tail comprises a terminal 3' nucleotide comprising (a) a 5' phosphorothioate linkage and (b) a nucleoside lacking a 3' hydroxyl group (in some instances, referred to herein as a free 3' hydroxyl, e.g., indicating that the 3' position is not hydroxyl and is not connected to another nucleotide). In certain embodiments, the poly-A tail further comprises (i) a 5' nucleotide comprising a monocyclic nucleobase. To be clear, in some instances, such a poly-A tail may be attached to an additional poly-A group, such as wherein the poly-A tail was attached via a ligation technique to an existing 3'-poly-A group of an mRNA. Also provided in certain embodiments herein are poly-A tails (e.g., wherein the poly-A tail is not connected to other mRNA elements, such as until after ligation) useful in the synthesis of an mRNA molecule via ligation, wherein the poly-A tails comprise (i) a terminal 5' nucleotide comprising a monocyclic nucleobase and free 5' phosphate, and (ii) a terminal 3' nucleotide comprising (a) a 5' phosphorothioate linkage and (b) a nucleoside lacking a free 3' hydroxyl.

[0256]

[0236] In certain embodiments, provided herein is an mRNA comprising any poly-A tail provided herein. In some embodiments, the mRNA comprises a second poly-A region. In some embodiments, the poly-A tail is connected (e.g., through the 5'-chain terminating nucleotide of the poly-A tail) to the second poly-A region. In certain embodiments, the combined poly-A tail and second poly-A region comprises any suitable number of adenine nucleobases. In certain embodiments, the poly-A tail comprises about 10-400 nucleotides (including, e.g., 10-200, 10- 150, 10-100, 10-50, 10-25, or 10-20 nucleotides). In certain embodiments, the poly-A tail comprises 10-250 nucleotides (including, e.g., 10-200, 10-150, 10-100, 10-50, 10-25, or 10-20). In some embodiments, any poly-A tail provided herein comprises a 5' (when not attached to a larger mRNA structure) nucleotide that comprises a monocyclic nucleobase. In certain embodiments, the monocyclic nucleobase is a pyrimidine nucleobase. In certain embodiments, the monocyclic nucleobase is cytidine or a modified cytidine (e.g., m5C, hm5C, f5C, etc.). In certain embodiments, the terminal 3' nucleotide also lacks a 2' hydroxyl (is a dideoxy nucleotide). In certain embodiments, the monocyclic nucleobase is cytidine.

[0257]

[0237] In some embodiments, any poly-A tail provided herein comprises a 5'-chain terminating (when not attached to a larger mRNA structure) abasic nucleotide (e.g., wherein the base is replaced with any suitable group, such as described herein). In some embodiments, the abasic nucleotide is substituted at the 1' position with a group that is smaller than a bicyclic base, such as a purine base. In some embodiments, the abasic nucleotide is substituted at the 1' position with H (at both 1' positions), OR0, NR°2, alkyl (saturated or unsaturated), heteroalkyl, aryl, heteroaryl, cycloaklyl, or heterocyclyl, such as wherein each R° is independently H, alkyl (saturated or unsaturated), heteroalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. In some embodiments, each alkyl (saturated or unsaturated), heteroalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl is substituted or unsubstituted. In some embodiments, such a substituent is a C1-C3 alkyl.

[0258]

[0238] Provided in some embodiments herein, are methods of RNA ligation. In certain embodiments, the method of RNA ligation comprises contacting an RNA ligase enzyme with (i) an acceptor RNA polynucleotide and (ii) a donor RNA polynucleotide.

[0259]

[0239] In certain embodiments, provided herein is a polynucleotide having a structure of N(pNA)nZ (pND)n. In some embodiments, ligating the acceptor RNA polynucleotide and the donor RNA polynucleotide forms a structure represented by the formula: (pN)nZ (pN)n. In some embodiments, ligating the acceptor RNA polynucleotide and the donor RNA polynucleotide forms a structure represented by the formula: (p-N)n-p-Z'-(p-N)n.

[0260]

[0240] In some embodiments, donor RNA polynucleotide provided herein comprises a structure represented by the formula Z (pN)n. In some embodiments, a donor RNA polynucleotide provided herein comprises a structure represented by the formula Z pZ2 (pN)n- In some embodiments, a donor RNA polynucleotide provided herein comprises a structure represented by the formula p'- Z'-p-Z2-(p-N)n. In some embodiments, (N) of the donor is ND.

[0261]

[0241] In some embodiments, each nucleoside described herein independently comprises a ribose or analog thereof.

[0262]

[0242] In some embodiments, a nucleoside (e.g., of any formula described herein) comprises a ribose. In some embodiments, each nucleoside (e.g., of any formula described herein) comprises a ribose.

[0263]

[0243] In some embodiments, any nucleoside provided herein (e.g., of any formula described herein), such as a 3' chain terminating nucleoside of a poly-A tail or N of formula (I), comprises a ribose analog, such as any suitable ribose analog (e.g., for the purposes provided herein). In some embodiments, the ribose analog is a locked ribose, an unlocked ribose, a spiro-ribose, or a deoxyribose (e.g, 3' deoxy, 2' deoxy). In some embodiments, the ribose analog is deoxyribose. In certain embodiments, the deoxy ribose is a 2' deoxyribose. In certain embodiments (e.g., in certain chain terminating nucleosides), the deoxyribose is a 3' deoxyribose, n certain embodiments (e.g., in certain chain terminating nucleosides), the ribose analog is dideoxyribose. In some embodiments, the ribose analog is a furanose or other cyclized sugar, such as a pyranose (e.g., glucose). In some embodiments, the ribose analog is a nitrogen-containing heterocycle (e.g., wherein the ring has a nitrogen instead of or in addition to the ribose oxygen). In some embodiments, the ribose analog a heteroribose (e.g., wherein the furanose of the ribose has the oxygen replaced with a nitrogen (e.g., NR°) group, and / or has a carbon replaced with a nitrogen (e.g., NR°) group), a locked ribose (e.g., wherein the 2' position of the ribose is bridged with the 4' position of the ribose), or an unlocked ribose (e.g., wherein the ribose ring is opened). In some embodiments, each nucleoside (e.g., of any formula described herein) independently comprises a heteroribose, a locked ribose, or an unlocked ribose. In some instances, an unlocked ribose comprises an acyclic structure group, rather than a cyclized group (e.g., ribose). In some instances, a ribose analog comprises a furanose structure and a methylene, irrespective of further hydroxyl substitution (but is not ribose).

[0264]

[0244] In some embodiments, any nucleoside provided herein, unless otherwise limited, may have any suitable structure. In some embodiments, a nucleoside provided herein is natural or modified. In some embodiments, a nucleoside provided here in comprises a nucleobase or may be abasic, such as having an abasic structure described herein.

[0265]

[0245] In some embodiments, a ribose analog described herein, unless otherwise limited, may include structures where a ribose ring methylene is added (e.g., glucose) or removed (e.g., and replaced or substituted with a heteroatom).

[0266]

[0246] In some embodiments, each nucleoside (e.g., of any formula described herein) independently comprises a ribose or a ribose analog. In specific embodiments, all nucleosides of an mRNA or poly-A tail or other structure provided herein comprises a ribose.

[0267]

[0247] In some embodiments, each nucleoside (e.g., of any formula described herein) independently comprises a ribose, a heteroribose, a locked ribose, or an unlocked ribose.

[0268]

[0248] In some embodiments, a Z described herein (e.g., Z, Z', or Z2) is a nucleoside. In some embodiments, a Z described herein (e.g., Z, Z', or Z2) is a nucleoside comprising a monocyclic base, such as a natural or modified pyrimidine base (e.g., cytosine or uracil). In some embodiments, a Z described herein (e.g., Z, Z', or Z2) is a natural nucleoside. In some embodiments, a Z described herein (e.g., Z, Z', or Z2) is a modified nucleoside. In some embodiments, a Z described herein (e.g., Z, Z', or Z2) is a monocyclic nucleoside. In some embodiments, a Z described herein (e.g., Z, Z', or Z2) is an abasic nucleoside.

[0269]

[0249]

[0270]

[0250] In some embodiments, Z is a nucleoside comprising a monocyclic nucleobase. In some embodiments, Z is cytidine (C) or an analog thereof. In some embodiments, Z is cytidine (C). In some embodiments, Z is a cytidine (C) analog.

[0271]

[0251] In some embodiments, the ribose of Z is substituted with a hydrogen and a second group R4, wherein R4is a hydrogen, a halogen, a hydroxyl, a substituted or unsubstituted alkoxy, a substituted or unsubstituted heteroalkoxy, or a substituted or unsubstituted alkoxy. In some embodiments, R4is hydroxyl.

[0272]

[0252] I n some embodiments, Z is a nucleoside substituted at the 1-position with a hydrogen and B, wherein B is hydrogen, Cz-Cealkynyl, C2-Csheterocycloalkyl, aryl, or heteroaryl, and wherein C2- Cealkynyl, C2-Csheterocycloalkyl, aryl, and heteroaryl are unsubstituted or substituted.

[0273]

[0253] In some embodiments, a Z described herein (e.g., Z, Z', or Z2) comprises any nucleobase described herein, such as (B).

[0274]

[0254] In some embodiments, a Z described herein (e.g., Z, Z', or Z2) comprises a nucleoside having a structure represented by the formula: iments, (Z) is a nucleotide having a structure represented by the formula:

[0275]

[0256] In some embodiments, (Z) is a modified nucleotide (e.g., the having a base modification, sugar modification, and / or inter-nucleotide linkage modification).

[0276]

[0257] In some embodiments, (Z) is a modified nucleotide represented by the formula: (pZ'). In some embodiments, (Z) is a modified nucleotide represented by the formula: (p'-Z'), wherein p' is a terminal phosphate. In some embodiments, (Z') is represented by the formula:

[0258] In some embodiments, Z' is cytidine or an analog thereof. In some embodiments, Z' is cytidine.

[0277]

[0259] In some embodiments, donor RNA polynucleotide provided herein comprises a structure represented by the formula p'-Z'-(p-N)n.

[0278]

[0260] I n some embodiments, (Z2) is a nucleoside having a structure represented by the formula:

[0279]

[0261]

[0280]

[0262] I n some embodiments, (Z2) is a nucleoside having a structure represented by the formula:

[0281]

[0263]

[0282]

[0264] In some embodiments, (Z2) is a modified nucleoside.

[0283]

[0265] In some embodiments, (Z) and (pZ2) are the same nucleotide or the same modified nucleotide.

[0284]

[0266] In some embodiments, (Z) and (Z2) are different nucleotides or modified nucleotides.

[0285]

[0267] In some embodiments, (Z') and (pZ2) are the same nucleotide or the same modified nucleotide.

[0286]

[0268] In some embodiments, (Z') and (Z2) are different nucleotides or modified nucleotides.

[0287]

[0269] In some embodiments, (B) is hydrogen, C2-C6alkynyl, Cz-Csheterocycloalkyl, aryl, or heteroaryl, wherein C2-C6alkynyl, C2-C8heterocycloalkyl, aryl, and heteroaryl are unsubstituted or substituted (e.g., with m R1). In some embodiments, B is 5- to 6-membered aryl or 5- to 6- membered heteroaryl, each of which is independently substituted or unsubstituted. In some embodiments, wherein B is unsubstituted C2-Cealkynyl, C2-Csheterocycloalkyl, aryl, or heteroaryl. In some embodiments, wherein B is substituted (e.g., with m R1) C2-Csalkynyl, C2- Csheterocycloalkyl, aryl, or heteroaryl. In some embodiments, B is a pyrimidine nucleobase. In some embodiments, B is cytosine.

[0288]

[0270] In some embodiments, B is monocyclic C2-C8heterocycloalkyl, monocyclic aryl, or monocyclic heteroaryl. In some embodiments, B is a monocyclic C2-C8heterocycloalkyl. In some embodiments, B is monocyclic monocyclic aryl. In some embodiments, B is a monocyclic heteroaryl.

[0289]

[0271] In some embodiments, B is a substituted or unsubstituted 5- or 6-membered aryl or a substituted or unsubstituted 5- or 6-membered heteroaryl. In some embodiments, B is a substituted or unsubstituted 5- or 6-membered aryl. In some embodiments, B is a substituted or unsubstituted 5- or 6-membered heteroaryl.

[0290]

[0272] In some embodiments, (B) is , 2,4-difluorotoluene, phenyl substituted with m R1, phenyl substituted with m R1, 5-nitroindole, or N4-hydrocytidine.

[0291]

[0273] In some embodiments, (B) is

[0292]

[0274] In some embodiments, B is a pyrimidine nucleobase. In some embodiments, B is cytosine.

[0293]

[0275] In some embodiments, (B) is any one structure of Table 1.

[0294] Table 1

[0295]

[0276] In some embodiments, each R1is independently hydrogen, halogen, -CN, -NO2, -NH-OH, -OH, -ORa, -NRcRd, -NRbC(=O)Ra, -C(=O)Ra, -C(=O)NRcRd, substituted or unsubstituted Ci-C6alkyl, orsubstituted or unsubstituted Ci-Ceheteroalkyl. In some embodiments, each R1is independently hydrogen, -NO2, -OH, -ORa, -NRcRd, -NRbC(=O)Ra, substituted or unsubstituted Ci-Cealkyl, or substituted or unsubstituted Ci-Ceheteroalkyl. In some embodiments, each R1is independently hydrogen, -NH2-NO2, -NH-OH, -OH, -OCH3, -CH3, -CH2OH, -N(H)CH3, or -N(H)C(=O)CH3. In some embodiments, two R1on the same atom are taken together to form an oxo.

[0296]

[0277] In some embodiments, m is 0, 1, 2, or 3.

[0297]

[0278] In some embodiments, R4is hydrogen, halogen, -OH, -ORa, or -NRcRd.

[0298]

[0279] In some embodiments, R4is OCH3.

[0299]

[0280] In some embodiments, R4is F.

[0300]

[0281] In some embodiments, each Rais independently substituted or unsubstituted Ci-C6alkyl.

[0301]

[0282] In some embodiments, each Rbis independently hydrogen or substituted or unsubstituted Ci-Cealkyl.

[0302]

[0283] In some embodiments, each Rcand Rdare independently hydrogen or substituted or unsubstituted Ci-Cealkyl. In some embodiments, Rcand Rdare taken together with the atom to which they are attached to form a substituted or unsubstituted heterocycloalkyl.

[0284] In some embodiments, each (p) is independently an inter-nucleoside linkage (e.g., phosphate or phosphorothioate linkage). In some embodiments, (p) is a phosphate or a modified phosphate, such as a modified phosphate from the group consisting of phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O-methylphosphonate, 3'-O- methylphosphonate, 5'- hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0303]

[0285] In some embodiments, each (N) is independently a nucleotide (or nucleoside, as applicable) or a modified nucleotide (or nucleoside, as applicable). In some embodiments, (N) is a natural nucleotide (or nucleoside, as applicable). In some embodiments, (N) is a modified nucleotide (or nucleoside, as applicable). In some embodiments, each nucleotide is independently selected from the group consisting of adenine (A), cytosine (C), guanine (G) and uracil (U). In some embodiments, any suitable modified nucleotide is used. In some embodiments, exemplary modified nucleotide includes those described herein.

[0304]

[0286] In some embodiments, each (ND) is independently nucleotide (or nucleoside, as applicable) or a modified nucleotide (or nucleoside, as applicable). In some embodiments, any suitable modified nucleotide is used. In some embodiments, at least one (ND) is adenosine (A). In some embodiments, at least 5 (ND) are adenosine (A). In some embodiments, at least 10 (ND) are adenosine (A). In some embodiments, at least 50% of (ND) are adenosine (A). In some embodiments, at least 80% of (ND) are adenosine (A). In some embodiments, at least 90% of (ND) are adenosine (A).

[0305]

[0287] In some embodiments, the 3' terminal (ND) is a dideoxy nucleotide or an inverted nucleotide.

[0306]

[0288] In some embodiments, each n is independently an integer of of 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 25, 10 to 20, or 10 to 15. In certain embodiments, each n is independently an integer of 10 to 200. In certain embodiments, each n is independently an integer of 10 to 150. In certain embodiments, each n is independently an integer of 10 to 100. In certain embodiments, each n is independently an integer of 10 to 50. In certain embodiments, each n is independently an integer of 10 to 25. In certain embodiments, each n is independently an integer of 10 to 20. In certain embodiments, each n is independently an integer of 10 to 15.

[0307]

[0289] In some embodiments, each n is independently an integer equal to or greater than 5. In some embodiments, each n is independently an integer equal to or greater than 6. In some embodiments, each n is independently an integer equal to or greater than 8. In some embodiments, each n is independently an integer equal to or greater than 10. In some embodiments, each n is independently an integer equal to or greater than 12. In some embodiments, each n is independently an integer equal to or greater than 14. In some embodiments, each n is independently an integer equal to or greater than 16.

[0308]

[0290] In some embodiments, each n is independently an integer equal to or less than 5. In some embodiments, each n is independently an integer equal to or less than 6. In some embodiments, each n is independently an integer equal to or less than 8. In some embodiments, each n is independently an integer equal to or less than 10. In some embodiments, each n is independently an integer equal to or less than 12. In some embodiments, each n is independently an integer equal to or less than 14. In some embodiments, each n is independently an integer equal to or less than 16. In some embodiments, each n is independently an integer equal to or less than 18. In some embodiments, each n is independently an integer equal to or less than 20.

[0309]

[0291] In some embodiments, n is an integer of 5 to 20. In some embodiments, n is an integer of 10 to 16. In some embodiments, n is an integer of 12 to 16.

[0310]

[0292] In some embodiments, n is 10. In some embodiments, n is 11. In some embodiments, n is 12. In some embodiments, n is 13. In some embodiments, n is 14. In some embodiments, n is 15. In some embodiments, n is 16.

[0311]

[0293] In some embodiments, donor RNA polynucleotide provided herein comprises a modified 3' nucleotide (located at the terminal 3' position).

[0312]

[0294] In some embodiments, a donor RNA polynucleotide provided herein comprises three or more modified nucleotides (e.g., located at 3' terminal nucleotide positions).

[0313]

[0295] I n some embodiments, the modified 3' nucleotide comprises a modification that prevents self-ligation of the donor RNA polynucleotide.

[0296] In some embodiments, the modified 3' nucleotide lacks a 3' hydroxyl group (e.g., an inverted nucleotide or dideoxy nucleotide). In some embodiments, the modified 3' nucleotide is a non-hydroxyl nucleoside. In some embodiments, the modified 3' nucleotide is a dideoxynucleoside (e.g., ddC or ddA). In some embodiments, the modified 3' nucleotide is an inverted nucleoside (e.g., wherein N is attached at the 3' position to the immediately upstream internucleotide linker). In some embodiments, the modified 3' nucleotide is an inverted 2'- deoxynucleoside (InvdN) (e.g., inverted 2'-deoxythymidine (InvdT), inverted 2'-deoxycytidine (InvdC) or inverted 2'-deoxyadenosine (InvdA)).

[0314]

[0297] In some embodiments, (Z') is cytidine, n is an integer of 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 25, 10 to 20, or 10 to 15, and the donor RNA polynucleotide comprises a modified 3' nucleotide located at the terminal 3' position. In some embodiments, (Z') is cytidine, n is an integer of 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 25, 10 to 20, or 10 to 15, and the donor RNA polynucleotide comprises a dideoxynucleoside (e.g., ddC or ddA) at the terminal 3' position. In some embodiments, (Z') is cytidine, n is an integer of 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 25, 10 to 20, or 10 to 15, and the donor RNA polynucleotide comprises ddC at the terminal 3' position.

[0315]

[0298] In some embodiments, (Z') is cytidine, n is an integer of 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 25, 10 to 20, or 10 to 15, the donor RNA polynucleotide comprises a dideoxynucleoside (e.g., ddC or ddA) located at the terminal 3' position, and each other N of the donor RNA polynucleotide is adenosine.

[0316]

[0299] In some embodiments, (Z') is cytidine, n is an integer of 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 25, 10 to 20, or 10 to 15, the donor RNA polynucleotide comprises a dideoxynucleoside (e.g., ddC or ddA) located at the terminal 3' position, each other N of the donor RNA polynucleotide is adenosine, and each p within 1 to 6 nucleosides of the terminal 3' position is a phosphorothioate linkage. In some embodiments, (Z') is cytidine, n is an integer of 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 25, 10 to 20, or 10 to 15, the donor RNA polynucleotide comprises a dideoxynucleoside (e.g., ddC or ddA) located at the terminal 3' position, each other N of the donor RNA polynucleotide is adenosine, and each p within 1 to 6 nucleosides of the terminal 3' position is a phosphorothioate linkage and the remaining p groups of the donor RNA polynucleotide are phosphate linkages.

[0317]

[0300] In some embodiments, the modified 3' nucleotide is inverted 2'-deoxythymidine (InvdT). In some embodiments, the modified 3' nucleotide is a nucleoside substituted at the 2'-position with a non-hydroxyl. In some embodiments, a nucleoside within six upstream nucleosides of the modified 3' nucleotide comprises a 2' hydroxyl. In some embodiments, the upstream nucleoside to which p is bonded is natural or modified, wherein the modified nucleoside is not: (i) a locked nucleic acid or (ii) substituted in the 2'-position with methoxy or methoxyethoxy (MOE).

[0318]

[0301] In some embodiments, a 3' chain terminating tail described herein comprises fifty nucleotides (50mer) or less. In some embodiments, a 3' chain terminating tail described herein comprises forty nucleotides (40mer) or less. In some embodiments, a 3' chain terminating tail described herein comprises thirty nucleotides (30mer) or less. In some embodiments, a 3' chain terminating tail described herein comprises twenty nucleotides (20mer) or less. In some embodiments, a 3' chain terminating tail described herein comprises fifteen nucleotides (15mer) or less. In some embodiments, a 3' chain terminating tail described herein comprises ten nucleotides (lOmer) or less.

[0319]

[0302] In some embodiments, the donor RNA polynucleotide comprises one or one more modified nucleotides (e.g., additionally located at non-terminal nucleotide positions).

[0320]

[0303] In some embodiments, the donor polynucleotide (e.g., RNA) comprises a poly-A polynucleotide, such as provided herein. In specific embodiments, the poly-A polynucleotide comprises a nucleobase sequence having 75% or greater adenine.

[0321]

[0304] In some embodiments, provided herein is a method comprising ligating an acceptor RNA polynucleotide provided herein and a donor RNA polynucleotide provided herein. In specific embodiments, contacting the acceptor RNA polynucleotide, the donor RNA polynucleotide and the ligation enzyme results in the ligation of the acceptor RNA polynucleotide with the donor RNA polynucleotide, such as when subject to suitable conditions for ligation.

[0322]

[0305] In some embodiments, acceptor RNA polynucleotide provided herein comprises a hydroxyl group at the 3' nucleotide. In some embodiments, contacting the acceptor RNA polynucleotide, the donor RNA polynucleotide and the ligation enzyme results in the ligation of the hydroxyl group of the 3' terminal nucleotide of the acceptor RNA polynucleotide with the p (e.g., phosphate) group of the 5' terminal nucleotide (Z) of the donor RNA polynucleotide, such as when subject to suitable conditions for ligation. In some instances, use of a (Z), such as using a single ring system or other small group, facilitates high ligation efficiency and / or improved reaction parameters and / or reduced amounts of PEG to allow post-ligation processing techniques.

[0323]

[0306] In certain embodiments, the acceptor RNA polynucleotide comprises a structure represented by the formula (pN)n. In some embodiments, (N) of the acceptor is NA.

[0324]

[0307] In some embodiments, each (NA) is independently a nucleotide (or nucleoside, as applicable) or a modified nucleotide (or nucleoside, as applicable). In some embodiments, any suitable modified nucleotide is used. In some embodiments, the (p) of the 5' terminal pN (pNA) is blocked, such as with a 5' CAP.

[0325]

[0308] In some embodiments, the acceptor RNA polynucleotide comprises a modified 5' nucleotide (located atthe terminal 5' position). In some embodiments, the modified 5' nucleotide lacks a 5' phosphate group. In some embodiments, the 5' terminal nucleotide comprises a CAP structure.

[0326]

[0309] In certain embodiments, the acceptor RNA polynucleotide comprises a modified 5' nucleotide, such as a modification that prevents self-ligation of the donor RNA polynucleotide.

[0327]

[0310] In some embodiments, the acceptor RNA polynucleotide comprises one or one more modified nucleotides (e.g., additionally located at non-terminal nucleotide positions).

[0328]

[0311] In some embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' cap structure, a 5' UTR, a protein coding sequence, or a 3' UTR sequence element. In specific embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' UTR. In specific embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' UTR and a protein coding sequence. In specific embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' UTR, a 3' UTR, and a protein coding sequence. In further embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' CAP.

[0329]

[0312] In certain embodiments, ligating the acceptor RNA polynucleotide and the donor RNA polynucleotide forms a structure represented by the formula (pN)nZ (pN)n. In certain embodiments, ligating the acceptor RNA polynucleotide and the donor RNA polynucleotide forms a structure represented by the formula (p-N)n-p-Z'-(p-N)n

[0330]

[0313] In some embodiments, provided herein is a method of making a (e.g., mRNA) polynucleotide. In some embodiments, the method provided herein comprises ligating any acceptor polynucleotide provided herein and any donor polynucleotide (e.g., RNA) provided herein. In some embodiments, the donor polynucleotide (e.g., RNA) comprises a poly-A polynucleotide. In specific embodiments, the poly-A polynucleotide comprises a nucleobase sequence having 75% or greater adenine. In some embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' cap structure, a 5' UTR, a protein coding sequence, or a 3' UTR sequence element. In certain embodiments, the acceptor polynucleotide (e.g., RNA) comprises a 5' cap structure, a 5' UTR, a protein coding sequence, and a 3' UTR sequence elements.

[0331]

[0314] In some embodiments, a donor polynucleotide (e.g., RNA) provided herein comprises a multi-terminus (e.g., branched) polynucleotide. In certain embodiments, a donor polynucleotide (e.g., RNA) provided herein comprise one or more (e.g., 1-8) poly-A polynucleotides (e.g., branches) linked to the donor poly-A polynucleotide (e.g., backbone polynucleotide). For example, a branch poly-A polynucleotide and the donor polynucleotide can each contain a functionalized nucleotide, wherein the functionalized nucleotides comprise a modification that permits linkage (e.g., conjugation). Suitable functionalized nucleotides include those known in the art, for example and not limited to, a first functionalized nucleotide having an azide modification and a second functionalized nucleotide having an alkyne modification, facilitating a linkage to the linker through a triazole linkage. Such functionalized nucleotides can comprise the functionalizing modification within the base, sugar-backbone (e.g., 2' or 3' position), or nucleotide phosphate (e.g., 5' or 3' modifications). In certain embodiments, a branch can have one or more poly-A polynucleotides linked to the branch polynucleotide, yielding a donor poly-A polynucleotide linked to one or more (e.g., 1-8) branch poly-A polynucleotides, wherein the branch poly-A polynucleotides are further linked to one or more (e.g., 1-8) poly-A polynucleotides.

[0332]

[0315] In some embodiments, a donor polynucleotide (e.g., RNA) provided herein comprises a circularized poly-A region. For example, a donor polynucleotide can contain two functionalized nucleotides (a first and a second functionalized nucleotide), wherein the functionalized nucleotides comprise a modification that permits linkage (e.g., conjugation). As described herein, suitable functionalized nucleotides include those known in the art, for example and not limited to, a first functionalized nucleotide having an azide modification and a second functionalized nucleotide having an alkyne modification, facilitating a linkage to the linker through a triazole linkage. Such functionalized nucleotides can comprise the functionalizing modification within the base, sugar-backbone (e.g., 2' or 3' position), or nucleotide phosphate (e.g., 5' or 3' modifications). In certain embodiments, the circularized poly-A region comprises about 10-50, about 10-25, about 10-50, about 10-100, or greater than 150 (e.g., 150-200) nucleotides between the two functionalized nucleotides. In such embodiments, the 5' terminal nucleotide (e.g., p Z described herein) is not a functionalized nucleotide (e.g., the first functionalized nucleotide. For example, non-limiting position of the first functionalized nucleotide can include positions 5-20 of the donor polynucleotide. Also in such embodiments, the 3' terminal nucleotide of the donor polynucleotide is the second functionalized nucleotide.

[0333]

[0316] In some embodiments, a ligation method provided herein performed in a composition comprising a vehicle, such as described herein. In some embodiments, the vehicle is an aqueous vehicle, such as comprising a buffer. In specific embodiments, a vehicle comprises a PEG, such as in any concentration described herein.

[0334]

[0317] In some embodiments, a ligation method provided herein is performed in a reaction vehicle comprising less than 20% polyethylene glycol (PEG). In some embodiments, a ligation method provided herein is performed in a reaction vehicle comprising less than 15% polyethylene glycol (PEG). In some embodiments, a ligation method provided herein is performed in a reaction vehicle comprising less than 10% polyethylene glycol (PEG). In some embodiments, a ligation method provided herein is performed in a reaction vehicle comprising less than 5% polyethylene glycol (PEG). In some embodiments, a ligation method provided herein is performed in a reaction vehicle comprising less than 2% polyethylene glycol (PEG). In some embodiments, a ligation method provided herein performed in a reaction vehicle is substantially free of PEG.

[0335]

[0318] In some embodiments, any suitable polyethylene glycol (PEG) is used in any methods or compositions provided herein. In some embodiments, the polyethylene glycol (PEG) is a high molecular weight PEG (e.g., having an average molecular mass > 1000). In specific embodiments, the polyethylene glycol (PEG) is PEG8000.

[0336]

[0319] In some embodiments, a ligation method provided herein is performed at a total polynucleotide concentration of at least 0.2 mg / mL (e.g., in the composition comprising the vehicle). In some embodiments, a ligation method provided herein is performed at a total polynucleotide concentration of at least 0.5 mg / mL (e.g., in the composition comprising the vehicle). In some embodiments, a ligation method provided herein is performed at a total polynucleotide concentration of at least 1 mg / mL (e.g., in the composition comprising the vehicle).

[0337]

[0320] In some embodiments, provided herein is a composition comprising any acceptor polynucleotide provided herein or donor polynucleotide provided herein. In specific embodiments, the composition comprises any acceptor polynucleotide provided herein and donor polynucleotide provided herein. In some embodiments, provided herein is a composition comprising a polynucleotide provided herein (e.g., a polynucleotide described herein producible by ligating a donor and an acceptor described herein). In certain embodiments, provided herein is a composition comprising a polynucleotide provided herein (e.g., a polynucleotide described herein producible by ligating a donorand an acceptor described herein), a donordescribed herein and an acceptor described herein. In certain embodiments, a composition provided herein comprises an enzyme.

[0338]

[0321] In some embodiments, a composition provided herein is performed in a reaction vehicle comprising less than 20% polyethylene glycol (PEG). In some embodiments, a composition provided herein is performed in a reaction vehicle comprising less than 15% polyethylene glycol (PEG). In some embodiments, a composition provided herein is performed in a reaction vehicle comprising less than 10% polyethylene glycol (PEG). In some embodiments, a composition provided herein is performed in a reaction vehicle comprising less than 5% polyethylene glycol (PEG). In some embodiments, a composition provided herein is performed in a reaction vehicle comprising less than 2% polyethylene glycol (PEG). In some embodiments, a composition provided herein performed in a reaction vehicle is substantially free of PEG.

[0322] In some embodiments, provided herein is a polynucleotide (e.g., RNA, such as a messenger ribonucleic acid (mRNA)) comprising a structure represented by the formula: (Xi) Z p(X2).

[0339]

[0323] In certain embodiments, the polynucleotide comprises a structure represented by the formula X^p'-Z'-p-X2. In some embodiments, (XI) is a 3' end of 5' CAP region. In some embodiments, the polynucleotide provided herein comprises a 5' CAP, wherein the 5' CAP is connected to Z at the 3' end of 5' CAP region. In some embodiments, the polynucleotide further comprises a 5' UTR, protein coding sequence region, 3' UTR. In some embodiments, the polynucleotide further comprises a poly-A region or tailing polynucleotide.

[0340]

[0324] In some embodiments, (XI) is a 3' end of a 5' UTR. In some embodiments, the polynucleotide provided herein comprises a 5' UTR, wherein the 5' UTR is connected to Z at the 3' end of 5' UTR. In some embodiments, the polynucleotide further comprises a protein coding sequence region, 3' UTR. In some embodiments, the polynucleotide further comprises a poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide further comprises a 5' CAP.

[0341]

[0325] In some embodiments, (XI) is a 3' end of a protein coding sequence region. In some embodiments, the polynucleotide provided herein comprises a protein coding sequence region, wherein the protein coding sequence region is connected to Z at the 3' end of the protein coding sequence region. In some embodiments, the polynucleotide further comprises a 5'UTR and 3' UTR. In some embodiments, the polynucleotide further comprises a poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide further comprises a 5' CAP.

[0342]

[0326] In some embodiments, (XI) is a 3' end of a 3' UTR. In some embodiments, the polynucleotide provided herein comprises a 3' UTR, wherein the 3' UTR is connected to Z at the 3' end of the 3' UTR. In some embodiments, the polynucleotide further comprises a 5'UTR and protein coding sequence region. In some embodiments, the polynucleotide further comprises a poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide further comprises a 5' CAP.

[0343]

[0327] In some embodiments, (XI) is a 3' end of a poly-A region ortailing polynucleotide. In some embodiments, the polynucleotide provided herein comprises a poly-A region or tailing polynucleotide, wherein the poly-A region or tailing polynucleotide is connected to Z at the 3' end of the poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide further comprises a 5'UTR, 3' UTR, and protein coding sequence region. In some embodiments, the polynucleotide further comprises a poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide further comprises a 5' CAP.

[0344]

[0328] In some embodiments, (X2) is a 5' end of a 5' UTR. In some embodiments, the polynucleotide provided herein comprises a 5' UTR, wherein the 5' UTR is connected to Z at the 5' end of the 5' UTR. In some embodiments, the polynucleotide further comprises a 5'UTR and protein coding sequence region. In some embodiments, the polynucleotide further comprises a poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide further comprises a 5' CAP.

[0345]

[0329] In some embodiments, (X2) is a 5' end of a protein coding sequence region. In some embodiments, the polynucleotide provided herein comprises a protein coding sequence region, wherein the protein coding sequence region is connected to Z at the 5' end of the protein coding sequence region. In some embodiments, the polynucleotide further comprises a 5'UTR and 3' UTR. In some embodiments, the polynucleotide further comprises a poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide further comprises a 5' CAP.

[0346]

[0330] In some embodiments, (X2) is a 5' end of a 3' UTR. In some embodiments, the polynucleotide provided herein comprises a 3' UTR, wherein the 3' UTR is connected to Z at the 5' end of the 3' UTR. In some embodiments, the polynucleotide further comprises a 5'UTR and protein coding sequence region. In some embodiments, the polynucleotide further comprises a poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide further comprises a 5' CAP.

[0347]

[0331] In some embodiments, (X2) is a 5' end of a poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide provided herein comprises a poly-A region or tailing polynucleotide, wherein the poly-A region or tailing polynucleotide is connected to Z at the 5' end of the poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide further comprises a 5'UTR, 3' UTR, and protein coding sequence region. In some embodiments, the polynucleotide further comprises a poly-A region or tailing polynucleotide. In some embodiments, the polynucleotide further comprises a 5' CAP.

[0348]

[0332] In some embodiments, provided herein is a polynucleotide (e.g., RNA, such as a messenger ribonucleic acid (mRNA)) comprising a poly-A tail (e.g., a poly-A region). In some embodiments, poly-A tail provided herein comprises a structure represented by the formula: (pA)nl Z (pA)m.

[0349]

[0333] In specific embodiments, poly-A tail provided herein comprises a structure represented by the formula (pA)ni-p-Z'-(pA)ni. In some embodiments, each (A) is independently an adenine nucleotide or a modified adenine nucleotide. In some embodiments, each (n1) is an integer equal to or greater than 1.

[0350]

[0334] In some embodiments, provided herein is a polynucleotide (e.g., RNA, such as a messenger ribonucleic acid (mRNA)) comprising a poly-A tail (e.g., a poly-A region). In some embodiments, poly-A tail provided herein comprises a structure represented by the formula: - (pA)ni-(p'A)n2-. In some embodiments, each (p) is independently a phosphate linkage. In some embodiments, each (p') is independently a phosphorothioate linkage. In some embodiments, each (A) is independently an adenine nucleotide or a modified adenine nucleotide. In some embodiments, (n1) is an integer equal to or greater than 1. In some embodiments, (n2) is an integer equal to or greater than 1.

[0351]

[0335] In some embodiments, the polyA tail comprises a dideoxynucleoside (e.g., ddC or ddA) located at the terminal 3' position. In some embodiments, the dideoxynucleoside is attached to the penultimate A. In some embodiments, the dideoxynucleoside is attached to the penultimate A via a phosphothioate linkage.

[0352]

[0336] In some embodiments, a polynucleotide described herein comprises a cytosine (C) attached to p. In some embodiments, a polynucleotide described herein comprises a cytosine (C) attached to the 5' terminal p (e.g., of the polyA tail).

[0353]

[0337] In some embodiments, (n1) is an integer equal to or greater than 1. In some embodiments, (n1) is an integer equal to or greater than 2. In some embodiments, (n1) is an integer equal to or greater than 3. In some embodiments, (n1) is an integer equal to or greater than 4. In some embodiments, (n1) is an integer equal to or greater than 5. In some embodiments, (n1) is an integer equal to or greater than 6. In some embodiments, (n1) is an integer equal to or greater than 8. In some embodiments, (n1) is an integer equal to or greater than 10. In some embodiments, (n1) is an integer equal to or greater than 12. In some embodiments, (n1) is an integer equal to or greater than 14. In some embodiments, (n1) is an integer equal to or greater than 16.

[0354]

[0338] In some embodiments, (n1) is an integer equal to or less than 5. In some embodiments, (n1) is an integer equal to or less than 6. In some embodiments, (n1) is an integer equal to or less than 8. In some embodiments, (n1) is an integer equal to or less than 10. In some embodiments, (n1) is an integer equal to or less than 12. In some embodiments, (n1) is an integer equal to or less than 14. In some embodiments, (n1) is an integer equal to or less than 16. In some embodiments, (n1) is an integer equal to or less than 18. In some embodiments, (n1) is an integer equal to or less than 20.

[0355]

[0339] In some embodiments, (n1) is an integer of 1 to 20. In some embodiments, (n1) is an integer of 3 to 15. In some embodiments, (n1) is an integer of 7 to 12.

[0356]

[0340] In some embodiments, (n1) is 5. In some embodiments, (n1) is 6. In some embodiments, (n1) is 7. In some embodiments, (n1) is 8. In some embodiments, (n1) is 9. In some embodiments, (n1) is 10. In some embodiments, (n1) is 11. In some embodiments, (n1) is 12. In some embodiments, (n1) is 13. In some embodiments, (n1) is 14.

[0357]

[0341] I n some embodiments, (n2) is an integer equal to or greater than 1. In some embodiments, (n2) is an integer equal to or greater than 2. In some embodiments, (n2) is an integer equal to or greater than 3. In some embodiments, (n2) is an integer equal to or greater than 4. In some embodiments, (n2) is an integer equal to or greater than 5. In some embodiments, (n2) is an integer equal to or greater than 6. In some embodiments, (n1) is an integer equal to or greater than 8. In some embodiments, (n2) is an integer equal to or greater than 10. In some embodiments, (n2) is an integer equal to or greater than 12. In some embodiments, (n2) is an integer equal to or greater than 14. In some embodiments, (n2) is an integer equal to or greater than 16.

[0358]

[0342] In some embodiments, (n2) is an integer equal to or less than 5. In some embodiments, (n2) is an integer equal to or less than 6. In some embodiments, (n2) is an integer equal to or less than 8. In some embodiments, (n2) is an integer equal to or less than 10. In some embodiments, (n2) is an integer equal to or less than 12. In some embodiments, (n2) is an integer equal to or less than 14. In some embodiments, (n2) is an integer equal to or less than 16. In some embodiments, (n2) is an integer equal to or less than 18. In some embodiments, (n2) is an integer equal to or less than 20.

[0359]

[0343] In some embodiments, (n2) is an integer of 1 to 20. In some embodiments, (n2) is an integer of 1 to 10. In some embodiments, (n2) is an integer of 1 to 6.

[0360]

[0344] In some embodiments, (n2) is 1. In some embodiments, (n2) is 2. In some embodiments, (n2) is 3. In some embodiments, (n2) is 4. In some embodiments, (n2) is 5. In some embodiments, (n2) is 6. In some embodiments, (n2) is 7. In some embodiments, (n2) is 8. In some embodiments, (n2) is 9. In some embodiments, (n2) is 10.

[0361]

[0345] In some embodiments, any suitable RNA ligase enzyme is used in any method provided herein. In specific embodiments, an RNA ligase enzyme is a T4 RNA ligase. In specific embodiments, the T4 RNA ligase is a T4 RNA ligase 1.

[0362]

[0346]

[0363] EXAMPLE 2: RNA ligation using the methods and modified nucleotides described herein exhibit good efficiency even when conducted in the absence at PEG8000 at larger scale.

[0364]

[0347] The scalability of the optimized ligation approach was demonstrated, as well as its compatibility with different chemistry in the oligos. Milligram level scale ligation reactions were performed with 4 different chemically modified oligos shown in TABLE 2 below.

[0365]

[0348] TABLE 2 RNA nucleotides: rN; RNA phosphorothioate nucleotide: rN*; 2'-0-methyl phosphorothioate nucleotide: mN*; locked nucleic acid [LNA] nucleotide: +N; Internal 2'-O-methoxyethyl RNA nucleotide: i2MOErN; Internal 2'-O-methoxyethyl RNA phosphorothioate nucleotide: i2MOErN*; 5' Phosphate modification: / 5Phos / ; 2'-3'-dideoxycytidine (ddC) nucleotide: / 3ddC / ; lnverted-2'- deoxythymidine [InvdT] nucleotide: / 3lnvdT /

[0366]

[0349] An RNase H digestion assay revealed efficient ligations for all constructs as shown in FIG.

[0367] 2. Thus, oligos bearing 5' rC facilitated ligation reaction scale up, eliminating the need for PEG8000.

[0368] EXAMPLE 3: Constructs ligated to modified polynucleotides provided herein provide durable expression

[0369]

[0350] Introducing modifications to polynucleotides or oligonucleotides can have detrimental effects on the efficacy of their gene expression and protein production. To establish that modifications described herein could be tolerated, polynucleotides described herein, such as bearing a modified penultimate nucleotide, were expressed in cells. In particular, a luciferase assay for mice cells (FIG. 3, panel A) and human cells (FIG. 3, panel B) was used to measure the expression activity of 3' chain terminating tails (e.g., poly-As tail having a 3' chain terminating nucleotide) described herein. Luciferase activity is measured at day 1-4 after transfection with MessangerMax, and the activity at each time point is normalized to process control (unligated mRNA) of the same time point. An increase of the normalized value from day 1-4 indicates an increased stability.

[0370]

[0351] For example, modified donor polynucleotides provided in TABLE 3 were ligated to the 3' end of a polynucleotide encoding luciferase, and the expression of luciferase by resulting ligated products was evaluated at days 1-4 in AML12 mice cells (FIG. 3, panel A) and HepG2 cells (FIG.

[0371] 3, panel B). The structures of the modified donor polynucleotides that were ligated to the polynucleotide encoding luciferase are provided in TABLE 3 below.

[0372]

[0352] TABLE 3

[0373] RNA nucleotides: rN; RNA phosphorothioate nucleotide: rN*; 5' Phosphate modification: / 5Phos / ; 2'-3'-dideoxycytidine (ddC) nucleotide: / 3ddC /

[0374]

[0353] Expression levels were measured in mice and human cells for oligomers comprising 3' chain terminating tails (e.g., poly-As tail having a 3' chain terminating nucleotide) described herein. Overall, 3' chain terminating tails described herein provided relatively good expression in mice and human cells. For example, donor polynucleotides bearing 5' cytidine (e.g., 6PSrA C ddC) provided durable expression, similar to an analogous polynucleotide bearing 5' adenosine in place of 5' cytidine (6PSrA ddC). Overall, this data demonstrates that the 3' chain terminating tails described herein are relatively stable and provide robust cellular expression.

[0375] EXAMPLE 4: Ligated construct expression in mice and non-human primates (NHPs)

[0376]

[0354] mRNAs having a 6PS rA tail (SEQ ID NO: 14 - rC(rA)?(rA*)6 / 3ddC) were tested in mice (male, n=4), and cynomolgus monkeys (female, n=3-4). hEPO were used as a reporter for these studies. The conventional mRNA (unmodified, poly-A tail) and 6PS rA tail mRNA were formulated with ALC315 based lipid nanoparticle (LNP) and delivered through IV injection to mice (0.5mg / kg) and NHP (0.2mg / kg). hEPO levels at different timepoints were measured using ELISA. The 6PS rA showed substantially increased duration of hEPO expression and the AUC was improved on conventional mRNA for 3.9- and 3.6-fold for mice and NHP, respectively (FIG. 4).

Claims

CLAIMS1. An mRNA polynucleotide comprising a 3' chain terminating polynucleotide according to Formula (I): p-Z-(p'-A)n-p"-(R-pc)m-R1-pd-N (I) wherein: p is an inter-nucleotide linker or a chain terminal group;Z is a natural or modified nucleoside; each p' is independently an inter-nucleotide linker; each A is independently an adenosine nucleoside, or an analog thereof; p" is an inter-nucleotide linker; n is an integer of 0 to 30; each R is independently a nucleoside; each pcis independently a phosphorothioate; m is an integer of 2 to 8;R1is a nucleoside; pdis a phosphorothioate; andN is a chain terminating nucleoside (e.g., comprising a ribose lacking a free 3' hydroxyl), wherein each nucleoside independently comprises a ribose or analog thereof.

2. The mRNA polynucleotide of claim 1, wherein p is a chain terminal group selected from the group consisting of phosphate and diphosphate.

3. The mRNA polynucleotide of claim 1, wherein p is an inter-nucleotide linker selected from the group consisting of phosphate and diphosphate.

4. The mRNA polynucleotide of claim 1, wherein p is a chain terminal group selected from the group consisting of hydroxyl, phosphate, phosphorothioate, phosphorodithioate, thiophosphate, phosphonate, phosphanate, phosphoroselenoate, selenophosphate, and phosphoramidate.

5. The mRNA polynucleotide of any one of the preceding claims, wherein each nucleoside independently comprises a ribose or a ribose analog that is a heteroribose, a locked ribose, or an unlocked ribose.

6. The mRNA polynucleotide of any one of the preceding claims, wherein Z is a nucleoside substituted at the 1-position with a hydrogen and B, wherein B is hydrogen, C2-Cealkynyl, C2-C8heterocycloalkyl, aryl, or heteroaryl, and wherein C2-Cealkynyl, C2- Cgheterocycloalkyl, aryl, and heteroaryl are unsubstituted or substituted.

7. The mRNA polynucleotide of claim 6, wherein B is monocyclic C2-C8heterocycloalkyl, monocyclic aryl, or monocyclic heteroaryl.

8. The mRNA polynucleotide of claim 6, wherein B is a substituted or unsubstituted 5- or 6- membered aryl or a substituted or unsubstituted 5- or 6-membered heteroaryl.

9. The mRNA polynucleotide of claim 6, wherein B is a pyrimidine nucleobase.

10. The mRNA polynucleotide of claim 9, wherein B is cytosine.

11. The mRNA polynucleotide of claim 6, wherein B is:wherein each Rxis independently hydrogen, -NH2 -NO2, -NH-OH, -OH, -OCH3, -CH3, -CH2OH, - N(H)CH3, -NH-OH, or -N(H)C(=O)CH3.

12. The mRNA polynucleotide of any one of claims 1-6, wherein Z is a nucleoside comprising a monocyclic nucleobase.

13. The mRNA polynucleotide of any one of claims 1-6, wherein Z is cytidine (C) or an analog thereof.

14. The mRNA polynucleotide of any one of claims 1-6, wherein Z is cytidine (C).

15. The mRNA polynucleotide of any one of the preceding claims, wherein the ribose of Z is substituted with a hydrogen and a second group R4, wherein R4is a hydrogen, a halogen, a hydroxyl, a substituted or unsubstituted alkoxy, a substituted or unsubstituted heteroalkoxy, or a substituted or unsubstituted alkoxy.

16. The mRNA polynucleotide of claim 15, wherein R4is hydroxyl.

17. The mRNA polynucleotide of any one of the preceding claims, wherein each p' is a phosphate or analog thereof.

18. The mRNA polynucleotide of claim 17, wherein p" is phosphate or a phosphorothioate.

19. The mRNA polynucleotide of any one of the preceding claims, wherein each p' is phosphate.

20. The mRNA polynucleotide according to claim 17 or 18, wherein p" is a phosphorothioate (PS) or diphosphorothioate (PS2).

21. The mRNA polynucleotide of claim 20, wherein p" is a phosphorothioate.

22. The mRNA polynucleotide of any one of the preceding claims, wherein each A is independently an adenosine.

23. The mRNA polynucleotide of any one of the preceding claims, wherein n is an integer of 2 to 25.

24. The mRNA polynucleotide of any one of the preceding claims, wherein n is an integer of 6 to 20.

25. The mRNA polynucleotide of any one of the preceding claims, wherein p" is a phosphate or analog thereof.

26. The mRNA polynucleotide of any one of the preceding claims, wherein p" is phosphate.

27. The mRNA polynucleotide of any one of the preceding claims, wherein each R and R1is independently a natural or modified nucleoside.

28. The mRNA polynucleotide of any one of the preceding claims, wherein each R and R1is independently adenosine or an analog thereof.

29. The mRNA polynucleotide of any one of the preceding claims, wherein each R and R1is independently adenosine.

30. The mRNA polynucleotide of any one of the preceding claims, wherein at least 50% of the total of R and R1are adenosine.

31. The mRNA polynucleotide of any one of the preceding claims, wherein at least 80% of the total of R and R1are adenosine.

32. The mRNA polynucleotide of any one of the preceding claims, wherein m is an integer of3 to 7.

33. The mRNA polynucleotide of any one of the preceding claims, wherein m is an integer of4 to 6.

34. The mRNA polynucleotide of any one of the preceding claims, wherein m is an integer of 5.

35. The mRNA polynucleotide of any one of the preceding claims, wherein the ribose of N does not have a 3'-hydroxyl.

36. The mRNA polynucleotide of any one of the preceding claims, wherein N is a non-hydroxyl nucleoside.

37. The mRNA polynucleotide of any one of the preceding claims, wherein N is a dideoxynucleoside (e.g., ddC or ddA).

38. The mRNA polynucleotide of any one of the preceding claims, wherein N is an inverted nucleoside (e.g., wherein N is attached at the 3' position, through pd, to the 3' position of R1).

39. The mRNA polynucleotide of claim 38, wherein N is an inverted 2'-deoxynucleoside (InvdN) (e.g., inverted 2'-deoxythymidine (InvdT), inverted 2'-deoxycytidine (InvdC) or inverted 2'-deoxyadenosine (InvdA)).

40. The mRNA polynucleotide of claim 39, wherein N is inverted 2'-deoxythymidine (InvdT).

41. The mRNA polynucleotide of any one of the preceding claims, wherein N is a nucleoside substituted at the 2'-position with a non-hydroxyl.

42. The mRNA polynucleotide of any one of claims 1-34, wherein N is a nucleoside substituted at the 2'-position with R7, wherein R7is hydrogen, a halogen, an alkoxy, a heteroalkoxy, an alkylthio, an amino, an alkyl, or a heteroalkyl, wherein the alkoxy, heteroalkoxy, alkylthio, amino, alkyl, or heteroalkyl is substituted or unsubstituted.

43. The mRNA polynucleotide of any one of the preceding claims, wherein N is a nucleoside substituted at the 3'-position with a non-hydroxyl.

44. The mRNA polynucleotide of any one of claims 1-34, wherein N is a nucleoside substituted at the 3'-position with R6, wherein R6is hydrogen, a halogen, an alkoxy, a heteroalkoxy, an alkylthio, an amino, an alkyl, or a heteroalkyl, wherein the alkoxy, heteroalkoxy, alkylthio, amino, alkyl, or heteroalkyl is substituted or unsubstituted.

45. The mRNA polynucleotide of any one of claims 1-34, wherein the ribose of nucleoside N is a spiro-ribose, a bridged-ribose, bicyclic ribose, or a locked ribose.

46. The mRNA polynucleotide of claim 1, wherein a nucleoside within six upstream nucleosides of R and R1(e.g., R, N, or a nucleoside of the polynucleotide) comprises a 2' hydroxyl.

47. The mRNA polynucleotide of claim 1, wherein R or R1comprises a 2' hydroxyl.

48. The mRNA polynucleotide of claim 1, wherein p' is bonded to an upstream nucleoside (e.g., Z or another nucleoside of the polynucleotide) that is natural or modified, wherein the modified nucleoside is not: (i) a locked nucleic acid or (ii) substituted in the 2'-position with methoxy or methoxyethoxy (MOE).

49. The mRNA polynucleotide of any one of the preceding claims, wherein N comprises a nucleobase.

50. The mRNA polynucleotide of claim 49, wherein the nucleobase of N is a modified or natural nucleobase.

51. The mRNA polynucleotide of any one of the preceding claims, wherein the nucleobase of N is a cytosine.

52. The mRNA polynucleotide of any one of the preceding claims, wherein N is 2' ,3'- dideoxycytosine.

53. The mRNA polynucleotide of any one of the preceding claims, wherein N is a nucleoside reverse transcriptase inhibitor (NRTI), such as Lamivudine.

54. The mRNA polynucleotide of any one of the preceding claims, wherein the ribose of N is substituted at the 1' position with Q, wherein:Q is hydrogen, alkyl, Cz-Cealkynyl, Cz-Csheterocycloalkyl, aryl, or heteroaryl, wherein C2- Cealkynyl, C2-C8heterocycloalkyl, aryl, and heteroaryl are unsubstituted or substituted (e.g., a substituted heteroaryl that is N6-methyladenosine (m6A)).

55. The mRNA polynucleotide of claim 1, wherein m is 5, each R is adenosine, and each pcis independently a phosphorothioate.

56. The mRNA polynucleotide of claim 1, wherein m is 5, each R is adenosine, each pcis independently a phosphorothioate, R1is adenosine, and pdis a phosphorothioate.

57. The mRNA polynucleotide of claim 1, wherein m is 5, each R is adenosine, each pcis independently a phosphorothioate, R1is adenosine, pdis a phosphorothioate, and N is a dideoxynucleoside (e.g., ddC or ddA) or an inverted 2'-deoxynucleoside (InvdN).

58. The mRNA polynucleotide of any one of the preceding claims, wherein N is abasic.

59. The mRNA polynucleotide of any one of the preceding claims, wherein N is unlocked.

60. The mRNA polynucleotide of claim 1, wherein the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 1.

61. The mRNA polynucleotide of claim 1, wherein the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 2.

62. The mRNA polynucleotide of claim 1, wherein the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 3.

63. The mRNA polynucleotide of claim 1, wherein the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 4.

64. The mRNA polynucleotide of claim 1, wherein the 3' chain terminating polynucleotide comprises a sequence according to SEQ ID NO: 5.

65. The mRNA polynucleotide of any one of the preceding claims, wherein the polynucleotide further comprises: at least one 5'-cap structure; a 5'-UTR; a coding region; and a 3'-UTR.

66. The mRNA polynucleotide of any one of the preceding claims, wherein the polynucleotide further comprises a poly-A.

67. The mRNA polynucleotide of claim 66, wherein the poly-A is connected 3' to 5' to the 3' chain terminating polynucleotide.

68. A composition comprising a polynucleotide of any one of the preceding claims and a lipid nanoparticle (LNP).

69. A method of ligating a polynucleotide tail to an mRNA polynucleotide, the method comprising:(a) providing a first (donor) polynucleotide, the first polynucleotide being a 3'-chain terminating polynucleotide of any one of the preceding claims;(b) providing a second (acceptor) polynucleotide, wherein the first polynucleotide has a 3' terminal hydroxyl group;(c) contacting an RNA ligase enzyme with the first polynucleotide and the second polynucleotide.

70. The method of claim 69, wherein the first polynucleotide has a 5'-terminal phosphate group.

71. The method of either one of claims 69 or 70, wherein the second polynucleotide comprises a poly A.

72. The method of any one of claims 69-71, wherein the second polynucleotide comprises a 5'-UTR, a coding region, or a 3'-UTR.

73. The method of any one of claims 69-71, wherein the second polynucleotide comprises a 5'-UTR, a coding region, and a 3'-UTR.

74. The method of any one of claims 69-73, wherein the RNA ligase enzyme is a T4 RNA ligase.

75. The method of any one of claims 69-74, wherein the contacting an RNA ligase enzyme with the first polynucleotide and the second polynucleotide occurs in a medium comprising less than 20% polyethylene glycol (PEG), less than 15% PEG, less than 10% PEG, less than 5% PEG, less than 2% PEG, or that is substantially free of PEG.

76. The method of any one of claims 69-74, wherein the contacting an RNA ligase enzyme with the first polynucleotide and the second polynucleotide occurs in a medium comprising less than 20% high molecular weight polyethylene glycol (PEG), less than 15% high molecular weight PEG, less than 10% high molecular weight PEG, less than 5% high molecular weight PEG, less than 2% high molecular weight PEG, or that is substantially free of high molecular weight PEG.

77. The method of claim 69, wherein the contacting an RNA ligase enzyme with the first polynucleotide and the second polynucleotide is conducted in a composition comprising a total polynucleotide concentration of at least 0.5 mg / mL.

78. A polyribonucleotide comprising a structure represented by the formula: pZ - (p'N)n - p"N* wherein :(p) is a free 5' terminal phosphate;(Z) is a nucleoside comprising a monocyclic nucleobase; each (p') is independently an inter-nucleotide linkage (e.g., phosphorothioate linkage or phosphate linkage);(p") is a phosphorothioate inter-nucleotide linkage; each (N) is independently a nucleoside;(N*) is a nucleoside lacking a 3' hydroxyl;(n) is an integer equal to or greater than 1; and the nucleobase content of the polyribonucleotide is 80% adenine or greater than 80% adenine.

79. The polyribonucleotide of claim 78, wherein (p') is a phosphate inter-nucleotide linkage and (p") is a phosphorothioate inter-nucleotide linkage.

80. A polyribonucleotide comprising a structure represented by the formula: pZ - (p'N)n1- (p''A)n2- p"'N* wherein :(p) is a free 5' terminal phosphate;(Z) is a nucleoside comprising a monocyclic nucleobase (e.g., cytosine); each (p') is independently a phosphate inter-nucleotide linkage; each (N) is independently a nucleoside; each (p") is independently a phosphorothioate inter-nucleotide linkage;each (A) is independently a nucleoside comprising an adenine nucleobase or modified adenine nucleobase;(p'") is a phosphorothioate inter-nucleotide linkage;(N*) is a nucleoside lacking a 3' hydroxyl;(n1) is an integer equal to or greater than 1;(n2) is 0, 1, 2, 3, 4, 5, 6, or 7; and the nucleobase content of the polyribonucleotide is 80% adenine or greater than 80% adenine.

81. The polyribonucleotide of claim 80, wherein (n2) is 6.

82. A poly-A polynucleotide comprising (i) a terminal 5' nucleotide comprising a monocyclic nucleobase and free 5' phosphate, and (ii) a terminal 3' nucleotide comprising (a) a 5' phosphorothioate linkage and (b) a nucleoside lacking a free 3' hydroxyl.

83. The poly-A polynucleotide of claim 82, wherein the last 2-8 inter-nucleotide linkages at the 3' terminus are phosphorothioate inter-nucleotide linkages.

84. The poly-A polynucleotide of claim 83, wherein the last 6 inter-nucleotide linkages at the 3' terminus are phosphorothioate inter-nucleotide linkages.

85. A messenger ribonucleic acid (mRNA) polynucleotide comprising a poly-A tail, wherein the poly-A tail comprises (i) a 5' nucleotide comprising a monocyclic nucleobase, and (ii) a terminal 3' nucleotide comprising (a) a 5' phosphorothioate linkage and (b) a nucleoside lacking a 3' hydroxyl.

86. The mRNA polynucleotide of claim 85, wherein the last 2-8 inter-nucleotide linkages at the 3' terminus are phosphorothioate inter-nucleotide linkages.

87. The mRNA polynucleotide of claim 86, wherein the last 6 inter-nucleotide linkages at the 3' terminus are phosphorothioate inter-nucleotide linkages.

88. The mRNA polynucleotide of any one of claims 85-87, wherein the RNA polynucleotide comprises, from 5' to 3', a 5' cap structure, a 5' UTR, a protein coding sequence, a 3' UTR, and the poly-A tail.

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