Modified RNA used to increase protein expression

Abstract

This document describes a modified RNA molecule in which a 3'-stable region is covalently linked to the RNA, and wherein the 3'-stable region comprises one or more modified nucleosides. This document also provides a method for synthesizing the RNA.

Description

[0001] Cross-reference of priority claims This application claims priority to U.S. Provisional Application No. 63 / 617,664, filed January 4, 2024, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] The field of this invention relates to modified RNA and methods for producing the same. Furthermore, this invention relates to the efficient production of proteins using said modified RNA. Background Technology

[0003] Eukaryotic mRNA has five important parts, including a 5' cap, a 5'-UTR, an open reading frame (ORF), a 3'-UTR, and a 3'-tail (poly-A tail) consisting of 100-250 adenosine residues, the length of which varies in different cell types (Youn, H. and Chung, JK, (2015)). Expert Opinion. Biol. Ther 15:1337-1348).

[0004] Although mRNA therapeutics are promising, they also have problems such as instability and high immunogenicity (Kormann et al., (2011)). Nature Biotech (29:154-157). Because mRNA degrades naturally in biological systems, high doses or repeated administrations are often required. The main pathway of mRNA degradation in eukaryotic cells occurs within a ribonucleic acid complex in the cytoplasm called the P-body, which contains 5'-3'-exonucleases, decapping enzymes, and deadenylate enzymes. Once the poly-A tail is shortened to 12 residues or less, degradation occurs via cap cleavage and 5'-exonuclease. 3' or 3' 5' cleavage leads to mRNA degradation (Melo et al., (2019)). Mol. Ther (27:2080-2090). Endonucleases can also participate in mRNA degradation.

[0005] Chemical modification of mRNA can increase its stability and improve its translational properties and immunogenicity (Anderson et al., (2010)). Nucleic Acids Res 38:5884-5892; Jemielity et al. , (2010) New J Chem 34:829-844; Kariko et al., (2012) Mol. Ther 20:948-953; Sahin et al., (2014) Nat. Rev. Drug Discov13:759-780).

[0006] One of the structural elements that influences mRNA half-life and translation is the 5'-terminal 7-methylguanosine cap (Topisirovic et al., (2011)). Interdiscip. Rev. RNA 2:277-298). Modification of the 5' cap can produce enhanced mRNA stability and expression in living cells (Ziemniak et al., (2013)). Future Med. Chem 5:1141-1172; Kowalska et al., (2014) Nucleic Acids Res. 42:10245-10264; WO2017 / 053297).

[0007] The poly-A tail is another key element responsible for efficient translation and increasing mRNA stability (Chang et al., (2014)). Mol. Cell 53:1044-1052). The role of the poly-A tail in translation consists of binding to a large amount of polyadenosine-binding protein (PABP), which in turn binds to eukaryotic translation initiation factor 4G (eIF4G). This forms a ring structure with a cap-eIF4E-eIF4G-PABP-polyadenosine nucleotide closed loop, which promotes ribosome binding and protects mRNA from nuclease degradation (Newbury, SF, (2006)). Biochem. Soc. Trans 34:30-34).

[0008] Woolf et al. described modifications to the poly-A tail that increased stability against nucleases (WO1999014346). They found that phosphate thioester bonds or other stabilizing modifications to RNA could be incorporated into the poly-A tail to further enhance the stability of the mRNA molecule, and that further modifications downstream of the poly-A tail could be made to retain the polyadenylate binding site and further block 3' exonucleases.

[0009] Despite recent successes in the clinical field, mRNA therapeutics still face challenges such as instability, toxicity, short-term efficacy, and potential immune responses. Therefore, improving the stability of mRNA to enhance its efficacy and reduce its immunogenicity in vivo remains a crucial issue that must be addressed to increase the feasibility of mRNA therapeutics in clinical applications. Summary of the Invention

[0010] This article describes RNA molecules covalently linked to a 3'-stabilizing region, wherein the 3'-stabilizing region contains one or more purification tags optionally covalently linked via a linker (L). While not wishing to be bound by theory, after purifying precursor RNA containing a 5'-cap, a protein-encoding ORF, and a 3' poly-A region located at the 3' end of the ORF (A in Formula I or II) on an oligo dT column, only precursor RNA containing at least a partial poly-A tail is linked to the 3'-stabilizing region containing one or more purification tags. Therefore, only full-length RNA molecules are stabilized by the 3'-stabilizing region and are readily separable from full-length RNA molecules not linked to the 3'-stabilizing region. Surprisingly, the RNA molecules described herein exhibit improved stability and improved translation efficiency.

[0011] In one aspect, this document provides an RNA molecule comprising a structure of Formula I: AB (Formula I), wherein A comprises: a) a 5'-cap; b) an open reading frame (ORF) encoding a protein; and c) a poly-A region, wherein the poly-A region is located at the 3' of the open reading frame; and B comprises a 3'-stable region containing 1 to 50 nucleotides, wherein one or more nucleotides within the 3'-stable region contain one or more purification tags.

[0012] In one aspect, this document provides an RNA molecule comprising a structure of Formula II: ABL (Formula II), wherein A comprises: a) a 5'-cap; b) an open reading frame (ORF) encoding a protein; and c) a poly-A region, wherein the poly-A region is located at the 3' of the open frame; and B comprises a 3'-stabilized region containing 1 to 50 nucleotides, wherein one or more nucleotides within the 3'-stabilized region contain one or more adapters (L), wherein the adapters (L) are capable of binding a purification tag.

[0013] In one embodiment, the 3'-stable region is covalently linked to the precursor RNA via a linker that can be formed by ligation. In another embodiment, the linker can be formed by enzymatic or chemical ligation. In yet another embodiment, the 3'-stable region is covalently linked to the precursor RNA via a linker that can be formed using polymerase. In yet another embodiment, the purification tag is linked to the 3'-stable region via a linker (L).

[0014] In one embodiment, one or more purification tags are covalently linked to one or more nucleosides or one or more adapters within the 3'-stabilizing region. In one embodiment, the purification tag comprises a lipid. In one embodiment, the 3'-stabilizing region forms a secondary structure. In one embodiment, the secondary structure is a hairpin ring.

[0015] In one embodiment, the 3'-stable region comprises one or more unmodified nucleosides and one or more unmodified nucleotide inter-bonds. In another embodiment, the 3'-stable region comprises one or more modified nucleosides and / or one or more modified nucleotide inter-bonds. In another embodiment, the modified nucleosides comprise modified nucleobases and / or modified sugars. In another embodiment, the modified nucleosides comprise modified nucleobases. In another embodiment, the modified nucleobase is modified uracil, modified cytosine, modified guanine, or modified adenine. In another embodiment, the modified nucleobase is pseudouracil (ψ), 2-thiouracil, 4-thiouracil, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuracil, 5-halouracil, 3-methyluracil, 5-azauracil, or 2-thio-5-azauracil. In embodiments, the modified nucleobases are 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-methyl-cytosine, 5-halo-cytosine, 2-thio-cytosine, or 2-thio-5-methyl-cytosine. In embodiments, the modified nucleobases are 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenine, 7-deadenine, N6-methyl-adenine, or 2-methylthio-N6-methyl-adenine. In embodiments, the modified nucleobase is inosine, 1-methyl-inosine, 7-cyano-7-deazo-guanine, 7-aminomethyl-7-deazo-guanine, 6-thio-guanine, 6-thio-7-deazo-guanine, or 6-methoxy-guanine. In embodiments, the modified nucleoside comprises a modified sugar. In embodiments, the modified sugar has a 5-membered ring or a 6-membered ring, or is a modified ribose. In embodiments, the modified ribose is 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, or 3'-amino-2',3'-dideoxyribose. In embodiments, the modified nucleoside comprises a morpholino ring. In this embodiment, the internucleotide bond comprises a modified phosphate ester. The modified phosphate ester has one or more modifications relative to the unmodified phosphate ester, such as substitution of oxygen with different atoms or groups.In embodiments, the modified phosphate ester is a phosphorothioate, dithiophosphate, thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphonate, phosphoroselenoate, selenophosphate, aminophosphate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate, methylphosphonate, or guanidinopropyl aminophosphate. In embodiments, the last nucleoside in the 3'-stable region does not contain a 3'-hydroxyl group. In embodiments, the last nucleoside in the 3'-stable region is ddC, reverse dT, 3'-phosphate nucleoside, 3'-oxime nucleoside, 3'-azidomethyl nucleoside, or 3'-methyl nucleoside.

[0016] In one embodiment, the poly-A region is 10 or more nucleotides long. In another embodiment, the poly-A region is 30 or more nucleotides long. In another embodiment, the poly-A region is 70 or more nucleotides long. In another embodiment, the poly-A region is 100 or more nucleotides long. In another embodiment, the poly-A region is 2 to 500 nucleotides long. In another embodiment, the poly-A region is 5 to 250 nucleotides long. In another embodiment, the poly-A region is 10 to 200 nucleotides long. In another embodiment, the poly-A region is 15 to 150 nucleotides long. In this embodiment, the RNA molecule is messenger RNA (mRNA).

[0017] In one aspect, this document provides a cell comprising any of the RNA molecules described herein. In an embodiment, the cell is an isolated cell. In another aspect, this document provides a cell comprising a protein or peptide translated from any of the RNA molecules described herein.

[0018] In one aspect, this document provides a pharmaceutical composition comprising any of the RNA molecules described herein and a pharmaceutically acceptable carrier. In embodiments, a pharmaceutically acceptable carrier is a solvent, dispersion medium, diluent, surfactant, isotonic agent, thickener or emulsifier, lipid, liposome, nanoparticle, lipid nanoparticle (LNP), polymer, lipid complex, protein, or mixture thereof. In one embodiment, a pharmaceutically acceptable carrier is an LNP. In another aspect, this document provides a pharmaceutical composition comprising cells, said cells comprising any of the RNA molecules described herein.

[0019] In one aspect, this document provides a method for increasing the expression of a target protein or peptide in cells, comprising contacting cells with any of the RNA molecules described herein, wherein the RNA molecule encodes the target protein or peptide, and wherein the expression is increased compared to the expression of an RNA molecule lacking a 3'-stabilizing region. In an embodiment, the cells are isolated in vitro or ex vivo. In another aspect, this document provides a method for expressing a target protein or peptide in cells, comprising contacting cells with any of the RNA molecules described herein, wherein the RNA molecule encodes the target protein or peptide, and the cells translating the target protein or peptide from the RNA molecule. In an embodiment, the cells are isolated in vitro or ex vivo. In another aspect, this document provides a method for increasing the half-life of RNA molecules in cells, comprising contacting cells with any of the RNA molecules described herein, wherein the RNA molecule encodes the target protein or peptide, and the cells translating the target protein or peptide from the RNA molecule. In an embodiment, the cells are isolated in vitro or ex vivo.

[0020] In one aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, an ORF encoding a protein, and a poly-A region located at the 3' end of the ORF, wherein the stable region is added to the 3' end of the poly-A region and the stable region comprises one or more purification tags and / or one or more adapters capable of binding purification tags. In an embodiment, the 3'-stable region is ligated. In an embodiment, the 3'-stable region is ligated by chemical or enzymatic ligation. In an embodiment, the 3'-stable region is ligated using a polymerase.

[0021] In one aspect, this document provides a method for treating a disease in a subject in need, comprising introducing an effective amount of any of the RNA molecules, cells, or pharmaceutical compositions described herein. In another aspect, this document provides a method for preventing a disease in a subject in need, comprising introducing an effective amount of any of the RNA molecules, cells, or pharmaceutical compositions described herein.

[0022] In one respect, this article provides a compound of formula (III) or (IV): Where N is a nucleoside; L is a adapter that can bind to the purification tag; P stands for purification tag; Q-L1 may optionally exist, where L1 is a connector covalently bonded to N and Q; and Q is hydrogen or a chain-terminating nucleoside.

[0023] In one embodiment, the adapter capable of binding the purification tag is linked to the nucleoside via the 3'-carbon or 2'-carbon of the nucleoside's sugar. In another embodiment, the adapter capable of binding the purification tag is linked to the nucleoside via the nucleobase, and the L1 adapter is linked to the nucleoside via the 3'-carbon or 2'-carbon of the nucleoside's sugar. In yet another embodiment, the purification tag is linked to the nucleoside via the 3'-carbon or 2'-carbon of the nucleoside's sugar. In yet another embodiment, the purification tag is linked to the nucleoside via the nucleobase.

[0024] In one aspect, this document provides a method for increasing the expression of a target protein or peptide in cells, comprising contacting cells with an RNA molecule comprising any one of the compounds of formula (III) or (IV) described herein, wherein the RNA molecule encodes the target protein or peptide, and wherein the expression is increased compared to the expression of an RNA molecule not comprising a compound of formula (III) or (IV). In an embodiment, the cells are isolated in vitro or ex vivo. In another aspect, this document provides a method for prolonging the half-life of RNA molecules in cells, comprising contacting cells with an RNA molecule comprising any one of the compounds of formula (III) or (IV) described herein, wherein the half-life is prolonged compared to the half-life of an RNA molecule not comprising a compound of formula (III) or (IV). In an embodiment, the cells are isolated in vitro or ex vivo. Attached Figure Description

[0025] Figure 1A -B shows the HPLC curves of sequence 1a formed by the reaction of (A) 15-meric oligonucleotide (SEQ ID NO: 1) and (B) with SEQ ID NO: 1 and 2,5-dioxopyrrolidine-1-yl ester of hexanoic acid.

[0026] Figure 2A -C shows the HPLC curves for the following: (a) 39-mer oligonucleotide (SEQ ID NO: 2); (B) sequence 2a formed by linking SEQ ID NO: 2 with compound 1; and (C) sequence 2b formed by linking SEQ ID NO: 2 with compound 2.

[0027] Figure 3A -D shows the HPLC curves for the following: (a) 15-mer oligonucleotide (SEQ ID NO: 1); (B) 39-mer oligonucleotide (SEQ ID NO: 2); (C) sequence 2c formed by ligating SEQ ID NO: 1 and SEQ ID NO: 2, and unreacted SEQ ID NO: 1; (D) sequence 2d formed by ligating sequence 1a and SEQ ID NO: 2, and unreacted SEQ ID NO: 2 and sequence 1a.

[0028] Figure 4A -C shows the HPLC curves of the following: (A) firefly luciferase (FLuc) mRNA; (B) sequence 3 formed by ligating FLuc mRNA with compound 1, and unreacted FLuc mRNA was also detected in the same peak; (C) sequence 5 formed by ligating FLuc mRNA with compound 2, and unreacted FLuc mRNA.

[0029] Figure 5A -C shows the HPLC curves of the following: (A) firefly luciferase (FLuc) mRNA; (B) sequence 4 formed by ligating FLuc mRNA with SEQ ID NO: 1, and unreacted FLuc mRNA was also detected in the same peak; (C) sequence 6 formed by ligating FLuc mRNA with sequence 1a, and unreacted FLuc mRNA.

[0030] Figure 6A -C shows the HPLC curves of the following: (A) firefly luciferase (FLuc) mRNA; (B) sequence 3 formed by linking FLuc mRNA with compound 1, and unreacted FLuc mRNA was also detected in the same peak; (C) sequence 7 formed by linking FLuc mRNA with compound 4, and unreacted FLuc mRNA.

[0031] Figure 7A -C shows the HPLC curves of the following: (A) firefly luciferase (FLuc) mRNA; (B) sequence 4 formed by ligating FLuc mRNA with SEQ ID NO: 1, and unreacted FLuc mRNA was also detected in the same peak; (C) sequence 8 formed by ligating FLuc mRNA with sequence 1b, and unreacted FLuc mRNA.

[0032] Figure 8A -C shows the HPLC curves of the following: (A) firefly luciferase (FLuc) mRNA; (B) sequence 3 formed by linking FLuc mRNA with compound 1, and unreacted FLuc mRNA was also detected in the same peak; (C) sequence 9 formed by linking FLuc mRNA with compound 6, and unreacted FLuc mRNA.

[0033] Figure 9A-C shows the HPLC curves for each of the following: (A) firefly luciferase (FLuc) mRNA; (B) sequence 4 formed by ligating FLuc mRNA with SEQ ID NO: 1, and unreacted FLuc mRNA also detected in the same peak; (C) sequence 10 formed by ligating FLuc mRNA with sequence 1c, and unreacted FLuc mRNA.

[0034] Figure 10A -B shows the HPLC curves for each of the following: (A) enhanced green fluorescent protein (eGFP) mRNA (SEQ ID NO: 21) and (B) eGFP mRNA with tail modification (Sequence 23).

[0035] Figure 11 HPLC curves showing co-injection of eGFP mRNA (SEQ ID NO: 21) and eGFP mRNA with tail modification (Sequence 12).

[0036] Figure 12 The translation of mRNA encoding eGFP (prepared with various tail modifications) in 293T cells over time (10 ng / well mRNA).

[0037] Figure 13 Total protein expression (10 ng / well mRNA) was shown 96 hours after transfection of T-293 cells with mRNA encoding eGFP (prepared with various tail modifications).

[0038] Figure 14 Shows the change in translation over time of mRNA encoding eGFP (prepared with various tail modifications) in A549 cells (10 ng / well mRNA).

[0039] Figure 15 Total protein expression (10 ng / well mRNA) was shown 96 hours after transfection of the mRNA encoding eGFP (prepared with various tail modifications) in A549 cells. Detailed Implementation

[0040] definition: Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents, applications, published applications, and other disclosures mentioned herein are incorporated herein by reference in their entirety. Where a definition set forth in this section contradicts or otherwise differs from the definition set forth in a patent, application, or other disclosure incorporated herein by reference, the definition set forth in this section shall prevail with respect to the definition set forth in this section.

[0041] As used herein, “a” or “an” means “at least one” or “one or more”. For example, references to “a transcript” may include multiple transcripts.

[0042] Unless the context clearly indicates otherwise, as used herein, “or” is used in an inclusive sense, that is, equivalent to “and / or”.

[0043] As used herein, “or mixtures thereof” means any combination of the components, including any amount of each component and any combination thereof. Components may exist individually or in combination with each other (in any ratio). For example, when a material is stated to consist of substances A, B, C, or mixtures thereof, it means that the material may consist of A alone, B alone, C alone, or A and B, A and C, B and C, or any combination (mixture) of all A, B, and C.

[0044] Unless otherwise required, the use of any and all instances or exemplary language (e.g., “for example”) provided herein is intended only to better illustrate the invention and does not limit the scope of the invention.

[0045] Unless the context clearly indicates otherwise, the terms “may,” “may be,” “can,” and “can be,” and related terms, are intended to convey that the subject matter is optional (i.e., the subject matter exists in some instances and not in others), and not to refer to the ability or probability of the subject matter.

[0046] The terms “optional” and “optionally” mean that the event, situation, or material described below may or may not occur or not exist, and the description includes both the occurrence and absence of the event, situation, or material.

[0047] As used herein, the term "about" refers to a range of values ​​that includes a specified value, which those skilled in the art will consider to be substantially similar to the specified value. In embodiments, "about" refers to a range within a standard deviation obtained using measurement methods generally acceptable in the art. In embodiments, "about" refers to a range extending to + / - 10% of the specified value. In embodiments, "about" includes the specified value.

[0048] The abbreviations used in this article have their conventional meanings in the fields of chemistry and biology. The chemical structures and formulas described in this article are constructed according to the standard rules of chemical valence known in the field of chemistry.

[0049] The range includes the endpoints of the range. For example, "between 0 and 2" includes 0, 1, 2, and (unless the context requires otherwise) fractions greater than 0 and less than 2.

[0050] Where substituents are specified by their conventional chemical formula written from left to right, they also encompass chemically identical substituents produced by structures written from right to left, such as -CH2O- being equivalent to -OCH2-.

[0051] Unless otherwise stated, the term "alkyl" itself, or as part of another substituent, refers to a straight-chain (i.e., unbranched) or branched carbon chain (or carbon) or combination thereof, which may be fully saturated, monounsaturated, or polyunsaturated, and may include monovalent, divalent, and polyvalent groups. An alkyl group may include a specified number of carbons (e.g., C1-C1). 10 (Refers to one to ten carbon atoms). Alkyl groups are uncyclic chains. Examples of saturated hydrocarbon groups include (but are not limited to) groups such as each of the following: methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, methyl, such as homologues and isomers of n-pentyl, n-hexyl, n-heptyl, and n-octyl. Unsaturated alkyl groups are groups having one or more double or triple bonds. Examples of unsaturated alkyl groups include (but are not limited to) vinyl, 2-propenyl, crotonyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl, and 3-propynyl, 3-butynyl, and high-carbon homologues and isomers. Alkoxy groups are alkyl groups attached to the rest of the molecule via an oxygen linker (-O-). The alkyl moiety can be an alkenyl moiety. The alkyl moiety can be an alkynyl moiety. The alkyl moiety can be fully saturated. In addition to one or more double bonds, an alkenyl group may also include more than one double bond and / or one or more triple bonds. An alkynyl group may include more than one triple bond and / or one or more double bonds as well as one or more triple bonds.

[0052] Unless otherwise stated, the term "alkylene" itself, or as part of another substituent, refers to a divalent group derived from an alkyl group, exemplified but not limited to -CH2CH2CH2CH2-. Typically, alkyl (or alkylene) groups have 1 to 24 carbon atoms, with those having 10 or fewer carbon atoms being preferred herein. "Lower alkyl" or "lower alkylene" generally refers to a shorter-chain alkyl or alkylene group having eight or fewer carbon atoms. Unless otherwise stated, the term "alkenyl" itself, or as part of another substituent, refers to a divalent group derived from an olefin.

[0053] Unless otherwise stated, the term "heteroalkyl," alone or in combination with another term, refers to a stable straight or branched chain, or a combination thereof, comprising at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. One or more heteroatoms (e.g., O, N, S, Si, or P) may be located at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. The heteroalkyl group is an uncyclic chain. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-S-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. At most two or three heteroatoms can be consecutive, such as -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. The heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may contain up to eight optionally different heteroatoms (e.g., O, N, S, Si, or P). Unless otherwise stated, the term "heteroalkenyl," alone or in combination with another term, refers to a heteroalkyl group containing at least one double bond. A heteroalkenyl group may optionally contain more than one double bond and / or one or more triple bonds in addition to one or more double bonds. Unless otherwise stated, the term "heteroyynyl," alone or in combination with another term, refers to a heteroalkyl group containing at least one triple bond. A heteroyynyl group may optionally contain more than one triple bond and / or one or more double bonds in addition to one or more triple bonds.

[0054] Similarly, unless otherwise stated, the term "heteroalkylene" itself, or as part of another substituent, refers to a divalent group derived from a heteroalkylene group, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the heteroatom can also occupy any or both of the chain ends (e.g., alkeneoxy, alkenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, the direction in which the linking group's chemical formula is written does not imply the orientation of the linking group. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As mentioned above, heteroalkylene groups as used herein include those groups that are linked to the remainder of the molecule via a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SO2R'. When referring to "heteroalkyl," and subsequently to a specific heteroalkyl group such as -NR'R'', it should be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, referring to a specific heteroalkyl group adds clarification. Therefore, the term "heteroalkyl" should not be interpreted herein as excluding a specific heteroalkyl group such as -NR'R''.

[0055] Unless otherwise stated, the terms "cycloalkyl" and "heterocycloalkyl" on their own or in combination with other terms refer to the cyclic form of "alkyl" and "heteroalkyl," respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, the heteroatom may occupy a position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. Examples of heterocycloalkyl groups include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and similar groups. "Cycloalkylene" and "heterocycloalkylene" alone or as part of another substituent refer to divalent groups derived from cycloalkylene and heterocycloalkylene, respectively.

[0056] In embodiments, the term "cycloalkyl" refers to a monocyclic, bicyclic, or polycyclic cycloalkyl ring system. In embodiments, a monocyclic system is a cycloalkyl group containing 3 to 8 carbon atoms, wherein such groups may be saturated or unsaturated, but are not aromatic. In embodiments, the cycloalkyl group is fully saturated. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclic or fused bicyclic rings. In embodiments, the bridged monocyclic ring contains a monocyclic cycloalkyl group, wherein two non-adjacent carbon atoms of the monocyclic ring are bridged by an alkylene bridge having one to three additional carbon atoms (i.e., in the form (CH2)). w The bridging group, wherein w is a 1, 2, or 3 bond. Representative examples of bicyclic systems include (but are not limited to) bicyclic [3.1.1]heptane, bicyclic [2.2.1]heptane, bicyclic [2.2.2]octane, bicyclic [3.2.2]nonane, bicyclic [3.3.1]nonane, and bicyclic [4.2.1]nonane. In embodiments, the fused bicyclic cycloalkyl ring system contains a monocyclic cycloalkyl ring fused to a phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocyclic, or monocyclic heteroaryl group. In embodiments, the bridging or fused bicyclic cycloalkyl group is connected to the parent molecule moiety via any carbon atom contained within the monocyclic cycloalkyl ring. In embodiments, the cycloalkyl group is optionally substituted with one or two groups that are independently oxo or thiazolyl. In embodiments, the fused bicyclic cycloalkyl group is a 5- or 6-membered monocyclic cycloalkyl ring fused to a benzene ring, a 5- or 6-membered monocyclic cycloalkyl group, a 5- or 6-membered monocyclic cycloalkenyl group, a 5- or 6-membered monocyclic heterocyclic group, or a 5- or 6-membered monocyclic heteroaryl group, wherein the fused bicyclic cycloalkyl group is optionally substituted by one or two independently oxo or thiazo groups. In embodiments, the polycyclic cycloalkyl ring system is a ring system fused to (i) a ring system selected from the group consisting of: bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclic groups; or (ii) two other ring systems independently selected from the group consisting of: phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclic groups (base ring). In embodiments, the polycyclic cycloalkyl group is connected to the parent molecule moiety through any carbon atom contained within the base ring. In embodiments, the polycyclic cycloalkyl ring system is fused to a monocyclic cycloalkyl ring (base ring) of the following: (i) a ring system selected from the group consisting of: bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclic; or (ii) two other ring systems independently selected from the group consisting of: phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclic. Examples of polycyclic cycloalkyl include (but are not limited to) tetradecylhydrophenanthrene, perhydrophenothiazin-1-yl, and perhydrophenotoxazin-1-yl.

[0057] In embodiments, the cycloalkyl group is a cycloalkenyl group. The term "cycloalkenyl" is used according to its common meaning. In embodiments, the cycloalkenyl group is a monocyclic, bicyclic, or polycyclic cycloalkenyl ring system. In embodiments, a monocyclic cycloalkenyl ring system is a cycloalkyl group containing 3 to 8 carbon atoms, wherein such groups are unsaturated (i.e., contain at least one cyclic carbon-carbon double bond) but are not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. In embodiments, the bicyclic cycloalkenyl ring is a bridged monocyclic or fused bicyclic ring. In embodiments, a bridged monocyclic ring contains a monocyclic cycloalkenyl ring, wherein the two non-adjacent carbon atoms of the monocyclic ring are bridged by an alkylene bridge having one to three additional carbon atoms (i.e., in the form (CH2)). w The bridging group, wherein w is a 1, 2, or 3-bond. Representative examples of bicyclic cycloalkenyl groups include (but are not limited to) norbornenyl and bicyclic [2.2.2]oct-2-enyl. In embodiments, the fused bicyclic cycloalkenyl ring system contains a monocyclic cycloalkenyl ring fused to a phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocyclic, or monocyclic heteroaryl group. In embodiments, the bridging or fused bicyclic cycloalkenyl group is connected to the parent molecule moiety via any carbon atom contained within the monocyclic cycloalkenyl ring. In embodiments, the cycloalkenyl group is optionally substituted with one or two groups that are independently oxo or thiazolyl. In embodiments, the polycyclic cycloalkenyl ring comprises a monocyclic cycloalkenyl ring (base ring) fused to: (i) a ring system selected from the group consisting of: bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclic groups; or (ii) two independently selected ring systems consisting of: phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclic groups. In embodiments, the polycyclic cycloalkenyl group is connected to the parent molecule moiety via any carbon atom contained within the base ring. In embodiments, the polycyclic cycloalkenyl ring comprises a monocyclic cycloalkenyl ring (base ring) fused to: (i) a ring system selected from the group consisting of: bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclic; or (ii) two ring systems independently selected from the group consisting of: phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclic.

[0058] In embodiments, heterocyclic alkyl groups are heterocyclic groups. As used herein, the term "heterocyclic group" refers to a monocyclic, bicyclic, or polycyclic heterocycle. A heterocyclic monocyclic heterocycle is a 3-, 4-, 5-, 6-, or 7-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, P, and S, wherein the ring is saturated or unsaturated, but not aromatic. A 3- or 4-membered ring contains one heteroatom selected from the group consisting of O, N, P, and S. A 5-membered ring may contain zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, P, and S. A 6- or 7-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, P, and S. The heterocyclic monocyclic heterocycle is connected to the parent molecule moiety by any carbon or nitrogen atom contained within the heterocyclic monocyclic heterocycle. Representative examples of heterocyclic monocyclic heterocycles include, but are not limited to, azahexacyclic butyl, azahexacyclic heptyl, aziridinyl, diazacyclic heptyl, 1,3-dioxyl, 1,3-dioxopentyl, 1,3-dithiopentyl, 1,3-dithiaalkyl, imidazolinyl, imidazolinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, morpholinyl, oxadiazolinyl, oxadiazolinyl, oxazolinyl, oxazolinyl, piperazine, piperidinyl, pyranyl, pyrazolinyl, pyrazolyl, pyrrololinyl, pyrrolyl, tetrahydrofuranyl, tetrahydrothiophenyl, thiadiazolinyl, thiadiazolinyl, thiazolinyl, thiazolinyl, thiazolinyl, thiomorpholinyl, 1,1-dioxothiomorpholinyl (thiomorpholinone), thiopyranyl, and trithiaalkyl. The heterocyclic bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocycle, or monocyclic heteroaryl group. The heterocyclic bicyclic heterocycle is connected to the parent molecule moiety via any carbon or nitrogen atom contained within the monocyclic heterocyclic portion of the bicyclic system. Representative examples of bicyclic heterocyclic groups include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indoline-1-yl, indoline-2-yl, indoline-3-yl, 2,3-dihydrobenzothiophene-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, and octahydrobenzofuranyl. In embodiments, the heterocyclic group is optionally substituted with one or two independently oxo or thiazo groups. In some embodiments, the bicyclic heterocyclic group is a 5- or 6-membered monocyclic heterocyclic group fused to a benzene ring, a 5- or 6-membered monocyclic cycloalkyl group, a 5- or 6-membered monocyclic cycloalkenyl group, a 5- or 6-membered monocyclic heterocyclic group, or a 5- or 6-membered monocyclic heteroaryl group, wherein the bicyclic heterocyclic group is optionally substituted with one or two groups that are independently oxo or thiazo groups.Polycyclic heterocyclic ring systems are fused to the following monocyclic heterocyclic rings (base rings): (i) a ring system selected from the group consisting of: bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclic groups; or (ii) two other ring systems independently selected from the group consisting of: phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclic groups. The polycyclic heterocyclic group is connected to the parent molecule moiety via any carbon or nitrogen atom contained within the base ring. In embodiments, the polycyclic heterocyclic base ring system is fused to the following monocyclic heterocyclic base rings (base rings): (i) a ring system selected from the group consisting of: bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclic; or (ii) two other ring systems independently selected from the group consisting of: phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclic. Examples of polycyclic heterocyclic groups include (but are not limited to) 10H-phenthiazin-10-yl, 9,10-dihydroacrylin-9-yl, 9,10-dihydroacrylin-10-yl, 10-phenoxazin-10-yl, 10,11-dihydro-5-dibenzo[b,f]azapheno-5-yl, 1,2,3,4-tetrahydropyridyl[4,3-g]isoquinoline-2-yl, 12H-benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazole-9-yl.

[0059] Unless otherwise stated, the term "halogen" or "halogen" itself, or as a substituent, refers to a fluorine, chlorine, bromine, or iodine atom. Additionally, the term "haloalkyl," for example, is intended to include both monohaloalkyl and polyhaloalkyl groups. For instance, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and other groups.

[0060] Unless otherwise stated, the term "acyl" means -C(O)R, where R is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0061] Unless otherwise stated, the term "aryl" refers to a polyunsaturated aromatic hydrocarbon substituent, which can be a single ring or fused together (i.e., a fused-ring aryl) or multiple rings covalently linked (preferably 1 to 3 rings). A fused-ring aryl refers to multiple rings fused together, wherein at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to an aryl (or ring) containing at least one heteroatom (e.g., N, O, or S), wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl (i.e., multiple rings fused together, wherein at least one of the fused rings is a heteroaromatic ring). 5,6-fused-ring heteroaryl refers to two fused rings, one ring having 5 members and the other ring having 6 members, and at least one of the rings being a heteroaryl ring. Similarly, 6,6-fused-ring heteroaryl refers to two fused rings, one with six members and the other with six members, and at least one of the rings is a heteroaryl ring. And 6,5-fused-ring heteroaryl refers to two fused rings, one with six members and the other with five members, and at least one of the rings is a heteroaryl ring. The heteroaryl group can be attached to the rest of the molecule via a carbon atom or a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, thiopheneyl, pyridinyl, pyrimidinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzofuran, isobenzofuranyl, indoleyl, isoindoleyl, benzothiaphenyl, isoquinolinyl, quinoxalinyl, quinolinyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl The substituents in the aryl and heteroaryl ring systems mentioned above are selected from the group of acceptable substituents described below. "Arylidene" and "heteroarylidene" refer, alone or as part of another substituent, to divalent groups derived from aryl and heteroaryl groups, respectively. Heteroaryl substituents can be bonded to the cyclic heteroatom nitrogen via -O-.

[0062] Fused-ring heterocyclic alkyl-aryl is an aryl group fused with a heterocyclic alkyl group. Fused-ring heterocyclic alkyl-heteroaryl is a heteroaryl group fused with a heterocyclic alkyl group. Fused-ring heterocyclic alkyl-cycloalkyl is a heterocyclic alkyl group fused with a cycloalkyl group. Fused-ring heterocyclic alkyl-heterocyclic alkyl is a heterocyclic alkyl group fused with another heterocyclic alkyl group. Fused-ring heterocyclic alkyl-aryl, fused-ring heterocyclic alkyl-heteroaryl, fused-ring heterocyclic alkyl-cycloalkyl, or fused-ring heterocyclic alkyl-heterocyclic alkyl may each be independently unsubstituted or substituted with one or more substituents described herein.

[0063] A spirocyclic ring is two or more rings, wherein adjacent rings are connected by a single atom. Individual rings within a spirocyclic ring may be the same or different. Individual rings within a spirocyclic ring may be substituted or unsubstituted and may have substituents different from those of other individual rings in a group of spirocyclic rings. When not part of a spirocyclic ring, the possible substituents of individual rings within a spirocyclic ring are possible substituents of the same ring (e.g., substituents of cycloalkyl or heterocyclic alkyl rings). A spirocyclic ring may be a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heteroalkyl, or a substituted or unsubstituted heteroalkylene, and the individual rings within a spirocyclic ring may be any of those just listed above, including all rings of one type (e.g., all rings are substituted heteroalkylene, wherein each ring may be the same or different substituted heteroalkylene). When referring to a spirocyclic system, a heterocyclic spirocyclic ring means a spirocyclic ring in which at least one ring is heterocyclic and each ring may be different. When referring to a spirocyclic system, a substituted spirocyclic ring means at least one ring is substituted and each substituent may optionally be different.

[0064] symbol" "" indicates the connection point between the chemical part and the rest of the molecule or chemical formula.

[0065] As used in this article, the term "oxo" refers to oxygen double-bonded to a carbon atom.

[0066] As used herein, the term "alkylsulfonyl" refers to a portion having the formula S(O2)-R', where R is a substituted or unsubstituted alkyl group as defined above. R' may have a specified number of carbons (e.g., C1-C4 alkylsulfonyl).

[0067] The term "alkylarylene" refers to an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, the alkylarylene has the following formula: or .

[0068] The alkylarylene moiety may be substituted (e.g., substituted with substituents) at the alkylene moiety or the arylene junction (e.g., at carbon 2, 3, 4, or 6) with the following: halogen, oxo, -N3, -CF3, -CCl3, -CBr3, -CI3, -CN, -CHO, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2CH3, -SO3H, -OSO3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, substituted or unsubstituted C1-C5 alkyl groups, or substituted or unsubstituted 2- to 5-membered heteroalkyl groups. In the embodiments, the alkylarylene moiety is unsubstituted.

[0069] Each of the above terms (e.g., "alkyl", "heteroalkyl", "cycloalkyl", "heterocycloalkyl", "aryl", and "heteroaryl") includes both the substituted and unsubstituted forms of the indicated group. Preferred substituents for each type of group are provided below.

[0070] Substituents in alkyl and heteroalkyl groups (including those commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, ynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) may be selected from (but not limited to) one or more of the following groups: -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O) The number of these groups ranges from zero to (2m'+1), where m' is the total number of carbon atoms in these groups. R, R', R'', R''', and R'''' each preferably independently refer to hydrogen, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted aryl group (e.g., an aryl group substituted with 1-3 halogens), a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkyl group, an alkoxy or thioalkoxy or aralkyl group. When the compounds described herein include more than one R group, each of the R groups is independently selected as an R', R'', R''', and R'''' group (e.g., when more than one of these groups is present). When R' and R'' are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. Based on the above discussion of substituents, those skilled in the art should understand that the term "alkyl" is intended to include groups containing a carbon atom bonded to a group other than a hydrogen group, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0071] Similar to the substituents described for alkyl groups, the substituents for aryl and heteroaryl groups are different and selected from, for example: -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R'', -NR''C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -NR'NR''R''', -ON R'R'', -NR'C(O)NR''NR'''R'''', -CN, -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy and fluoro(C1-C4)alkyl, -NR'SO2R'', -NR'C(O)R'', -NR'C(O)-OR'', -NR'OR'', wherein the number is in the range of zero to the total number of open valence atoms in the aromatic ring system; and wherein R', R'', R''' and R'''' are preferably independently selected independently from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When the compounds described herein include more than one R group, each of the R groups is independently selected as an R', R'', R''', and R'''' group (e.g., when more than one of these groups is present).

[0072] Substituents in the ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) can be described as substituents on the ring rather than on a specific atom of the ring (often referred to as floating substituents). In this case, the substituent can be attached to any atom of the ring (following the valence rules), and in the case of fused or spirocyclic rings, the substituent is described as associated with a member of the fused or spirocyclic ring (floating substituent on a single ring), and can be a substituent on any of the fused or spirocyclic rings (floating substituents on multiple rings). When the substituent is attached to the ring rather than a specific atom (floating substituent) and the substituent subscript is an integer greater than one, multiple substituents can be on the same atom, the same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent can optionally be different. Where the connection point between the ring and the rest of the molecule is not limited to a single atom (floating substituent), the connection point can be any atom of the ring, and in the case of fused or spirocyclic rings, the connection point can be any atom of either the fused or spirocyclic ring, while following the valence rules. In the case where a ring, fused ring, or spirocyclic ring contains one or more cyclic heteroatoms and the ring, fused ring, or spirocyclic ring is shown to have another floating substituent (including, but not limited to, a connection point with the rest of the molecule), the floating substituent may be bonded to the heteroatom. When the cyclic heteroatom in a structure or chemical formula with a floating substituent is shown to be bonded to one or more hydrogen atoms (e.g., a cyclic nitrogen with two bonds to the ring atom and a third bond to the hydrogen), the substituent should be understood as replacing the hydrogen atom when the heteroatom is bonded to the floating substituent, while obeying the rules of chemical valence.

[0073] Two or more substituents may optionally be linked to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Although not always necessary, it is commonly found that such so-called cyclizing substituents are linked to a cyclic base structure. In one embodiment, the cyclizing substituent is linked to an adjacent member of the base structure. For example, two cyclizing substituents linked to an adjacent member of the cyclic base structure produce a fused ring structure. In another embodiment, the cyclizing substituent is linked to a single member of the base structure. For example, two cyclizing substituents linked to a single member of the cyclic base structure produce a spirocyclic structure. In yet another embodiment, the cyclizing substituent is linked to a non-adjacent member of the base structure.

[0074] The two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form the formula -TC(O)-(CRR'). q A -U- ring, where T and U are independently -NR-, -O-, -CRR'-, or single bonds, and q is an integer from 0 to 3. Alternatively, the two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be cyclic with the formula -A-(CH2). rThe substituent substitution of -B-, where A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer from 1 to 4. One single bond in the resulting new ring may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by the formula -(CRR'). s -X'-(C''R''R''') d The substituents are replaced by -, wherein s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituents R, R', R'', and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0075] As used herein, the term "heteroatom" or "cyclic heteroatom" refers to oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0076] As used herein, "substituent" refers to a group selected from the following: (A) Oxide, Halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3 -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl groups (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl groups (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl groups (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl groups (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl groups (e.g., C6-C... 10 Aryl, C 10Aryl or phenyl) or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (B) Alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C4 alkyl). 10 Aryl, C 10 aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), wherein the group is substituted with at least one substituent selected from the following: (i) Oxygenated, Halogenated, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3 -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl groups (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl groups (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl groups (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl groups (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl groups (e.g., C6-C... 10 Aryl, C 10 Aryl or phenyl) or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (ii) Alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C4 alkyl).10 Aryl, C 10 aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), wherein the group is substituted with at least one substituent selected from the following: (a) Oxygenated, Halogenated, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3 -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl groups (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl groups (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl groups (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl groups (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl groups (e.g., C6-C... 10 Aryl, C 10 Aryl or phenyl) or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (b) Alkyl groups (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl groups (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl groups (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl groups (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl groups (e.g., C6-C4 alkyl). 10 Aryl, C 10aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), wherein the group is substituted by at least one of the following substituents: oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O) ... -H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl groups (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl groups (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl groups (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl groups (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl groups (e.g., C6-C... 10 Aryl, C 10 Aryl or phenyl) or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl or 5- to 6-membered heteroaryl).

[0077] As used herein, “size-restricted substituent” or “size-restricted substituent group” refers to a group selected from all the substituents described above for “substituent group”, wherein each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C2 alkyl group. 20 Alkyl groups, each substituted or unsubstituted heteroalkyl group being a substituted or unsubstituted 2- to 20-membered heteroalkyl group; each substituted or unsubstituted cycloalkyl group being a substituted or unsubstituted C3-C8 cycloalkyl group; each substituted or unsubstituted heterocycloalkyl group being a substituted or unsubstituted 3- to 8-membered heterocycloalkyl group; and each substituted or unsubstituted aryl group being a substituted or unsubstituted C6-C8 cycloalkyl group. 10 Aryl group, and each substituted or unsubstituted heteroaryl group is a substituted or unsubstituted 5- to 10-membered heteroaryl group.

[0078] As used herein, “lower substituent” or “lower substituent group” refers to a group selected from all the substituents described above for “substituent group”, wherein each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C8 alkyl group, each substituted or unsubstituted heteroalkyl group is a substituted or unsubstituted 2- to 8-membered heteroalkyl group, each substituted or unsubstituted cycloalkyl group is a substituted or unsubstituted C3- to 7-membered cycloalkyl group, each substituted or unsubstituted heterocycloalkyl group is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl group, each substituted or unsubstituted aryl group is a substituted or unsubstituted phenyl group, and each substituted or unsubstituted heteroaryl group is a substituted or unsubstituted 5- to 6-membered heteroaryl group.

[0079] In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein is substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-restricted substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.

[0080] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl group may be a substituted or unsubstituted C1-C2 group. 20 Alkyl groups, each substituted or unsubstituted heteroalkyl group being a substituted or unsubstituted 2- to 20-membered heteroalkyl group; each substituted or unsubstituted cycloalkyl group being a substituted or unsubstituted C3-C8 cycloalkyl group; each substituted or unsubstituted heterocycloalkyl group being a substituted or unsubstituted 3- to 8-membered heterocycloalkyl group; and each substituted or unsubstituted aryl group being a substituted or unsubstituted C6-C8 cycloalkyl group. 10 The aryl group, and / or each substituted or unsubstituted heteroaryl group, is a substituted or unsubstituted 5- to 10-membered heteroaryl group. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene group is a substituted or unsubstituted C1-C... 20Alkylenes, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2- to 20-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 3- to 8-membered heteroalkylene, and each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C 10 A aryl group, and / or each substituted or unsubstituted heteroaryl group is a substituted or unsubstituted 5- to 10-membered heteroaryl group.

[0081] In some embodiments, each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C8 alkyl group, each substituted or unsubstituted heteroalkyl group is a substituted or unsubstituted 2- to 8-membered heteroalkyl group, each substituted or unsubstituted cycloalkyl group is a substituted or unsubstituted C3-C7 cycloalkyl group, each substituted or unsubstituted heterocycloalkyl group is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl group, and each substituted or unsubstituted aryl group is a substituted or unsubstituted C6-C8 cycloalkyl group. 10 The aryl group, and / or each substituted or unsubstituted heteroaryl group is a substituted or unsubstituted 5- to 9-membered heteroaryl group. In some embodiments, each substituted or unsubstituted alkylene group is a substituted or unsubstituted C1-C8 alkylene group, each substituted or unsubstituted heteroalkylene group is a substituted or unsubstituted 2- to 8-membered heteroalkylene group, each substituted or unsubstituted cycloalkylene group is a substituted or unsubstituted C3-C7 cycloalkylene group, each substituted or unsubstituted heteroalkylene group is a substituted or unsubstituted 3- to 7-membered heteroalkylene group, and each substituted or unsubstituted aryl group is a substituted or unsubstituted C6-C8 alkylene group. 10 The aryl group, and / or each substituted or unsubstituted heteroaryl group is a substituted or unsubstituted 5- to 9-membered heteroaryl group. In some embodiments, the compound is a chemical species illustrated in the Examples section, figures, or tables below.

[0082] In the embodiments, the substituted or unsubstituted portions (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and / or substituted or unsubstituted heteroaryl) are unsubstituted (e.g., unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkyl, unsubstituted aryl and / or unsubstituted heteroaryl). In the embodiments, the substituted or unsubstituted portions (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene and / or substituted or unsubstituted heteroaryl) are substituted (e.g., respectively substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroaryl).

[0083] In embodiments, the substituted portion (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent group, wherein if the substituted portion is substituted with multiple substituent groups, then each substituent group may optionally be different. In embodiments, if the substituted portion is substituted with multiple substituent groups, then each substituent group is different.

[0084] In embodiments, the substituted portion (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one size-restricted substituent, wherein if the substituted portion is substituted with multiple size-restricted substituents, then each size-restricted substituent may optionally be different. In embodiments, if the substituted portion is substituted with multiple size-restricted substituents, then each size-restricted substituent is different.

[0085] In embodiments, the substituted portion (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one lower substituent group, wherein if the substituted portion is substituted with multiple lower substituent groups, then each substituent group may optionally be different. In embodiments, if the substituted portion is substituted with multiple lower substituent groups, then each lower substituent group is different.

[0086] In embodiments, the substituted portion (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent group, a size-restricted substituent group, or a lower substituent group; wherein if the substituted portion is substituted with multiple substituent groups selected from substituent groups, size-restricted substituent groups, and lower substituent groups, each substituent group, size-restricted substituent group, and / or lower substituent group may optionally be different. In embodiments, if the substituted portion is substituted with multiple groups selected from substituent groups, size-restricted substituent groups, and lower substituent groups, then each substituent group, size-restricted substituent group, and / or lower substituent group is different.

[0087] Certain compounds disclosed herein have asymmetric carbon atoms (optical or chiral chiral centers) or double bonds; in absolute stereochemistry, they may refer to enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomers, and individual isomers defined as (R)- or (S)- or (D)- or (L)- for amino acids, and are covered within the scope of this invention. The compounds of this invention do not include those known in the art that are too unstable to be synthesized and / or isolated. This disclosure is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)- or (D)- and (L)- isomers can be prepared using chiral chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain an alkene bond or other geometrically asymmetric centers, and unless otherwise specified, it is desirable for the compounds to include E and Z geometric isomers. In some embodiments, "optical activity" and "enantiomeric activity" refer to a collection of molecules in which the enantiomers are in excess of not less than about 50%, not less than about 70%, not less than about 80%, not less than about 90%, not less than about 91%, not less than about 92%, not less than about 93%, not less than about 94%, not less than about 95%, not less than about 96%, not less than about 97%, not less than about 98%, not less than about 99%, not less than about 99.5%, or not less than about 99.8%. In some embodiments, based on the total weight of the racemic mixture, the compound comprises about 95% or more of one enantiomer and about 5% or less of another enantiomer.

[0088] As used herein, the term "isomer" refers to compounds that have the same number and type of atoms and therefore the same molecular weight, but different atomic arrangements or configurations.

[0089] As used in this article, a "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are readily convertible from one isomer to another.

[0090] It will be apparent to those skilled in the art that some of the compounds disclosed herein may exist in tautomeric forms, and all such tautomeric forms of the compounds are within the scope of this disclosure.

[0091] Unless otherwise stated, the structures described herein are also intended to include all stereochemical forms of the structures; that is, the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers of the compounds disclosed herein, as well as mixtures of enantiomers and diastereomers, are within the scope of this disclosure.

[0092] Unless otherwise stated, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, those with hydrogen replaced by deuterium or tritium, or carbon replaced by...13 C- or 14 Compounds having the structure disclosed herein, other than those enriched by carbon substitution, are all within the scope of this disclosure.

[0093] The compounds disclosed herein may also contain atomic isotopes in non-natural proportions at one or more of the atoms constituting the compound. For example, the compounds may be derived from, for instance, tritium ( 3 H), Iodine-125 ( 125 I) or carbon-14 ( 14 C) Radiolabeling with radioactive isotopes. All isotopic variants of the compounds disclosed herein, whether or not radioactive, are intended to be covered within the scope of this disclosure.

[0094] It should be noted that throughout this application, substitutes are written into the Markush group, for example, containing more than one possible amino acid at each amino acid position. Specifically, each member of the Markush group should be considered separately to include another embodiment, and the Markush group is not understood as a single unit.

[0095] For any group containing one or more substituents disclosed herein, it should be understood that such groups do not contain any substitution or substitution pattern that is sterically impractical and / or synthetically infeasible. Furthermore, the compounds of this invention include all stereochemical isomers resulting from the substitution of these compounds.

[0096] The term "its salt" refers to a compound formed when the proton of an acid is replaced by a cation (e.g., a metal cation or an organic cation). Where applicable, the salt is a pharmaceutically acceptable salt, but this is not necessary for salts of intermediate compounds not intended for administration to a patient. For example, salts of the compounds of the present invention include those salts in which the compound is protonated by an inorganic or organic acid to form a cation, wherein the conjugate base of the inorganic or organic acid is the anionic component of said salt.

[0097] The term "pharmaceutically acceptable salt" refers to a salt (a salt containing a relative ion that has acceptable mammalian safety for a given dosing regimen) acceptable for administration to patients, such as mammals (e.g., humans). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound derived from various organic and inorganic relative ions well known in the art, and, when the molecule contains basic functionality, a salt of an organic or inorganic acid. Pharmaceutically acceptable salts of the compounds described herein are suitable for contact with the tissues of a subject without adverse toxicity, irritation, anaphylactic reactions, etc., commensurate with a reasonable benefit / risk ratio, and effective for their intended use, and, where possible, in the zwitterionic form of the compounds described herein. These salts can be prepared in situ during the isolation and purification of the compounds, or by reacting the purified compound, in its free base form, with a suitable organic or inorganic acid and isolating the resulting salt. Representative salts include hydrobromide, hydrochloride, sulfate, hydrogen sulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthalenedicarboxylate, methanesulfonate, glucono-p-ethyl, lactobionate, methanesulfonate, and laurylsulfonate. Salts can include alkali metal and alkaline earth metal-based cations, such as sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. (See SM Barge et al., J. Pharm. Sci. (1977) 66, 1; and Remington: The Science and Practice of Pharmacy, 23rd ed., Adejare et al., Academic Press (2020), which are incorporated herein by reference in their entirety.)

[0098] As used herein, the term "cap analogue" refers to a structural derivative of an RNA cap. A "natural 5'-cap" is a cap structure found at the 5' end of an mRNA molecule and is typically composed of guanosine 5'-triphosphate (Gppp), which is linked to the 5' end of the next nucleotide in the mRNA via its triphosphate portion (i.e., guanosine is linked to the rest of the mRNA via a 5'-to-5' triphosphate bond). Guanosine can be present at position N. 7 Methylation (producing cap structure m) 7(GPPP). Cap analogues include those described in International Patent Publications WO2017 / 053297, WO2023 / 147352, WO2021 / 162566, WO2021 / 162567, WO2022 / 006368, WO2022 / 086140, WO2023 / 033551, WO2018 / 075827, WO2023 / 07019 and U.S. Provisional Applications 63 / 528,990 and 63 / 536844, the respective cap structures of which are incorporated herein by reference. In embodiments, the term 5'-cap, as used herein, refers to a cap analogue as described herein or any part having the biological function of a cap.

[0099] As used herein, the terms “complement,” “complementary,” or “complementarity” refer to specific base pairings between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U), and G and C. For example, the complementarity between a capped oligonucleotide primer and a DNA template can be “complete” or “holistic,” where all nucleotide bases of the two nucleic acid strands match according to accepted base pairing rules; complementarity can be “partial,” where only some nucleotide bases of the initiating capped oligonucleotide primer and the DNA template match according to accepted base pairing rules; or complementarity can be “absent,” where none of the nucleotide bases of the two nucleic acid strands match according to accepted base pairing rules. Complementarity can also be “substantially complementary,” where the nucleotide bases of the two nucleic acids match according to accepted base pairing rules, but the total complementarity includes one or more mismatches (e.g., 1, 2, 3, 4).

[0100] As used in this article, "deoxyribonuclease" (abbreviated as "DNAase") is an enzyme that catalyzes the hydrolysis and cleavage of phosphodiester bonds in the DNA backbone, thereby degrading DNA.

[0101] As used in this article, the term "impurity" refers to a substance whose chemical composition differs from that of the target material (e.g., mRNA transcript). Impurities are also known as contaminants.

[0102] "Inorganic pyrophosphatase" refers to an enzyme that catalyzes the conversion of one pyrophosphate ion into two phosphate ions, thereby inhibiting aggregation and, in some cases, preventing the interaction of pyrophosphate with magnesium ions during the T7 transcription reaction.

[0103] As used herein, the term "in vitro" refers to a process that occurs outside a living organism (e.g., a multicellular organism, such as a human or non-human animal), for example, in a test tube, a petri dish, or elsewhere outside the living organism.

[0104] As used in this article, the term "in vivo" refers to events that occur within a living organism.

[0105] As used herein, the term "in vivo assay" refers to a method for detecting and / or measuring the ability of one or more of a compound or a molecule comprising the compound (e.g., mRNA molecules, e.g., at a therapeutic dose) to increase or decrease properties relative to a control (e.g., biomarker levels). Optionally, the in vivo assays described herein can be used to determine a subject's level of tolerance to a given compound or molecule. Exemplary measurements for assessing tolerance include one or more of the following: body weight, organ weight, aspartate aminotransferase (AST) levels, alanine aminotransferase (ALT) levels, C-reactive protein (CRP) levels, procalcitonin (PCT) levels, interleukin-6 (IL-6) levels, erythrocyte sedimentation rate (ESR), serum amyloid A levels, and serum ferritin levels.

[0106] As used in this article, a "locked nucleic acid" (LNA) ring refers to a ribonucleotide with a bridge between the 2'O and 4'C methylene bicyclic nucleotide monomers. The LNA moiety can have the following structures: .

[0107] As used in this article, an "unlocked nucleic acid" (UNA) ring refers to a ribonucleotide containing a non-cyclic structure in which the bond between the 2'C and 3'C is absent. The UNA moiety can have the following structures: .

[0108] As used herein, "messenger RNA transcript" or "mRNA transcript" refers to a transcript transcribed from a DNA template encoding a desired polypeptide. The mRNA transcript may contain coding and non-coding regions. For example, the DNA template may contain an RNA polymerase promoter sequence, a 5' UTR sequence, an open reading frame, and a 3' UTR sequence. In some instances, the DNA template may also contain a nucleic acid sequence encoding a poly-A tail. In this embodiment, the DNA template may contain a 5' UTR sequence, an open reading frame, and a 3' UTR sequence.

[0109] As used herein, the term "nucleoside" refers to a nitrogenous base linked to a 5-carbon sugar (e.g., ribose or deoxyribose). The term encompasses all nucleosides, including the nucleoside base and all forms of furanose. Five naturally occurring, unmodified nucleosides exist: adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U). According to Aduri et al. (Aduri, R. et al., AMBER force field parameters for the naturally occurring modified nucleotides in RNA. Journal of Chemical Theory and Computation. 2006.) 3(4):1464-75), containing 107 naturally occurring modified nucleosides, including 1-methyl adenosine, 2-methylthio-N6-hydroxyn-valinecarbamoyl adenosine, 2-methyl adenosine, 2-O-ribosylphosphoadenosine, N6-methyl-N6-threonylcarbamoyl adenosine, N6-acetyl adenosine, N6-glycylcarbamoyl adenosine, N6-isopentenyl adenosine, N6-methyl adenosine, N6-threonylcarbamoyl adenosine, N6,N6-dimethyl N6-(cis-hydroxyisopentenyl)adenosine, N6-hydroxyn-valine aminoformyl adenosine, 1,2-O-dimethyl adenosine, N6,2-O-dimethyl adenosine, 2-O-methyl adenosine, N6,N6,O-2-trimethyl adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyl adenosine, 2-methylthio-N6-isopentenyl adenosine, 2-methylthio-N6-threonyl aminoformyl adenosine, 2-thiocytidine 3-Methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, lysidine, N4-acetyl-2-O-methylcytidine, 5-formyl-2-O-methylcytidine, 5,2-O-dimethylcytidine, 2-O-methylcytidine, N4,2-O-dimethylcytidine, N4,N4,2-O-trimethylcytidine, 1-methylguanosine, N2,7-dimethylguanosine, N2-methyl Guanosine, 2-O-ribosyl phosphate guanosine, 7-methylguanosine, undermodified hydroxyweiningoside, 7-aminomethyl-7-deazoguanosine, 7-cyano-7-deazoguanosine, N2,N2-dimethylguanosine, 4-demethylweiningoside, epoxy guanosine, hydroxyweiningoside, isoweiningoside, N2,7,2-O-trimethylguanosine, N2,2-O-dimethylguanosine, 1,2-O-dimethylguanosine, 2-O-methylguanosine, N2,N2,2-O-trimethylguanosine, N2,N2,7-Trimethylguanosine, Peroxywaitoside, Galactosylwaitoside, Mannosylwaitoside, Waitoside, Archipelagopurin, Waitoside, Methylwaitoside, Waitoside, 2-Thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3-methyluridine, 4-Thiouridine, 5-Methyl-2-thiouridine, 5-Methylaminomethyluridine, 5-Carboxymethyluridine, 5-Carboxymethylaminomethyluridine, 5-Hydroxyuridine, 5-Methyluridine 5-Taurine methyluridine, 5-aminoformyl methyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5-methyldihydrouridine, 5-methylaminomethyl-2-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 3,2-O-dimethyluridine, 5-carboxymethylaminomethyl-2-O-methyluridine, 5-amine Formylate-2-O-methyluridine, 5-methoxycarbonylmethyl-2-O-methyluridine, 5-(isopentenylaminomethyl)-2-O-methyluridine, 5,2-O-dimethyluridine, 2-O-methyluridine, 2-thio-2-O-methyluridine, uridine 5-oxyacetic acid, 5-methoxycarboxymethyluridine, uridine 5-oxyacetic acid methyl ester, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5 -Carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-tauratemethyl-2-thiouridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine, 2-O-methylpseudouridine, inosine, 1-methylinosine, 1,2-O-dimethylinosine, and 2-O-methylinosine. Each of these or its modified nucleobases can be a component of the nucleic acid of the present invention.

[0110] All other nucleosides (excluding those described above as natural or modified natural nucleosides) are non-natural nucleosides.

[0111] As used herein, the term "nucleoside base" refers to a nitrogenous base. "Natural nucleoside bases" include purine and pyrimidine rings. Purine rings include, for example, adenine and guanine. Pyrimidine rings include, for example, cytosine, thymine, and uracil.

[0112] As used herein, the term "modified nucleoside base" describes naturally occurring modified nucleoside bases, including but not limited to, for example, pseudouracil, 5-methylcytosine, N6-methyladenine, inosine, 5-hydroxymethylcytosine, 5-carboxycytosine, N4-acetylcytosine, N4-methylcytosine, N1-methyladenine, N2,N2-dimethylguanine, etc. See also the above-mentioned naturally occurring modified nucleosides.

[0113] As used herein, the term "non-natural nucleoside base" refers to all nucleoside bases that are not naturally occurring (whether modified or unmodified; see above for naturally occurring modified nucleosides), including but not limited to, for example, 7-deadenine, 2-aminoadenine, 5-methylisocytosine, 5-fluorouracil, 5-bromouracil, 5-iodouracil, 2-thiouracil, 2-methylthioadenine, 2-thio-5-methyluracil, 2-amino-6-methylthiopurine, etc. Natural nucleosides are as described above.

[0114] As used herein, the terms “nucleoside analog,” “modified nucleoside,” or “nucleoside derivative” include the synthetic nucleosides described herein. Nucleoside derivatives also include nucleosides having modified bases and / or sugar moieties, with or without protecting groups, and include, for example, 2'-deoxy-2'-fluorouridine, 5-fluorouridine, etc. The compounds and methods provided herein include these base rings and their synthetic analogs, as well as non-natural heterocyclic substituted basic sugars and acyclic substituted basic sugars. Other nucleoside derivatives that can be used in this disclosure include, for example, LNA nucleosides, halogenated purines (e.g., 6-fluoropurines), halogenated pyrimidines, N… 6 -Ethyl adenine, N 4 -(alkyl)-cytosine, 5-ethylcytosine, etc. (US Patent No. 6,762,298).

[0115] As used herein, the terms “nucleoside triphosphate,” “5’-nucleoside triphosphate,” or “NTP” refer to nucleosides linked to three phosphate groups. The terms encompass both native NTPs (e.g., adenosine triphosphate (ATP), uridine triphosphate (UTP), guanine triphosphate (GTP), and cytosine triphosphate (CTP)) and modified NTPs.

[0116] As used herein, the term “modified NTP” refers to a nucleoside 5'-triphosphate having a chemical moiety group that is either bound at any position or substituted at any position, where any position includes a sugar, a base, a triphosphate chain, or any combination of these three positions. Optionally, the chemical moiety group can be any group compatible with the nature of the transcription process. Examples of such NTPs include inosine triphosphate, dihydrouridine triphosphate, 2'-fluoro-2'-deoxycytidine triphosphate, pseudouridine triphosphate, N1-methylpseudouridine triphosphate, and 5-methyluridine triphosphate, and can be found, for example, in “Nucleoside Triphosphates and Their Analogs: Chemistry, Biotechnology and Biological Applications”, edited by Vaghefi, M., Taylor and Francis, Boca Raton (2005).

[0117] As used herein, the terms "modified RNA" or "modified mRNA" include, for example, RNA containing modified nucleosides, modified nucleotide inter-bonds, or any combination of modified nucleosides and nucleotide inter-bonds. Non-limiting examples of nucleotide inter-bond modifications include, but are not limited to, thiophosphates, triphosphates, and methylphosphonate derivatives (Stec, WJ et al.). Chem. Int. Ed. Engl., 33:709-722 (1994); Lebedev, AV et al. , E., Perspect. Drug Discov. Des. , 4:17-40 (1996); and Zon et al. , (US Patent Application No. 20070281308). Other examples of nucleotide inter-bond modifications can be found in Waldner et al. Bioorg. Med. Chem. Letters 6:2363-2366 (1996)

[0118] As used herein, the term "nucleotide bond" refers to one or more bonds linking two nucleosides of an oligonucleotide or nucleic acid, and can be a natural phosphodiester bond or a modified bond. Some non-limiting examples of modified nucleotide bonds include, for example, phosphorothioates, dithiophosphates, thiophosphates, 5'-O-methylphosphonates, 3'-O-methylphosphonates, 5'-hydroxyphosphonates, hydroxyphosphonates, phosphoroselenoates, selenophosphates, aminophosphates, carbophosphonates, phenylphosphonates, ethylphosphonates, H-phosphonates, guanidinium rings, triazole rings, boron phosphates, and methylphosphonates. As used herein, "phosphodiester bond" refers to a bond between nucleosides in which the phosphodiester bond is modified by replacing one of the oxygen atoms linked to a phosphorus atom with a sulfur atom.

[0119] As used herein, “oligo dT purification” is an affinity chromatography method for purifying mRNA containing or including a poly-A tail. This method is based on the specific targeting and separation of RNA molecules by the poly-A tail (RNA molecules with a poly-A tail bind to a solid support containing oligo dT, enabling their separation from RNA molecules without a poly-A tail).

[0120] As used herein, the term "prematurely terminated RNA transcript" refers to an incomplete product of an in vitro transcription reaction. Prematurely terminated RNA sequences can be of any length smaller than the expected length of the desired transcript.

[0121] As used herein, the term "promoter" refers to a nucleotide sequence in a DNA template that guides and controls the initiation of transcription of a specific DNA sequence. A promoter is typically adjacent to (or partially overlaps with) the DNA sequence to be transcribed. The promoter sequence is usually located directly upstream of or at the 5' end of the transcription start site. The nucleotide positions in the promoter are indicated relative to the transcription start site (position + 1) where DNA transcription begins.

[0122] As used herein, the terms "purified" or "purify" refer to the separation of a substance from at least some of the components (e.g., impurities or contaminants) that were initially bound to it. For example, RNA transcripts are purified by removing contaminating proteins or other unwanted nucleic acid substances (e.g., double-stranded RNA, DNA, and / or incomplete or terminated RNA transcripts). The purified substance (e.g., capped mRNA transcripts) can be separated from 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% of the other components that were initially bound to it.

[0123] As used herein, the term "RNase inhibitor" or "ribonuclease inhibitor" refers to a protein that, for example, inhibits RNase activity during in vitro transcription reactions.

[0124] As used herein, the term "RNA polymerase" refers to an enzyme that synthesizes RNA using a DNA template. For in vitro transcription methods, single-unit phage RNA polymerases derived from T7, T3, SP6, K1-5, K1E, K1F, or K11 phages, or variants thereof, are typically used. This family of polymerases has a simple, minimal promoter sequence of approximately 17 nucleotides, requires no accessory proteins, and has minimal restrictions on the starting nucleotide sequence.

[0125] As used in this article, "self-amplifying RNA" or "saRNA" is a straight-stranded single-stranded RNA molecule that encodes a target gene. saRNA is a type of mRNA that also includes a non-structural protein encoding a viral replicase. The viral replicase enables the RNA to replicate itself once it is delivered to the cell.

[0126] As used herein, the term "substantially free" means that a sample contains relatively small amounts or no unwanted substances (e.g., prematurely terminated RNA sequences, DNA, and / or double-stranded RNA). "Substantially free of impurities" means that impurities are present in the sample at levels of approximately 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less (w / w). For example, "substantially free of double-stranded RNA" means that double-stranded RNA is present in the sample at levels of approximately 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less (w / w).

[0127] As used in this article, "tangential flow filtration (TFF)" is a type of filtration in which the material to be filtered passes tangentially through the filter rather than through it. In TFF, unwanted permeate passes through the filter, while desired residue flows along the filter and is collected downstream. In TFF, the residue typically contains the desired material, which is the opposite of what is encountered in conventional membrane or dead-end filtration.

[0128] As used herein, the term "transcription" refers to the enzymatic preparation or synthesis of RNA complementary to a DNA template, thereby producing multiple RNA copies of the DNA sequence. The RNA molecules synthesized in a transcription reaction are referred to as "RNA transcripts," "primary transcripts," or simply "transcripts." Transcription reactions involving the compositions and methods provided herein employ the initiating capped oligonucleotide primers described herein. Transcription of a DNA template can be exponential, nonlinear, or linear. The DNA template can be double-stranded linear DNA, partially double-stranded linear DNA, circular double-stranded DNA, DNA plasmids, PCR amplification products, or modified nucleic acid templates compatible with RNA polymerase.

[0129] As used herein, "ligase" refers to an enzyme capable of forming a covalent bond between two nucleotides, and the "ligation" process refers to the formation of a covalent bond between two nucleotides. When two nucleotides are ligated, a linker is formed due to the ligation process. The linker can be formed by enzymatic ligation or chemical ligation.

[0130] As used herein, the terms “universal base,” “degenerate base,” “universal base analog,” and “degenerate base analog” include, for example, nucleoside analogs having artificial bases that, in some embodiments, can be recognized by RNA polymerase as a substitute for one of natural NTPs (e.g., ATP, UTP, CTP, and GTP) or other specific NTPs. Universal or degenerate bases are disclosed in: Loakes, D., Nucleic Acids Res., 29:2437-2447 (2001); Crey-Desbiolles, C. et al. , Nucleic Acids Res ., 33:1532-1543 (2005); Kincaid, K. et al. , Nucleic Acids Res. , 33:2620-2628 (2005); Preparata, FP, Oliver, JS, J. Comput. Biol 753-765 (2004); and Hill, F. et al. , Proc Natl Acad. Sci. USA , 95:4258-4263(1998)).

[0131] As used herein, the terms "subject" or "patient" can refer to vertebrates, such as mammals, fish, birds, reptiles, or amphibians. Therefore, subjects of the methods disclosed herein can be humans, non-human primates, horses, pigs, rabbits, dogs, sheep, goats, cattle, cats, guinea pigs, or rodents. The terms do not indicate a specific age or sex. Therefore, adult and neonatal subjects, as well as fetuses, whether male or female, are intended to be included. In one aspect, the subject is a mammal. A patient refers to a subject suffering from a disease or ailment. The term "patient" includes both human and veterinary subjects.

[0132] The terms "effective amount," "therapeutic effective amount," or "effective dose," or related terms, are used interchangeably and refer to an amount of therapeutic agent sufficient to achieve the desired therapeutic outcome or to have an effect on undesired symptoms but generally insufficient to cause adverse side effects when administered to a subject. Therapeutic effective amounts of the therapeutic agents provided herein will vary depending on the relative activity of the therapeutic agent and will vary depending on the following: the subject and the condition being treated, the subject's weight, age, and sex, the severity of the subject's condition, the method of administration, and any drugs used in combination with or concurrently with the specific compound employed, which can be readily determined by those skilled in the art. In one embodiment, the therapeutic effective amount will be determined based on certain aspects of the subject to be treated and the condition to be treated and can be determined by those skilled in the art using known techniques. Additionally, as is known in the art, adjustments may be necessary based on age and weight, general health, sex, diet, time of administration, drug interactions, and disease severity. For example, within the skill level of the art, the initial dose of the compound is typically lower than those necessary to achieve the desired therapeutic effect, and the dose is gradually increased until the desired efficacy is achieved. Where necessary, the effective daily dose may be divided into multiple doses for the purpose of administration. Therefore, a single-dose composition may contain such an amount or an approximation thereof constituting a daily dose. In any contraindication, the dosage may be adjusted by an individual physician. Dosage can be varied and may be administered once or multiple times daily for one or more days. Guidelines regarding appropriate dosages for a given class of pharmaceutical products can be found in the literature. In various other respects, formulations may be administered in a “preventatively effective amount”; that is, an amount that effectively prevents disease or symptoms.

[0133] As used herein, “dosage form” means a pharmacologically active material in a medium, carrier, mediator, or device suitable for administration to a subject. Dosage forms may comprise the compounds disclosed in this invention, products of the disclosed methods of preparation, or salts, solvates, or polymorphs thereof, in combination with pharmaceutically acceptable excipients (e.g., preservatives, buffer solutions, saline solutions, or phosphate-buffered saline solutions). Dosage forms can be prepared using conventional pharmaceutical manufacturing and compounding techniques. Dosage forms may contain inorganic or organic buffer solutions (e.g., sodium or potassium salts of phosphate, carbonate, acetic acid, or citric acid) and pH adjusters (e.g., salts of hydrochloric acid, sodium or potassium hydroxide, citric acid or acetic acid, amino acids and their salts), antioxidants (e.g., ascorbic acid, α-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), solution and / or freeze / lyophilize stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjusters (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), defoamers (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymer stabilizers, and viscosity modifiers (e.g., polyvinylpyrrolidone, poloxamer 488). 488), carboxymethyl cellulose), and co-solvents (e.g., glycerol, polyethylene glycol, ethanol). Formulations for injectable applications may contain the disclosed compounds, products of the disclosed preparation methods, or salts, solvates, or polymorphs thereof, suspended together with a preservative in a sterile saline solution for injection.

[0134] As used herein, a "kit" refers to a collection of at least two components that constitute a kit. Together, these components form a functional unit for a given purpose. Individual component components may be physically packaged together or separately. For example, a kit that includes instructions for use may or may not physically include instructions for other individual component components. In practice, instructions may be provided as a separate component, in paper or electronic form, the electronic form being available on a computer-readable storage device or downloadable from an Internet website, or in the form of a recorded presentation.

[0135] The terms “administering,” “administered,” and grammatical variations refer to the physical introduction of a pharmaceutical agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. The phrase “parenteral administration,” as used herein, refers to a mode of administration other than enteral and local administration, typically by injection, and includes (but is not limited to) intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcystic, subarachnoid, spinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. In one embodiment, the formulation is administered via a non-parenteral route, such as oral administration. Other non-parenteral routes include local, epidermal, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. Application may be performed, for example, once, multiple times, and / or over one or more extended cycles. Application may be continuous or intermittent. In various respects, the preparation may be administered therapeutically; that is, administered to treat an existing disease or symptom. In other respects, the preparation may be administered prophylactically; that is, administered to prevent a disease or symptom.

[0136] "Treatment" should be broadly understood and encompass any beneficial effect, including, for example, delaying, mitigating, or curbing the worsening of symptoms associated with viral diseases, or at least partially remedying such symptoms. The term is intended to include curing or eliminating a disease, condition, or symptom. Subjects requiring treatment include those already suffering from a disease or condition, as well as those for whom prevention of disease or condition is necessary. Patients to be treated are preferably mammals, particularly humans.

[0137] As used herein, the terms “prevent” or “preventing” refer to the elimination, prevention, avoidance, preemption, suppression, or hindrance of something, especially through proactive action. It should be understood that, unless otherwise specifically indicated, the use of “reduce,” “suppress,” or “prevent” herein requires explicit disclosure of the use of the other two terms.

[0138] As used herein, the term "therapeutic agent" includes any synthetic or naturally occurring biologically active compound or composition of substances that, when administered to an organism (human or non-human animal), induce desired pharmacological, immunogenic, and / or physiological effects through local and / or systemic action. Therefore, the term encompasses those compounds or chemical substances traditionally considered as drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, etc. In addition to the RNA molecules described herein, several non-limiting examples of other therapeutic agents are provided below. The following therapeutic agents are described in well-known references such as the Merck Index (14th edition), Physicians' Desk Reference (64th edition), and Pharmacological Basis of Therapeutics (12th edition), and include, but are not limited to: medicines; vitamins; mineral supplements; substances intended to treat, prevent, diagnose, cure, or alleviate diseases or ailments; substances that affect the structure or function of the body, or prodrugs that become biologically active or more active when placed in a physiological environment. For example, the term "therapeutic agent" includes compounds or compositions used in all major therapeutic areas, including but not limited to: adjuvants; anti-infective agents, such as antibiotics and antiviral agents; analgesics and combinations of analgesics, anorexia nervosa, anti-inflammatory agents, antiepileptic drugs, local and general anesthetics, hypnotics, sedatives, antipsychotics, tranquilizers, antidepressants, anxiolytics, antagonists, neuronal blocking agents, anticholinergic and cholinergic agents, antimuscarinic and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensives, hormones and nutrients, antiarthritis drugs, bronchodilators, anticonvulsants, antihistamines, antinausea drugs, antitumor drugs, antipruritics, antipyretics; antispasmodics, cardiovascular agents (including calcium channel blockers, beta-blockers, beta-promoters) Pharmaceuticals include: antiarrhythmics, antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostic agents; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppression; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides and their fragments (whether naturally occurring, chemically synthesized or recombinant); and nucleic acid molecules (polymers of two or more nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)), including double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin), and other bioactive macromolecules such as proteins and enzymes. Pharmaceuticals can be bioactive agents used in medical (including veterinary) applications and in agriculture (e.g., plants) and other fields.The term "therapeutic agent" also includes, but is not limited to: medicines; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure or alleviate a disease or ailment; or substances that affect the structure or function of the body; or prodrugs that become biologically active or more active when placed in a predetermined physiological environment.

[0139] As used herein, the term "derivative" refers to a compound having a structure derived from a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to that disclosed herein, and based on said structural similarity, is expected by those skilled in the art to exhibit the same or similar activity and utility as the claimed compound, or to induce the same or similar activity and utility as the claimed compound as a precursor. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of parent compounds.

[0140] The term "analog" or "analogue" is used in its ordinary, general sense within *Chemistry and Biology* and refers to a compound that is structurally similar to another compound (the so-called "reference compound") but has a different composition, such as through atomic substitution of one atom with a different element, the presence of a particular functional group, substitution of one functional group with another, or the absolute stereochemistry of one or more chiral centers of the reference compound. Thus, an analog is a compound that is functionally or morphologically similar to or equivalent to the reference compound but structurally or originally dissimilar or non-equivalent.

[0141] As used herein, the term "bioconjugate" refers to the association between atoms or molecules of a "bioconjugate reactive group" or a "bioconjugate reactive moiety." A "bioconjugate connector" refers to a link in a bioconjugate formed by a bioconjugate reactive group or a bioconjugate reactive moiety. The association can be direct or indirect. For example, a conjugate between a first bioconjugate reactive group (e.g., -NH₂, -C(O)OH, -N-hydroxysuccinimide, or -cis-butenedimide) and a second bioconjugate reactive group (e.g., a thioglycolic acid group, a sulfur-containing amino acid, an amine, or an amine side chain containing an amino acid or carboxylic acid ester) can be direct, for example, through a covalent bond or connector (e.g., the first connector of the second connector), or indirect, for example, through non-covalent bonds (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi interaction), hydrophobic interactions, etc.). In embodiments, bioconjugate chemical reactions (i.e., the association of two bioconjugate reactive groups) are used to form bioconjugates or bioconjugate linkers, including (but not limited to) nucleophilic substitution (e.g., reactions of amines with alcohols with acyl halides, reactive esters), electrophilic substitution (e.g., enamine reactions), and additions of carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed, for example, in March, Advanced Organic Chemistry, 3rd edition, John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney et al., Modification of Proteins; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982. In one embodiment, a first bioconjugate reactive group (e.g., the maleic anhydride moiety) is covalently linked to a second bioconjugate reactive group (e.g., a thioglycolic acid group). In another embodiment, a first bioconjugate reactive group (e.g., the haloacetyl moiety) is covalently linked to a second bioconjugate reactive group (e.g., a thioglycolic acid group). In yet another embodiment, a first bioconjugate reactive group (e.g., the pyridyl moiety) is covalently linked to a second bioconjugate reactive group (e.g., a thioglycolic acid group).In one embodiment, a first bioconjugate reactive group (e.g., the -N-hydroxysuccinimide moiety) is covalently linked to a second bioconjugate reactive group (e.g., an amine). In another embodiment, a first bioconjugate reactive group (e.g., the maleic anhydride moiety) is covalently linked to a second bioconjugate reactive group (e.g., a thioglycolic acid group). In yet another embodiment, a first bioconjugate reactive group (e.g., the -sulfonyl-N-hydroxysuccinimide moiety) is covalently linked to a second bioconjugate reactive group (e.g., an amine).

[0142] The applicable bioconjugate reactivity portion used for the bioconjugate chemical reactions in this article includes, for example: (a) Carboxyl groups and their various derivatives include (but are not limited to) N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acylimidazolium, thioesters, p-nitrobenzene esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) Hydroxyl group, which can be converted into esters, ethers, aldehydes, etc.

[0143] (c) Haloalkyl, wherein the halide may subsequently be replaced by a nucleophilic group, such as an amine, carboxylate anion, thiol anion, carboanion or alkoxide ion, thereby covalently linking the new group at the site of the halogen atom. (d) Dienephilic groups capable of participating in the Diels-Ade reaction, such as maleimido or maleimide group; (e) Aldehydes or ketones, such that subsequent derivatization may occur by forming carbonyl derivatives, such as imines, hydrazones, hemicarbazones, or oximes, or by mechanisms such as Grinner addition or alkyllithium addition. (f) A sulfonyl halide group, which is used for subsequent reactions with amines, for example, to form sulfonamides; (g) Thiol group, which can be converted into disulfide, react with acyl halide or bonded to metals such as gold, or react with maleic diimide; (h) Amine or thiolated group (e.g., present in cysteine), which may be, for example, acylated, alkylated or oxidized; (i) Alkenes, which may undergo processes such as cycloaddition, acylation, Michael addition, etc.; (j) Epoxides that can react with, for example, amines and hydroxyl compounds; (k) phosphorous amide and other standard functional groups suitable for nucleic acid synthesis; (l) Metal-silicon oxide bonding; and (m) Metals are bonded to reactive phosphorus groups (e.g., phosphine) to form, for example, phosphate diester bonds.

[0144] (n) Coupling of azides with alkynes using copper-catalyzed cycloaddition click chemistry.

[0145] (o) Biotin conjugates can react with avidin or streptavidin to form avidin-biotin complexes or streptavidin-biotin complexes.

[0146] The reactive groups of a bioconjugate can be selected such that they do not participate in or interfere with the chemical stability of the conjugate described herein. Alternatively, the reactive functional groups can be protected from participating in cross-linking reactions by the presence of protecting groups. In embodiments, the bioconjugate comprises a molecular entity derived from the reaction of an unsaturated bond (e.g., maleic anhydride) with a thioglycolic acid group.

[0147] As used herein, the term "connector" refers to a chemical group that links two molecules or portions together (the term "connector" includes the term "bioconjugate connector," but is more broadly defined). In embodiments, a connector can be formed by a linker (e.g., where a phosphate ester reacts with a hydroxyl group to create a link between oxygen and phosphorus). In other embodiments, the connector can be a connector (L) capable of binding a purification tag. The linker (L) may contain a combination of one or more groups, such as -S(O)2-, -N(R)-, -O-, -S-, -C(O)-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)NH-, -NHC(O)N(R)-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; wherein R is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or any combination thereof.

[0148] As used herein, the term "precursor RNA" refers to "A" in Formula I: AB or Formula II: ABL. Precursor RNA comprises a 5'-cap, an open reading frame (ORF), and a poly-A region. In embodiments, the precursor RNA comprises a 5'-cap, a 5'-UTR, an open reading frame (ORF), a 3'-UTR, and a poly-A region. In embodiments, the precursor RNA comprises a 5'-cap, a 5'-UTR, an open reading frame (ORF), a 3'-UTR, and a poly-A region. In embodiments, the precursor RNA comprises a 5'-cap, a 5'-UTR, an open reading frame (ORF), and a poly-A region. The terms poly-A region and poly-A tail are used interchangeably herein. In embodiments, the term "poly-A region" refers to an ATP string. In embodiments, the term "poly-A region" refers to an ATP string followed by several GTPs, or several UTPs, or several CTPs. In embodiments, the term "poly-A region" refers to an ATP string interspersed with several GTPs, or several UTPs, or UTPCTPs, and followed by more ATP.

[0149] As used herein, the term "3'-stable region" or "stable region" refers to "B" in Formula I: AB or Formula II: ABL. These stable regions increase the stability of RNA molecules compared to molecules without such stable regions. In embodiments, the stable region inhibits the degradation of the RNA molecule. In embodiments, the 3'-stable region is covalently attached to the 3' end of the precursor RNA (an RNA molecule containing a 5'-cap, an open reading frame (ORF), and a poly-A region, or an RNA molecule containing a 5'-cap, a 5'-UTR, an open reading frame (ORF), a 3'-UTR, and a poly-A region) via a linker that can be formed by ligation. In embodiments, the linker is a bioconjugate linker. In embodiments, the linker can be formed by enzyme ligation, splinting, or chemical ligation. In embodiments, the 3'-stable region is covalently attached to the 3' end of the precursor RNA via a linker that can be formed using polymerase. In embodiments, the 3'-stable region comprises one or more unmodified nucleosides and one or more unmodified nucleotide inter-bonds. The unmodified nucleosides are naturally occurring unmodified nucleosides. In one embodiment, the 3'-stable region comprises one or more modified nucleosides and / or one or more modified nucleotide inter-bonds. In another embodiment, the modified nucleosides contain modified nucleobases and / or modified sugars. In another embodiment, the 3'-stable region forms a secondary structure. In another embodiment, any combination of one or more modified nucleosides and / or one or more modified nucleotide inter-bonds and / or one or more secondary structures is considered for stabilizing the RNA molecule. In another embodiment, one or more nucleosides within the 3'-stable region contain one or more purification tags. In another embodiment, one or more nucleosides within the 3'-stable region contain one or more linkers (L) capable of binding purification tags. In another embodiment, the last nucleoside of the 3'-stable region does not contain a 3'-hydroxyl group. In another embodiment, the last nucleoside of the 3'-stable region is blocked and cannot react with any other NTPs.

[0150] As used herein, the term "secondary structure" refers to the 3'-stabilizing region capable of forming secondary structures, such as G-quadruplexes (secondary structures formed from guanine-rich nucleic acid sequences), triplexes, protrusions, kissing hairpins, loops, e-loops, branched polyloops, stem loops (also known as "hairpin loops"), tetraloops, helices, or pseudoknots, which prevent exonucleases from approaching the 3' terminal nucleotides of the RNA molecule and protect the RNA from degradation. Therefore, secondary structures improve the stability and half-life of said RNA molecules.

[0151] As used herein, the term "purification tag" refers to a hydrophobic group covalently linked to the 3'-stable region or nucleoside of the RNA molecule described herein, for example, through a linker; such groups may be removable or non-removable. Purification tags enable HPLC separation of RNA molecules containing a 3'-stable region (which includes a covalently linked purification tag) from RNA molecules lacking a 3'-stable region. In embodiments, the purification tag may be a protecting group. In embodiments, the purification tag includes, for example, but not limited to, C6-C... 24 Alkyl, C4-C 24 alkenyl, C4-C 24 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, silyl, triphenylmethyl, lipid, dye, steroid, vinyl ether compounds, modified and unmodified Fmoc compounds, and any combination thereof. Fluorine substituents or fluorinated groups can be used to increase the hydrophobicity of the hydrophobic group. As used herein, the terms “hydrophobic moiety” or “hydrophobic group” are used interchangeably and refer to a carbon-rich hydrophobic substituent or a combination of hydrophobic substituents. The hydrophobicity of a substituent can be determined, measured, or calculated by the value of its partition coefficient (log P). The partition coefficient (log P) of a substance defines the ratio of its solubility in two immiscible solvents (typically octanol:water). When this value is calculated rather than measured, it is referred to as cLog P. In embodiments, the hydrophobic group has at least 2 cLog P, or a combination of two, three, or four “partially hydrophobic groups” has a collective value of at least 2 cLog P. Nucleoside bases with cLog P less than 2, such as cytosine, thymine, uracil, adenine, and guanine, are not considered “hydrophobic groups” as defined herein.

[0152] Purification labels can be, for example, (ethyl)carbonyl (azadibenzocyclooctyne) (DBCO). Purification labels can be, for example, 4-ethylphenol. The purification tag can be, for example, two isomers of dibenzohexyltriazolazine octene. and A mixture. The purification label can be, for example, 1'-O-butyl-3',4',6'-triacetylGalNAc. .

[0153] Alternatively, "purification tag" can refer to an affinity tag, such as biotin, polyhistidine, Myc-Tag, MBP-Tag, or GST-Tag.

[0154] As used herein, the terms “cleavable purification tag” or “removable purification tag” are used interchangeably and refer to hydrophobic groups, such as C3-C. 20Longer saturated alkyl, cycloalkyl, aryl, silyl, etc., can be chemically or thermally removed from the RNA molecules described herein under mild conditions. In embodiments, such groups can be removed under mildly acidic conditions (at room temperature for 1 hour) with a pH not lower than about 5. In embodiments, such groups can be removed under weakly alkaline conditions with a pH not higher than about 9 (at room temperature for 1 hour). In embodiments, such groups can be removed by mild heating at a temperature not higher than about 65°C for 1 hour. In embodiments, such groups can be removed by reductive amination. In embodiments, such groups can be removed by desilylation. In embodiments, such groups can be removed by oxidation. In embodiments, such groups can be removed by photolysis. In embodiments, such groups are photocleavable (photolabile) groups. In this application, a purification tag is considered "cleavable" or "removable" if it can be removed under conditions that do not alter the properties of the RNA molecule.

[0155] As used herein, the terms "uncuttable purification tag" or "unremovable purification tag" are used interchangeably and refer to hydrophobic groups, such as C3-C. 20 Or longer saturated alkyl groups, C3-C8 cycloalkyl groups, C6-C 10 Aryl groups, etc., cannot be easily removed from the RNA molecules described herein. In embodiments, the hydrophobic groups cannot be removed using the mild conditions described above for cleavable purification tags. In embodiments, uncleavable purification tags cannot be removed at a pH of about 5 to about 9 (for 1 hour at room temperature). In embodiments, uncleavable purification tags cannot be removed by heating at a temperature below about 65°C for 1 hour. In embodiments, up to 5% of the uncleavable hydrophobic groups are cleaved from the RNA molecule. In embodiments, up to 10% of the uncleavable hydrophobic groups are cleaved from the RNA molecule. In embodiments, up to 15% of the uncleavable hydrophobic groups are cleaved from the RNA molecule. In embodiments, up to 20% of the uncleavable hydrophobic groups are cleaved from the RNA molecule.

[0156] The term "protecting group" is used according to its common meaning in organic chemistry and refers to a portion covalently bonded to a heteroatom, heterocyclic alkyl group, or heteroaryl group to prevent the reactivity of the heteroatom, heterocyclic alkyl group, or heteroaryl group during one or more chemical reactions prior to the removal of the protecting group. Typically, during a portion of a multipart synthesis, the protecting group is bonded to a heteroatom (e.g., O or N) where the heteroatom is undesirable to react with a reagent (e.g., chemical reduction). After protection, the protecting group can be removed (e.g., by adjusting pH or temperature). In embodiments, the protecting group is an alcohol protecting group. Non-limiting examples of alcohol protecting groups include acyl, acetyl, benzoyl, benzyl, methoxymethyl ether (MOM), tetrahydropyranyl (THP), tert-butyldimethylsilyl (TBDMS), and silyl ethers (e.g., trimethylsilyl (TMS)). In embodiments, the protecting group is an amine protecting group. Non-limiting examples of amine protecting groups include triphenylmethyl, monomethoxytriphenylmethyl (MMT), dimethoxytriphenylmethyl (DMT) or other modified triphenylmethyl, dimethylbenzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), acyl, acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl ether (PMB), tert-butyldiphenylsilyl (TBDPS), and toluenesulfonyl (Ts).

[0157] "Pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that facilitate administration of the active agent to and absorption by a subject, and can be included in the compositions of this disclosure without producing significant adverse toxicological effects on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, standard saline solutions, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (e.g., Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and pigments. Such formulations may be sterilized and, as needed, mixed with adjuvants (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, and / or aromatic substances, etc.) that do not adversely react with the compounds of this disclosure. Those skilled in the art will recognize that other pharmaceutical excipients are applicable to this disclosure.

[0158] Combination therapy or “in combination with” refers to the use of more than one therapeutic agent to treat a specific condition or symptom. “In combination with” is not intended to imply that therapeutic agents must be administered simultaneously and / or mixed for delivery, but such delivery methods are also within the scope of this disclosure. The therapeutic agent may be administered simultaneously with, before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks prior to) or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks after) a particular condition or symptom. Therapeutic agents in combination therapy may also be administered in alternating dosing schedules, with or without a withdrawal period (e.g., the agent is not administered on certain days of the dosing schedule). Administration of a therapeutic agent "in combination with another therapeutic agent" includes, but is not limited to, sequential and simultaneous administration of two agents. Generally, each therapeutic agent will be administered at a dose and / or schedule determined according to the specific agent.

[0159] In this disclosure, terms such as “comprises,” “comprising,” “containing,” and “having” may have the meanings applicable under U.S. patent law and may refer to “includes,” “including,” etc. “Consisting essentially of” or “consists essentially” also have the meanings applicable under U.S. patent law, and these terms are open-ended, allowing for the presence of content other than those listed, as long as the basis or novel features of the listed content do not change due to the presence of content other than those listed, but excluding prior art embodiments.

[0160] As used herein, the term "nucleophile" refers to a chemical substance that provides an electron pair to form a reaction-related chemical bond with an electrophile. All molecules or ions having a free electron pair or at least one π bond can act as nucleophiles.

[0161] RNA molecules This document describes RNA molecules covalently linked to a 3'-stable region, wherein the 3'-stable region contains one or more purification tags optionally covalently linked via a linker (L). Additionally, this document describes pharmaceutical compositions comprising said RNA molecules and methods for preparing said RNA molecules.

[0162] In one respect, this paper provides an RNA molecule comprising the structure of formula I: , Where A includes: a) 5'-cap; b) Open reading frames (ORFs) encoding proteins; and c) The poly-A region, wherein the poly-A region is located at 3' of the open reading frame; and B contains a 3'-stable region containing 1 to 50 nucleosides, wherein one or more nucleosides within the 3'-stable region contain one or more purification tags.

[0163] In an embodiment, B includes a 3'-stable region containing 1 to 50 nucleosides, wherein one or more nucleosides within the 3'-stable region contain one or more purification tags and one or more adapters (L) capable of binding the purification tags.

[0164] In one embodiment, B includes a 3'-stable region containing a nucleoside, wherein the nucleoside within the 3'-stable region contains one or more purification tags. In another embodiment, B includes a 3'-stable region containing a nucleoside, wherein the nucleoside within the 3'-stable region contains a purification tag. In yet another embodiment, B includes a 3'-stable region containing a nucleoside, wherein the nucleoside within the 3'-stable region contains one or more purification tags and one or more linkers (L) capable of binding to the purification tags.

[0165] In one embodiment, B comprises a 3'-stable region containing two nucleosides, wherein one or both nucleosides within the 3'-stable region contain one or more purification tags. In another embodiment, B comprises a 3'-stable region containing two nucleosides, wherein one or both nucleosides within the 3'-stable region contain a purification tag. In yet another embodiment, B comprises a 3'-stable region containing two nucleosides, wherein one or both nucleosides within the 3'-stable region contain one or more purification tags and one or more linkers (L) capable of binding to the purification tags.

[0166] In one embodiment, the purification tag is placed on the last nucleotide of the RNA molecule containing the Formula I structure. In another embodiment, the purification tag is placed on the penultimate nucleotide of the RNA molecule containing the Formula I structure.

[0167] In one respect, this paper provides an RNA molecule containing the structure of formula II: Where A includes: a) 5'-cap; b) Open reading frames (ORFs) encoding proteins; and c) The poly-A region, wherein the poly-A region is located at 3' of the open reading frame; and B contains a 3'-stable region containing 1 to 50 nucleosides, wherein one or more nucleosides within the 3'-stable region contain one or more adapters (L), wherein the adapters (L) are capable of binding to the purification tag.

[0168] In one embodiment, B includes a 3'-stable region containing a nucleoside, wherein the nucleoside within the 3'-stable region contains one or more adapters (L) capable of binding to the purification tag. In another embodiment, B includes a 3'-stable region containing a nucleoside, wherein the nucleoside within the 3'-stable region contains an adapter (L) capable of binding to the purification tag.

[0169] In one embodiment, B comprises a 3'-stable region containing two nucleosides, wherein one or both nucleosides within the 3'-stable region contain one or more adapters (L) capable of binding to the purification tag. In another embodiment, B comprises a 3'-stable region containing two nucleosides, wherein one or both nucleosides within the 3'-stable region contain one adapter (L) capable of binding to the purification tag.

[0170] In one embodiment, the adapter (L) capable of binding the purification tag is located on the last nucleotide of the RNA molecule containing the formula II structure. In another embodiment, the adapter (L) capable of binding the purification tag is located on the penultimate nucleotide of the RNA molecule containing the formula II structure.

[0171] In this embodiment, the RNA molecule is an mRNA molecule. In this embodiment, the RNA molecule is a linear RNA molecule. In this embodiment, the RNA molecule is a circular RNA molecule. In this embodiment, the RNA molecule is a linear mRNA molecule. In this embodiment, the RNA molecule is a circular mRNA molecule.

[0172] In one embodiment, the precursor RNA includes a 5' cap, an open reading frame (ORF) encoding a protein, and a poly-A region, wherein the poly-A region is located at the 3' end of the ORF. In another embodiment, the precursor RNA includes a 5' cap, a 5'-UTR region, an ORF encoding a protein, a 3'-UTR region, and a poly-A region, wherein the poly-A region is located at the 3' end of the 3'-UTR region. In yet another embodiment, the precursor RNA includes a 5' cap, a 5'-UTR region, an ORF encoding a protein, and a poly-A region, wherein the poly-A region is located at the 3' end of the ORF. In yet another embodiment, the precursor RNA includes a 5' cap, an ORF encoding a protein, a 3'-UTR region, and a poly-A region, wherein the poly-A region is located at the 3' end of the 3'-UTR region.

[0173] In one embodiment, the precursor RNA comprises a 5' cap, a 5'-UTR region, an open reading frame (ORF) encoding a protein, a 3'-UTR region, and a poly-A region, wherein at least one of the 5'-cap structure, the 5'-UTR, the coding region, the 3'-UTR, and / or the poly-A region includes at least one modified nucleotide. In another embodiment, the precursor RNA comprises a 5' cap, a 5'-UTR region, an open reading frame (ORF) encoding a protein, a 3'-UTR region, and a poly-A region, wherein at least one of the 5'-cap structure, the 5'-UTR, the coding region, the 3'-UTR, and / or the poly-A region includes at least two modified nucleotides. In yet another embodiment, the precursor RNA comprises a 5' cap, a 5'-UTR region, an open reading frame (ORF) encoding a protein, a 3'-UTR region, and a poly-A region, wherein at least one of the 5'-cap structure, the 5'-UTR, the coding region, the 3'-UTR, and / or the poly-A region includes at least three modified nucleotides. In one embodiment, the precursor RNA comprises a 5' cap, a 5'-UTR region, an open reading frame (ORF) encoding a protein, a 3'-UTR region, and a poly-A region, wherein at least one of the 5'-cap structure, the 5'-UTR, the coding region, the 3'-UTR, and / or the poly-A region comprises at least four modified nucleotides. In another embodiment, the precursor RNA comprises a 5' cap, a 5'-UTR region, an open reading frame (ORF) encoding a protein, a 3'-UTR region, and a poly-A region, wherein at least one of the 5'-cap structure, the 5'-UTR, the coding region, the 3'-UTR, and / or the poly-A region comprises at least five modified nucleotides.

[0174] In embodiments, the modified nucleotide may include modified nucleosides and / or modified nucleotide inter-bonds. In embodiments, the modified nucleotide may include modified nucleosides and modified nucleotide inter-bonds. In embodiments, the modified nucleotide may include modified nucleosides or modified nucleotide inter-bonds. In embodiments, the modified nucleotide may include modified nucleobases and / or modified sugars and / or modified nucleotide inter-bonds. In embodiments, the modified nucleotide may include modified nucleobases, modified sugars, and modified nucleotide inter-bonds. In embodiments, the modified nucleotide may include modified nucleobases. In embodiments, the modified nucleotide may include modified sugars. In embodiments, the modified nucleotide may include modified nucleotide inter-bonds. In embodiments, the modified nucleotide may include modified nucleobases and modified sugars. In embodiments, the modified nucleotide may include modified nucleobases and modified nucleotide inter-bonds. In embodiments, the modified nucleotide may include modified nucleotide inter-bonds and modified sugars. Some non-limiting examples of modified nucleosides include 5-methylcytidine, 5'-methoxyuridine, pseudouridine, N1-methylpseudouridine, etc.

[0175] In embodiments, the modified nucleobase is modified uracil, modified cytosine, modified guanine, or modified adenine. In embodiments, the modified nucleobase is modified uracil. In embodiments, the modified nucleobase is modified cytosine. In embodiments, the modified nucleobase is modified guanine. In embodiments, the modified nucleobase is modified adenine. In embodiments, the modified nucleobase is pseudouracil (ψ), 2-thiouracil, 4-thiouracil, 4-thio-pseuuridine, 2-thio-pseuuridine, 5-hydroxyuracil, 5-halouracil, 3-methyluracil, 5-azauracil, or 2-thio-5-azauracil. In embodiments, the modified nucleobases are 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-methyl-cytosine, 5-halo-cytosine, 2-thio-cytosine, or 2-thio-5-methyl-cytosine. In embodiments, the modified nucleobases are 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenine, 7-deadenine, N6-methyl-adenine, or 2-methylthio-N6-methyl-adenine. In the embodiments, the modified nucleobases are inosine, 1-methyl-inosine, 7-cyano-7-denitro-guanine, 7-aminomethyl-7-denitro-guanine, 6-thio-guanine, 6-thio-7-denitro-guanine, or 6-methoxy-guanine.

[0176] Some non-limiting examples of modified nucleosides and nucleobases include pseudouridine (ψ), pyridin-4-one nucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, and 2-thio-uracil (s). 2 U), 4-thiouracil (s) 4 U), 4-thio-pseuuridine, 2-thio-pseuuridine, 5-hydroxy-uracil (ho) 5 U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m 3 U), 5-methoxy-uracil (mo) 5 U), uracil 5-oxyacetic acid (cmo) 5 U), uridine 5-oxyacetic acid methyl ester (mcmo) 5 U), 5-carboxymethyl-uracil (cm) 5 U), 1-carboxymethyl-pseuuridine, 5-carboxyhydroxymethyl-uracil (chm) 5 U), 5-Carboxyhydroxymethyl-uracil methyl ester (mchm) 5 U), 5-methoxycarbonylmethyl-uracil (mcm) 5 U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm) 5 s 2 U), 5-aminomethyl-2-thio-uracil (nm) 5 s 2 U), 5-methylaminomethyluracil (mnm) 5 U), 5-methylaminomethyl-2-thio-uracil (mnm) 5 s 2 U), 5-methylaminomethyl-2-seleno-uracil (mnm) 5 se 2 U), 5-aminocarbamoylmethyluracil (ncm) 5 U), 5-Carboxymethylaminomethyl-uracil (cmnm) 5 U), 5-Carboxymethylaminomethyl-2-thio-uracil (cmnm) 5 s 2 U), 5-propynyl-uracil, 1-propynyl-pseuuridine, 5-tauronic acid methyl-uracil (τm) 5 U), 1-Taurate methyl-pseuuridine, 5-Taurate methyl-2-thio-uracil (τm) 5 s 2 U), 1-Tauratemethyl-4-thio-pseuuridine, 5-methyl-uracil (m 5 U, i.e., having nucleobase deoxythymidine), 1-methyl-pseudouridine (m1 ψ), 5-methyl-2-thiouracil (m) 5 s 2 U), 1-methyl-4-thio-pseuuridine (m) 1 s 4 ψ), 4-thio-1-methyl-pseuuridine, 3-methyl-pseuuridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-1-deazo-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m) 5 D), 2-Thio-dihydrouracil, 2-Thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp) 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp) 3 ψ), 5-(isopentenylaminomethyl)uracil (inm) 5 U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm) 5 s 2 U), 5,2'-O-dimethyluridine (m) 5 Um), 2-thio-2'-O-methyluridine (s) 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm) 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm) 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm) 5 Um), 3,2'-O-dimethyluridine (m) 3 Um) and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm) 5Um), 1-thiouracil, deoxythymidine, 5-(2-methoxycarbonylvinyl)uracil, 5-(carbamoylhydroxymethyl)uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyluracil, 5-methoxy-2-thiouracil, 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methylcytosine (m4C), 5-methylcytosine Pyridine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudo-cytosine, pyrrolo-cytosine, pyrrolo-pseudo-cytosine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio-pseudo-cytosine, 4-thio-1-methyl-pseudo-cytosine, 4-thio-1-methyl-1-deazo-pseudo-cytosine, 1-methyl-1-deazo-pseudo-cytosine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio -Zebrane, 2-Thio-Zebrane, 2-Methoxy-cytosine, 2-Methoxy-5-methyl-cytosine, 4-Methoxy-pseudo-cytosine, 4-Methoxy-1-methyl-pseudo-cytosine, Lysicillin (k2C), 5,2'-O-dimethyl-cytosine (m5Cm), N4-acetyl-2'-O-methyl-cytosine (ac4Cm), N4,2'-O-dimethyl-cytosine (m4Cm), 5-formyl-2'-O-methyl-cytosine (f5Cm), N4,N4,2'-O-trimethyl-cytosine (m42Cm), 1-Thio-cytosine, 5-hydroxy -Cytosine, 5-(3-azidopropyl)-cytosine, 5-(2-azidoethyl)-cytosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deadenine, 7-deadenine-8-aza-adenine, 7-deadenine-2-amino-purine, 7-deadenine-8-aza-2-amino-purine, 7-deadenine-2,6-diaminopurine, 7-deadenine-8-aza-2,6-Diaminopurine, 1-Methyl-Adenine (m1A), 2-Methyl-Adenine (m2A), N6-Methyl-Adenine (m6A), 2-Methylthio-N6-Methyl-Adenine (ms2m6A), N6-Isopentenyl-Adenine (i6A), 2-Methylthio-N6-Isopentenyl-Adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-Methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-Glycylcarbamoyl-Adenine (g6A), N6-Threonylcarbamoyl-Adenine (t6A), N6-Methyl-N6-Threonylcarbamoyl-Adenine (m6A) t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxyn-valinecarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxyn-valinecarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenine (m6Am), N6,N6,2'-O-trimethyl-adenine (m62Am), 1,2'-O-dimethyl-adenine (mlAm), 2-amino-N6-methyl-purine, 1- Thio-adenine, 8-azido-adenine, N6-(19-amino-pentaenodecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, N6-hydroxymethyl-adenine, inosine (I), 1-methyl-inosine (m1I), wyoside (imG), methyl wyoside (mimG), 4-demethyl-wyoside (imG-14), isowyoside (imG2), wyoside (yW), peroxywyoside (o2yW), hydroxywyoside (OHyW), undermodified hydroxywyoside (OHyW*), 7-deazo-guanine, wyoside (Q), epoxy wyoside (oQ), galactosyl wyoside (galQ), mannosyl wyoside (manQ) 7-Cyano-7-deazo-guanine (preQ0), 7-aminomethyl-7-deazo-guanine (preQ1), archapurin (G+), 7-deazo-8-aza-guanine, 6-thio-guanine, 6-thio-7-deazo-guanine, 6-thio-7-deazo-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G)The modified nucleobases include 7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,n2-dimethyl-6-thio-guanine, N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 1-thio-guanine, and O-6-methyl-guanine. In embodiments, the modified nucleobase can be any of the aforementioned nucleobases.

[0177] In embodiments, the modified sugar has a 5-membered ring or a 6-membered ring, or is a modified ribose. In embodiments, the ribose is replaced by a morpholino ring. In embodiments, the modified nucleoside contains a morpholino ring. In embodiments, the modified ribose is 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, or 3'-amino-2',3'-dideoxyribose.

[0178] Some non-limiting examples of sugar modification include modification of the 2'-hydroxyl group of the ribocycle, substitution of oxygen in the ribocycle, and expansion or contraction of the ribocycle. In embodiments, the modified sugar may contain any of the foregoing modifications. In embodiments, the 2'-hydroxyl group of the ribocycle may be substituted with each of the following: hydrogen, halogroup, methoxy group, azide group, substituted or unsubstituted alkyl group, substituted or unsubstituted heteroalkyl group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocycloalkyl group, substituted or unsubstituted aryl group, and substituted or unsubstituted heteroaryl group.

[0179] In some embodiments, the 2'-hydroxyl group of the ribocycle may be hydrogen-substituted. In some embodiments, the 2'-hydroxyl group of the ribocycle may be halogen-substituted. In some embodiments, the 2'-hydroxyl group of the ribocycle may be azide-substituted. In some embodiments, the 2'-hydroxyl group of the ribocycle may be methoxy-substituted.

[0180] In one embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted alkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted alkyl group (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted alkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent). In yet another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by an unsubstituted alkyl group (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In yet another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted heteroalkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In one embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by an unsubstituted heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In yet another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted cycloalkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In one embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by an unsubstituted cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In yet another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted heterocyclic alkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heterocyclic alkyl group (e.g., 3- to 8-membered heteroalkyl, 3- to 6-membered heteroalkyl, or 5- to 6-membered heterocyclic alkyl). In one embodiment, the 2'-hydroxyl group of the ribose ring is replaced by a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heterocyclic alkyl group (e.g., 3- to 8-membered heterocyclic alkyl group, 3- to 6-membered heterocyclic alkyl group, or 5- to 6-membered heterocyclic alkyl group). In another embodiment, the 2'-hydroxyl group of the ribose ring is replaced by an unsubstituted heterocyclic alkyl group (e.g., 3- to 8-membered heterocyclic alkyl group, 3- to 6-membered heterocyclic alkyl group, or 5- to 6-membered heterocyclic alkyl group).In embodiments, the 2'-hydroxyl group of the ribose ring is replaced by a substituted aryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted aryl group (e.g., C6-C). 10 Aryl, C 10 Aryl or phenyl group is substituted. In embodiments, the 2'-hydroxyl group of the ribose ring is replaced by an aryl group (e.g., C6-C) that has been substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent). 10 Aryl, C 10 Aryl or phenyl group is substituted. In embodiments, the 2'-hydroxyl group of the ribose ring is replaced by an unsubstituted aryl group (e.g., C6-C). 10 Aryl, C 10 The 2'-hydroxyl group of the ribocycle is replaced by a substituted heteroaryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, the 2'-hydroxyl group of the ribocycle is replaced by a substituted heteroaryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) (e.g., 5- to 10-membered heteroalkyl, 5- to 9-membered heteroalkyl, or 5- to 6-membered heteroalkyl). In embodiments, the 2'-hydroxyl group of the ribocycle is replaced by an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl).

[0181] In embodiments, the oxygen in the ribocycle may be replaced by -S-, -Se-, -NH-, or -CH2-. In embodiments, the oxygen in the ribocycle may be replaced by -S-. In embodiments, the oxygen in the ribocycle may be replaced by -Se-. In embodiments, the oxygen in the ribocycle may be replaced by -NH-. In embodiments, the oxygen in the ribocycle may be replaced by -CH2-. In embodiments, the ribocycle may be substituted with another ring, for example, the ring may be a cyclobutene, mannitol, cyclohexyl, or morpholinyl ring. In embodiments, the ribocycle may be replaced by a locked nucleic acid ring (LNA). In embodiments, the ribocycle may be replaced by an unlocked nucleic acid ring (UNA).

[0182] In embodiments, the internucleotide bond comprises a modified phosphate ester. In embodiments, the modified phosphate ester is a phosphorothioate, dithiophosphate, thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphonate, phosphoroselenoate, selenophosphate, aminophosphate, carbophosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boron phosphate, methylphosphonate, or guanidinopropyl aminophosphate.

[0183] In embodiments, the modified nucleotide inter-bonds include, for example, thiophosphates, selenophosphates, boranophosphates, boranophosphate esters, hydrophosphonates, aminophosphates, diaminophosphates, alkyl or aryl phosphonates, and phosphate triesters. In embodiments, the modified nucleotide inter-bonds may be dithiophosphates, wherein both unlinked oxygen atoms are sulfur-substituted. In embodiments, the modified nucleotide inter-bonds may include replacing the linking oxygen with -HN-, -S-, or -CH2-. In embodiments, the modified nucleotide inter-bonds may include replacing the unlinked oxygen (single bond with phosphorus) with methyl, ethyl, methoxy, -SH, or -BH3, or any combination thereof.

[0184] Various 5'-cap structures can be used to synthesize the RNA molecules described herein (RNA molecules of Formula I: AB or Formula II: ABL). The 5'-cap structure increases the stability of RNA and its resistance to degradation by exonucleases. The 5'-cap is also essential for translation initiation, where it acts as a recognition site for the translation initiation complex.

[0185] The 5'-cap structure includes those described in International Patent Publication Nos. WO2017 / 053297, WO2023 / 147352, WO2021 / 162566, WO2021 / 162567, WO2022 / 006368, WO2022 / 086140, WO2023 / 033551, WO2018 / 075827, WO2023 / 07019 and U.S. Provisional Application Nos. 63 / 528,990 and 63 / 536844, the cap structure of each of which is incorporated herein by reference.

[0186] In embodiments, a 5'-cap structure (cap analogue) can cap RNA molecules during in vitro transcription (IVT) reactions. In embodiments, RNA molecules can be capped using an enzyme after the transcription reaction. In embodiments, the RNA molecules described herein may contain cap analogues. In embodiments, cap analogues can increase the stability of RNA molecules. In embodiments, cap analogues can increase the half-life of RNA molecules. In embodiments, cap analogues can increase the translation efficiency of RNA molecules. In embodiments, cap analogues may include purification tags. In embodiments, cap analogues may include adapters capable of binding purification tags. In embodiments, the purification tag is a cleavable purification tag. In embodiments, the purification tag is an incleavable purification tag.

[0187] Some non-limiting examples of hat-like objects include, but are not limited to, m7 G 3'OMe pppA 2'OMe pG, m7 G 3'OMe ppp (N-6-methyladenine) 2'OMe pG, m7 G 3'OMe pppApG, m7 G 3'OMe ppp (N-6-methyladenine) pG, m7 G 3'OMe pppG 2'OMe pG, m7 G 3' OMe pppGpG, m7 GpppA 2'OMe pG, m7 Gppp (N-6-methyladenine) 2'OMe pG, m7 GpppApG, m7 Gppp (N-6-methyladenine) pG, m7 GpppG 2'OMe pG, m7 GpppGpG m7 GpppA 2'OMe pU, m7 GpppApU m7 G 3'OMe pppA 2'OMe pU and m7 G 3' OMe pppApU.

[0188] In this embodiment, the 5'-UTR is upstream of the translation initiation site. In this embodiment, the 5'-UTR is adjacent to the 5' end of the open reading frame (ORF) encoding the protein and downstream of the 5'-cap.

[0189] In one embodiment, the 3'-UTR is downstream of the translation initiation site. In another embodiment, the 3'-UTR is adjacent to the 3' end of the open reading frame (ORF) encoding the protein. In yet another embodiment, the 3'-UTR is upstream of the poly-A region.

[0190] In one embodiment, the length of the poly-A region comprises approximately 2 to approximately 500 nucleotides. In another embodiment, the length of the poly-A region is 10 or more nucleotides. In another embodiment, the length of the poly-A region is 20 or more nucleotides. In another embodiment, the length of the poly-A region is 30 or more nucleotides. In another embodiment, the length of the poly-A region is 40 or more nucleotides. In another embodiment, the length of the poly-A region is 50 or more nucleotides. In another embodiment, the length of the poly-A region is 60 or more nucleotides. In another embodiment, the length of the poly-A region is 70 or more nucleotides. In another embodiment, the length of the poly-A region is 80 or more nucleotides. In another embodiment, the length of the poly-A region is 90 or more nucleotides. In another embodiment, the length of the poly-A region is 100 or more nucleotides. In another embodiment, the length of the poly-A region is 200 or more nucleotides. In another embodiment, the length of the poly-A region is 300 or more nucleotides. In another embodiment, the length of the poly-A region is 400 or more nucleotides. In this implementation, the length of the poly-A region is 500 or more nucleosides.

[0191] In one embodiment, the length of the poly-A region is 2 to 500 nucleotides. In another embodiment, the length of the poly-A region is 5 to 500 nucleotides. In another embodiment, the length of the poly-A region is 5 to 400 nucleotides. In another embodiment, the length of the poly-A region is 5 to 300 nucleotides. In another embodiment, the length of the poly-A region is 5 to 350 nucleotides. In another embodiment, the length of the poly-A region is 10 to 300 nucleotides. In another embodiment, the length of the poly-A region is 10 to 250 nucleotides. In another embodiment, the length of the poly-A region is 10 to 200 nucleotides. In another embodiment, the length of the poly-A region is 10 to 150 nucleotides. In another embodiment, the length of the poly-A region is 15 to 150 nucleotides. In another embodiment, the length of the poly-A region is 15 to 100 nucleotides. In another embodiment, the length of the poly-A region is 15 to 90 nucleotides. In another embodiment, the length of the poly-A region is 15 to 80 nucleotides. In one embodiment, the length of the poly-A region is 15 to 70 nucleotides. In another embodiment, the length of the poly-A region is 15 to 60 nucleotides. In yet another embodiment, the length of the poly-A region is 15 to 50 nucleotides. In still another embodiment, the length of the poly-A region is 15 to 40 nucleotides.

[0192] As used in this paper, “B” in Equation I: AB or Equation II: ABL contains the 3'-stable region.

[0193] In an embodiment, B includes a 3'-stable region comprising 1 to 1000 nucleosides, 1 to 900 nucleosides, 1 to 800 nucleosides, 1 to 700 nucleosides, 1 to 600 nucleosides, 1 to 500 nucleosides, 1 to 450 nucleosides, 1 to 400 nucleosides, 1 to 350 nucleosides, 1 to 300 nucleosides, 1 to 250 nucleosides, 1 to 240 nucleosides, 1 to 230 nucleosides, 1 to 220 nucleosides, 1 to 210 nucleosides, 1 to 200 nucleosides, 1 to 190 nucleosides, 1 to 180 nucleosides, 1 to 170 nucleosides, and 1 Up to 160 nucleosides, 1 to 150 nucleosides, 1 to 140 nucleosides, 1 to 130 nucleosides, 1 to 120 nucleosides, 1 to 110 nucleosides, 1 to 100 nucleosides, 1 to 90 nucleosides, 1 to 80 nucleosides, 1 to 70 nucleosides, 1 to 60 nucleosides, 1 to 50 nucleosides, 1 to 45 nucleosides, 1 to 40 nucleosides, 1 to 35 nucleosides, 1 to 30 nucleosides, 1 to 25 nucleosides, 1 to 20 nucleosides, 1 to 15 nucleosides, 1 to 10 nucleosides, 1 to 5 nucleosides, 1 to 4 nucleosides, 1 to 3 nucleosides, or 1 to 2 nucleosides.

[0194] In an embodiment, B includes a 3'-stable region comprising 1 to 100 nucleosides, 1 to 90 nucleosides, 1 to 80 nucleosides, 1 to 75 nucleosides, 1 to 70 nucleosides, 1 to 65 nucleosides, 1 to 60 nucleosides, 1 to 55 nucleosides, 1 to 50 nucleosides, 1 to 45 nucleosides, 1 to 40 nucleosides, 1 to 35 nucleosides, 1 to 30 nucleosides, 1 to 25 nucleosides, 1 to 20 nucleosides, 1 to 15 nucleosides, 1 to 10 nucleosides, 1 to 5 nucleosides, or 1 to 2 nucleosides.

[0195] In one embodiment, B includes a 3'-stable region containing one nucleoside. In another embodiment, B includes a 3'-stable region containing two nucleosides.

[0196] In one embodiment, the 3'-stable region comprises one or more unmodified nucleosides and one or more unmodified nucleotide inter-bonds. In another embodiment, the 3'-stable region comprises one or more modified nucleosides and / or one or more modified nucleotide inter-bonds. In yet another embodiment, the 3'-stable region comprises one or more modified nucleosides and one or more modified nucleotide inter-bonds. In yet another embodiment, the 3'-stable region comprises one or more modified nucleosides or one or more modified nucleotide inter-bonds.

[0197] In one embodiment, the 3'-stable region comprises one unmodified nucleoside and one unmodified nucleotide interbond. In another embodiment, the 3'-stable region comprises one unmodified nucleoside and more than one unmodified nucleotide interbond. In yet another embodiment, the 3'-stable region comprises more than one unmodified nucleoside and one unmodified nucleotide interbond. In a third embodiment, the 3'-stable region comprises two unmodified nucleosides and two unmodified nucleotide interbonds. In a fourth embodiment, the 3'-stable region comprises four unmodified nucleosides and four unmodified nucleotide interbonds. In a fifth embodiment, the 3'-stable region comprises five unmodified nucleosides and five unmodified nucleotide interbonds. In a sixth embodiment, the 3'-stable region comprises six unmodified nucleosides and six unmodified nucleotide interbonds. In a seventh embodiment, the 3'-stable region comprises seven unmodified nucleosides and seven unmodified nucleotide interbonds. In a fifth embodiment, the 3'-stable region comprises eight unmodified nucleosides and eight unmodified nucleotide interbonds. In one embodiment, the 3'-stable region comprises nine unmodified nucleosides and nine unmodified nucleotide inter-bonds. In another embodiment, the 3'-stable region comprises ten unmodified nucleosides and ten unmodified nucleotide inter-bonds.

[0198] In one embodiment, the 3'-stable region comprises a modified nucleoside and a modified nucleotide inter-bond. In another embodiment, the 3'-stable region comprises a modified nucleoside and more than one modified nucleotide inter-bond. In yet another embodiment, the 3'-stable region comprises more than one modified nucleoside and a modified nucleotide inter-bond. In yet another embodiment, the 3'-stable region comprises two modified nucleosides and two modified nucleotide inter-bonds. In yet another embodiment, the 3'-stable region comprises more than one modified nucleoside and more than one modified nucleotide inter-bond.

[0199] In one embodiment, the 3'-stable region comprises one modified nucleoside or one modified nucleotide interbond. In another embodiment, the 3'-stable region comprises two modified nucleosides or two modified nucleotide interbonds. In another embodiment, the 3'-stable region comprises three modified nucleosides or three modified nucleotide interbonds. In another embodiment, the 3'-stable region comprises four modified nucleosides or four modified nucleotide interbonds. In another embodiment, the 3'-stable region comprises five modified nucleosides or five modified nucleotide interbonds. In another embodiment, the 3'-stable region comprises six modified nucleosides or six modified nucleotide interbonds. In another embodiment, the 3'-stable region comprises seven modified nucleosides or seven modified nucleotide interbonds. In another embodiment, the 3'-stable region comprises eight modified nucleosides or eight modified nucleotide interbonds. In another embodiment, the 3'-stable region comprises nine modified nucleosides or nine modified nucleotide interbonds. In another embodiment, the 3'-stable region comprises ten modified nucleosides or ten modified nucleotide interbonds.

[0200] In one embodiment, the 3'-stable region comprises one or more unmodified nucleosides and / or one or more unmodified nucleotides bonded together and / or forming a secondary structure. In another embodiment, the 3'-stable region comprises one or more unmodified nucleosides or one or more unmodified nucleotides bonded together or forming a secondary structure. In another embodiment, the 3'-stable region comprises one or more unmodified nucleosides and one or more unmodified nucleotides bonded together and forming a secondary structure. In another embodiment, the 3'-stable region comprises one or more unmodified nucleosides and one or more unmodified nucleotides bonded together or forming a secondary structure. In another embodiment, the 3'-stable region comprises one or more unmodified nucleosides or one or more unmodified nucleotides bonded together and forming a secondary structure. In another embodiment, the 3'-stable region forms a secondary structure that prevents exonucleases from accessing the 3' terminal nucleotide and protects the 3' end from degradation. In another embodiment, the 3'-stable region contains an aptamer that protects RNA from degradation.

[0201] In one embodiment, the 3'-stable region comprises one or more modified nucleosides and / or one or more modified nucleotides and / or forms a secondary structure. In another embodiment, the 3'-stable region comprises one or more modified nucleosides or one or more modified nucleotides and forms a secondary structure. In another embodiment, the 3'-stable region comprises one or more modified nucleosides and one or more modified nucleotides and forms a secondary structure. In another embodiment, the 3'-stable region comprises one or more modified nucleosides and one or more modified nucleotides and forms a secondary structure. In another embodiment, the 3'-stable region comprises one or more modified nucleosides or one or more modified nucleotides and forms a secondary structure. In another embodiment, the 3'-stable region forms a secondary structure that prevents exonucleases from accessing the 3' terminal nucleotide and protects the 3' end from degradation. In another embodiment, the 3'-stable region contains an aptamer that protects RNA from degradation.

[0202] In one embodiment, the 3'-stabilizing region forms a secondary structure. In another embodiment, the secondary structure can be, for example, a G-quadruplex (a secondary structure formed from a guanine-rich nucleic acid sequence), a triplex, a protrusion, a kissing hairpin, a loop (e-loop), a branched multiloop, a stem loop (also called a "hairpin loop"), a tetraloop, a helix, or a pseudoknot. In one embodiment, the secondary structure can be a G-quadruplex. In another embodiment, the secondary structure can be a triplex. In another embodiment, the secondary structure can be a hairpin loop (stem loop). In another embodiment, the secondary structure can be a tetraloop. In another embodiment, the secondary structure can be a helix. In another embodiment, the secondary structure can be a protrusion. In another embodiment, the secondary structure can be a kissing hairpin. In another embodiment, the secondary structure can be a loop (e-loop). In another embodiment, the secondary structure can be a branched multiloop. In another embodiment, the secondary structure can be a pseudoknot.

[0203] In one embodiment, the modified nucleoside comprises modified nucleobases and / or modified sugars. In another embodiment, the modified nucleoside comprises modified nucleobases and modified sugars. In yet another embodiment, the modified nucleoside comprises modified nucleobases. In yet another embodiment, the modified nucleoside comprises modified sugars.

[0204] In embodiments, the modified nucleobase is modified uracil, modified cytosine, modified guanine, or modified adenine. In embodiments, the modified nucleobase is modified uracil. In embodiments, the modified nucleobase is modified cytosine. In embodiments, the modified nucleobase is modified guanine. In embodiments, the modified nucleobase is modified adenine. In embodiments, the modified nucleobase is pseudouracil (ψ), 2-thiouracil, 4-thiouracil, 4-thio-pseuuridine, 2-thio-pseuuridine, 5-hydroxyuracil, 5-halouracil, 3-methyluracil, 5-azauracil, or 2-thio-5-azauracil. In embodiments, the modified nucleobases are 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-methyl-cytosine, 5-halo-cytosine, 2-thio-cytosine, or 2-thio-5-methyl-cytosine. In embodiments, the modified nucleobases are 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenine, 7-deadenine, N6-methyl-adenine, or 2-methylthio-N6-methyl-adenine. In the embodiments, the modified nucleobases are inosine, 1-methyl-inosine, 7-cyano-7-denitro-guanine, 7-aminomethyl-7-denitro-guanine, 6-thio-guanine, 6-thio-7-denitro-guanine, or 6-methoxy-guanine.

[0205] Some non-limiting examples of modified nucleosides and nucleobases include pseudouridine (ψ), pyridin-4-one nucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, and 2-thio-uracil (s). 2 U), 4-thiouracil (s) 4 U), 4-thio-pseuuridine, 2-thio-pseuuridine, 5-hydroxy-uracil (ho) 5 U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m 3 U), 5-methoxy-uracil (mo) 5 U), uracil 5-oxyacetic acid (cmo) 5 U), uridine 5-oxyacetic acid methyl ester (mcmo) 5 U), 5-carboxymethyl-uracil (cm) 5 U), 1-carboxymethyl-pseuuridine, 5-carboxyhydroxymethyl-uracil (chm) 5 U), 5-Carboxyhydroxymethyl-uracil methyl ester (mchm) 5U), 5-methoxycarbonylmethyl-uracil (mcm) 5 U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm) 5 s 2 U), 5-aminomethyl-2-thio-uracil (nm) 5 s 2 U), 5-methylaminomethyluracil (mnm) 5 U), 5-methylaminomethyl-2-thio-uracil (mnm) 5 s 2 U), 5-methylaminomethyl-2-seleno-uracil (mnm) 5 se 2 U), 5-aminocarbamoylmethyluracil (ncm) 5 U), 5-Carboxymethylaminomethyl-uracil (cmnm) 5 U), 5-Carboxymethylaminomethyl-2-thio-uracil (cmnm) 5 s 2 U), 5-propynyl-uracil, 1-propynyl-pseuuridine, 5-tauronic acid methyl-uracil (τm) 5 U), 1-Taurate methyl-pseuuridine, 5-Taurate methyl-2-thio-uracil (τm) 5 s 2 U), 1-Tauratemethyl-4-thio-pseuuridine, 5-methyl-uracil (m 5 U, i.e., having nucleobase deoxythymidine), 1-methyl-pseudouridine (m 1 ψ), 5-methyl-2-thiouracil (m) 5 s 2 U), 1-methyl-4-thio-pseuuridine (m) 1 s 4 ψ), 4-thio-1-methyl-pseuuridine, 3-methyl-pseuuridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-1-deazo-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m) 5 D), 2-Thio-dihydrouracil, 2-Thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp) 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp) 3 ψ), 5-(isopentenylaminomethyl)uracil (inm)5 U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm) 5 s 2 U), 5,2'-O-dimethyluridine (m) 5 Um), 2-thio-2'-O-methyluridine (s) 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm) 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm) 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm) 5 Um), 3,2'-O-dimethyluridine (m) 3 Um) and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm) 5Um), 1-thiouracil, deoxythymidine, 5-(2-methoxycarbonylvinyl)uracil, 5-(carbamoylhydroxymethyl)uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyluracil, 5-methoxy-2-thiouracil, 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methylcytosine (m4C), 5-methyl 5-Cytosine (m5C), 5-Hallo-cytosine (e.g., 5-iodo-cytosine), 5-Hydroxymethyl-cytosine (hm5C), 1-Methyl-pseudo-cytosine, Pyrrolo-cytosine, Pyrrolo-pseudo-cytosine, 2-Thio-cytosine (s2C), 2-Thio-5-methyl-cytosine, 4-Thio-pseudo-cytosine, 4-Thio-1-methyl-pseudo-cytosine, 4-Thio-1-methyl-1-deazo-pseudo-cytosine, 1-Methyl-1-deazo-pseudo-cytosine, zabran, 5-aza-zabran, 5-methyl-zabran, 5-aza-2-thio-zabran 2-Thio-Zebrane, 2-Methoxy-Cytosine, 2-Methoxy-5-Methyl-Cytosine, 4-Methoxy-Pseudoisocytosine, 4-Methoxy-1-Methyl-Pseudoisocytosine, Lysictosine (k2C), 5,2'-O-Dimethyl-Cytosine (m5Cm), N4-Acetyl-2'-O-Methylcytosine (ac4Cm), N4,2'-O-Dimethyl-Cytosine (m4Cm), 5-Formyl-2'-O-Methyl-Cytosine (f5Cm), N4,N4,2'-O-Trimethyl-Cytosine (m42Cm), 1-Thio-Cytosine, 5-Hydroxy-Cytosine Pyridine, 5-(3-azidopropyl)-cytosine, 5-(2-azidoethyl)-cytosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deadenine, 7-deadenine-8-aza-adenine, 7-deadenine-2-amino-purine, 7-deadenine-8-aza-2-amino-purine, 7-deadenine-2,6-diaminopurine, 7-deadenine-8-aza-2,6-Diaminopurine, 1-methyl-adenine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-) N6-hydroxyisopentenyl)adenine (ms2io6A), N6-glycylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxyn-valine Acylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxyn-valinecarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenine (m6Am), N6,N6,2'-O-trimethyl-adenine (m62Am), 1,2'-O-dimethyl-adenine 2-Amino-N6-methyl-purine, 1-Thio-adenine, 8-Azide-adenine, N6-(19-amino-pentaenolide)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, N6-hydroxymethyl-adenine, inosine (I), 1-methyl-inosine (m1I), wyoside (imG), methyl wyoside (mimG), 4-demethyl wyoside (imG) -14), isowyoside (imG2), weitingin (yW), peroxyweitingin (o2yW), hydroxyweitingin (OHyW), undermodified hydroxyweitingin (OHyW*), 7-denitro-guanine, guanine (Q), epoxy-guanine (oQ), galactosyl-guanine (galQ), mannosyl-guanine (manQ), 7-cyano-7-denitro-guanine (preQ0), 7-aminomethyl-7-denitro-guanine (preQ1), archaenoside (G+), 7-denitro-8-aza- Guanine, 6-thio-guanine, 6-thio-7-deazo-guanine, 6-thio-7-deazo-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,The modified nucleobases include 7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl-6-thio-guanine, N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 1-thio-guanine, and O-6-methyl-guanine. In embodiments, the modified nucleobase can be any of the aforementioned nucleobases.

[0206] In embodiments, the modified sugar has a 5-membered ring or a 6-membered ring, or is a modified ribose. In embodiments, the ribose is replaced by a morpholino ring. In embodiments, the modified nucleoside contains a morpholino ring. In embodiments, the modified ribose is 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, or 3'-amino-2',3'-dideoxyribose.

[0207] Some non-limiting examples of sugar modification include modification of the 2'-hydroxyl group of the ribose ring, substitution of oxygen in the ribose ring, and expansion or contraction of the ribose ring. In embodiments, the modified sugar may contain any of the foregoing modifications. In embodiments, the 2'-hydroxyl group of the ribose ring may be substituted with each of the following: hydrogen, halogroup, methoxy group, azide group, substituted or unsubstituted alkyl group, substituted or unsubstituted heteroalkyl group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocycloalkyl group, substituted or unsubstituted aryl group, and substituted or unsubstituted heteroaryl group.

[0208] In some embodiments, the 2'-hydroxyl group of the ribocycle may be hydrogen-substituted. In some embodiments, the 2'-hydroxyl group of the ribocycle may be halogen-substituted. In some embodiments, the 2'-hydroxyl group of the ribocycle may be azide-substituted. In some embodiments, the 2'-hydroxyl group of the ribocycle may be methoxy-substituted.

[0209] In one embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted alkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted alkyl group (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted alkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent). In yet another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by an unsubstituted alkyl group (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In yet another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted heteroalkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In one embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by an unsubstituted heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In yet another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted cycloalkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In one embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by an unsubstituted cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In yet another embodiment, the 2'-hydroxyl group of the ribocycle is replaced by a substituted heterocyclic alkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heterocyclic alkyl group (e.g., 3- to 8-membered heteroalkyl, 3- to 6-membered heteroalkyl, or 5- to 6-membered heterocyclic alkyl). In one embodiment, the 2'-hydroxyl group of the ribose ring is replaced by a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heterocyclic alkyl group (e.g., 3- to 8-membered heterocyclic alkyl group, 3- to 6-membered heterocyclic alkyl group, or 5- to 6-membered heterocyclic alkyl group). In another embodiment, the 2'-hydroxyl group of the ribose ring is replaced by an unsubstituted heterocyclic alkyl group (e.g., 3- to 8-membered heterocyclic alkyl group, 3- to 6-membered heterocyclic alkyl group, or 5- to 6-membered heterocyclic alkyl group).In embodiments, the 2'-hydroxyl group of the ribose ring is replaced by a substituted aryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted aryl group (e.g., C6-C). 10 Aryl, C 10 Aryl or phenyl group is substituted. In embodiments, the 2'-hydroxyl group of the ribose ring is replaced by a substituted aryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an aryl group (e.g., C6-C). 10 Aryl, C 10 The 2'-hydroxyl group of the ribose ring is replaced by an unsubstituted aryl group (e.g., C6-C). In embodiments, the 2'-hydroxyl group of the ribose ring is replaced by an unsubstituted aryl group (e.g., C6-C). 10 Aryl, C 10 The 2'-hydroxyl group of the ribocycle is replaced by a substituted heteroaryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, the 2'-hydroxyl group of the ribocycle is replaced by a substituted heteroaryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) (e.g., 5- to 10-membered heteroalkyl, 5- to 9-membered heteroalkyl, or 5- to 6-membered heteroalkyl). In embodiments, the 2'-hydroxyl group of the ribocycle is replaced by an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl).

[0210] In embodiments, the oxygen in the ribocycle may be replaced by -S-, -Se-, -NH-, or -CH2-. In embodiments, the oxygen in the ribocycle may be replaced by -S-. In embodiments, the oxygen in the ribocycle may be replaced by -Se-. In embodiments, the oxygen in the ribocycle may be replaced by -NH-. In embodiments, the oxygen in the ribocycle may be replaced by -CH2-. In embodiments, the ribocycle may be substituted with another ring, for example, the ring may be a cyclobutene, mannitol, cyclohexyl, or morpholinyl ring. In embodiments, the ribocycle may be substituted with a morpholinyl ring. In embodiments, the ribocycle may be substituted with a mannitol ring. In embodiments, the ribocycle may be substituted with a cyclohexyl ring. In embodiments, the ribocycle may be substituted with a locked nucleic acid ring (LNA). In embodiments, the ribocycle may be substituted with an unlocked nucleic acid ring (UNA).

[0211] In an embodiment, the 3'-stabilizing region comprises one or more modified internucleotide bonds. In an embodiment, the internucleotide bonds comprise modified phosphate esters. In an embodiment, the modified phosphate ester is a phosphorothioate, dithiophosphate, thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphonate, phosphoroselenoate, selenophosphate, aminophosphate, carbophosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boron phosphate, methylphosphonate, or guanidinopropyl aminophosphate.

[0212] In embodiments, the modified nucleotide inter-bonds include, for example, thiophosphates, selenophosphates, boranophosphates, boranophosphate esters, hydrophosphonates, aminophosphates, diaminophosphates, alkyl or aryl phosphonates, and phosphate triesters. In embodiments, the modified nucleotide inter-bonds may be dithiophosphates, wherein both unlinked oxygen atoms are sulfur-substituted. In embodiments, the modified nucleotide inter-bonds may include replacing the linking oxygen with -HN-, -S-, or -CH2-. In embodiments, the modified nucleotide inter-bonds may include replacing the unlinked oxygen (single bond with phosphorus) with methyl, ethyl, methoxy, -SH, or -BH3, or any combination thereof.

[0213] In embodiments, one or more unmodified nucleosides and one or more unmodified nucleotide inter-bonds and one or more secondary structures are considered for stabilizing RNA molecules. In embodiments, one or more unmodified nucleosides and one or more unmodified nucleotide inter-bonds are considered for stabilizing RNA molecules. In embodiments, any combination of one or more modified nucleosides and / or one or more modified nucleotide inter-bonds and / or one or more secondary structures is considered for stabilizing RNA molecules. For example, the 3'-stabilizing region may include at least one PS modification (of the nucleotide inter-bond) and / or at least one modification of the 2'-hydroxyl group of the ribose ring. In embodiments, the 3'-stabilizing region may, for example, include at least one PS modification (of the nucleotide inter-bond) and / or at least one modification of the 2'-hydroxyl group of the ribose ring and / or at least one secondary structure.

[0214] In one embodiment, the last nucleoside of the 3'-stable region does not contain a 3'-hydroxyl group. In another embodiment, the last nucleoside of the 3'-stable region is a chain-terminating nucleoside. In another embodiment, the last nucleoside of the 3'-stable region is blocked and cannot react with any other NTP. In another embodiment, the last nucleoside of the 3'-stable region is ddC, reverse dT, a 3'-phosphate nucleoside, a 3'-oxime nucleoside, a 3'-azidomethyl nucleoside, or a 3'-methyl nucleoside. In another embodiment, the last nucleoside of the 3'-stable region is ddC. In another embodiment, the last nucleoside of the 3'-stable region is reverse dT. In another embodiment, the last nucleoside of the 3'-stable region is a 3'-phosphate nucleoside. In another embodiment, the last nucleoside of the 3'-stable region is a 3'-oxime nucleoside. In another embodiment, the last nucleoside of the 3'-stable region is a 3'-methyl nucleoside.

[0215] In one embodiment, one or more nucleosides within the 3'-stable region contain one or more purification tags. In another embodiment, one nucleoside within the 3'-stable region contains one or more purification tags. In yet another embodiment, one nucleoside within the 3'-stable region contains one purification tag. In a third embodiment, two nucleosides within the 3'-stable region contain two purification tags. In yet another embodiment, three nucleosides within the 3'-stable region contain three purification tags. In yet another embodiment, four nucleosides within the 3'-stable region contain four purification tags. In yet another embodiment, five nucleosides within the 3'-stable region contain five purification tags. In yet another embodiment, six nucleosides within the 3'-stable region contain six purification tags. In yet another embodiment, seven nucleosides within the 3'-stable region contain seven purification tags. In yet another embodiment, eight nucleosides within the 3'-stable region contain eight purification tags. In yet another embodiment, nine nucleosides within the 3'-stable region contain nine purification tags. In yet another embodiment, ten nucleosides within the 3'-stable region contain ten purification tags.

[0216] In one embodiment, a nucleoside within the 3'-stable region contains one purification tag. In another embodiment, a nucleoside within the 3'-stable region contains two purification tags. In yet another embodiment, a nucleoside within the 3'-stable region contains three purification tags. In yet another embodiment, a nucleoside within the 3'-stable region contains four purification tags. In yet another embodiment, a nucleoside within the 3'-stable region contains five purification tags. In yet another embodiment, a nucleoside within the 3'-stable region contains at least five purification tags. In yet another embodiment, a nucleoside within the 3'-stable region contains ten purification tags.

[0217] In one embodiment, one or more nucleosides within the 3'-stable region contain one or more adapters (L), wherein the adapters (L) are capable of binding to the purification tag. In another embodiment, one nucleoside within the 3'-stable region contains one or more adapters (L). In yet another embodiment, one nucleoside within the 3'-stable region contains one adapter (L). In yet another embodiment, two nucleosides within the 3'-stable region contain two adapters (L). In yet another embodiment, three nucleosides within the 3'-stable region contain three adapters (L). In yet another embodiment, four nucleosides within the 3'-stable region contain four adapters (L). In yet another embodiment, five nucleosides within the 3'-stable region contain five adapters (L). In yet another embodiment, six nucleosides within the 3'-stable region contain six adapters (L). In yet another embodiment, seven nucleosides within the 3'-stable region contain seven adapters (L). In yet another embodiment, eight nucleosides within the 3'-stable region contain eight adapters (L). In yet another embodiment, nine nucleosides within the 3'-stable region contain nine adapters (L). In yet another embodiment, ten nucleosides within the 3'-stable region contain ten adapters (L).

[0218] In one embodiment, a nucleoside within the 3'-stable region contains one linker (L). In another embodiment, a nucleoside within the 3'-stable region contains two linkers (L). In yet another embodiment, a nucleoside within the 3'-stable region contains three linkers (L). In yet another embodiment, a nucleoside within the 3'-stable region contains four linkers (L). In yet another embodiment, a nucleoside within the 3'-stable region contains five linkers (L). In yet another embodiment, a nucleoside within the 3'-stable region contains at least five linkers (L). In yet another embodiment, a nucleoside within the 3'-stable region contains ten linkers (L).

[0219] In this embodiment, the purification tag is connected to the 3'-stable region via a connector (L). In this embodiment, L is a bond, -S(O)2-, -N(R)-, -O-, -S-, -C(O)-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)NH-, -NHC(O)N(R)-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or any combination thereof; and R is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC( O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or any combination thereof.

[0220] In embodiments, L is a substituted alkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is an unsubstituted alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is a substituted heteroalkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene). In embodiments, L is an unsubstituted heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene). In embodiments, L is a substituted cycloalkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, L is an unsubstituted heterocyclic alkylene (e.g., C3-C8 heterocyclic alkylene, C3-C6 heterocyclic alkylene, or C5-C6 heterocyclic alkylene). In embodiments, L is a substituted heterocyclic alkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heterocyclic alkylene (e.g., 3- to 8-membered heterocyclic alkylene, 3- to 6-membered heterocyclic alkylene, or 5- to 6-membered heterocyclic alkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heterocyclic alkylene (e.g., 3- to 8-membered heterocyclic alkylene, 3- to 6-membered heterocyclic alkylene, or 5- to 6-membered heterocyclic alkylene). In embodiments, L is an unsubstituted heterocyclic alkylene (e.g., 3- to 8-membered heterocyclic alkylene, 3- to 6-membered heterocyclic alkylene, or 5- to 6-membered heterocyclic alkylene). In the implementation, L is a substituted arylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted arylene (e.g., C6-C). 10 Alpha-aryl, C 10(Arylidene or phenylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) arylene (e.g., C6-C). 10 Alpha-aryl, C 10 (Arylidene or phenylene). In embodiments, L is an unsubstituted arylidene (e.g., C6-C). 10 Alpha-aryl, C 10 (Arylidene or phenylene). In embodiments, L is a substituted heteroaryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, L is an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl).

[0221] In the embodiments, R independently represents hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH 2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, substituted or unsubstituted alkyl groups (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), substituted or unsubstituted heteroalkyl groups (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), substituted or unsubstituted cycloalkyl groups (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), substituted or unsubstituted heterocycloalkyl groups (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), substituted or unsubstituted aryl groups (e.g., C6-C... 10 Aryl, C 10 Aryl or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl).

[0222] In embodiments, R is a substituted alkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted alkyl group (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) alkyl group (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R is an unsubstituted alkyl group (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R is a substituted heteroalkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In embodiments, R is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In embodiments, R is an unsubstituted heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In embodiments, R is a substituted cycloalkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R is an unsubstituted cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R is a substituted heterocyclic alkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heterocyclic alkyl group (e.g., 3- to 8-membered heterocyclic alkyl, 3- to 6-membered heterocyclic alkyl, or 5- to 6-membered heterocyclic alkyl). In embodiments, R is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heterocyclic alkyl group (e.g., 3- to 8-membered heterocyclic alkyl, 3- to 6-membered heterocyclic alkyl, or 5- to 6-membered heterocyclic alkyl). In embodiments, R is an unsubstituted heterocyclic alkyl group (e.g., 3- to 8-membered heterocyclic alkyl, 3- to 6-membered heterocyclic alkyl, or 5- to 6-membered heterocyclic alkyl). In embodiments, R is a substituted aryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted aryl group (e.g., C6-C). 10 Aryl, C 10 Aryl or phenyl). In embodiments, R is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) aryl group (e.g., C6-C). 10 Aryl, C 10Aryl or phenyl). In embodiments, R is an unsubstituted aryl group (e.g., C6-C). 10 Aryl, C 10 Aryl or phenyl). In embodiments, R is a substituted heteroaryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, R is a substituted heteroaryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, R is an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl).

[0223] In embodiments, L is a substituted or unsubstituted alkylene or a substituted or unsubstituted heteroalkylene. In embodiments, L is a substituted alkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is an unsubstituted alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is a substituted heteroalkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene). In embodiments, L is an unsubstituted heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene).

[0224] In this embodiment, L stands for -CH2CH(CH2OH)(CH2). m NH-, -CH=CHC(O)NH(CH2) p NH-, , or Where m is an integer from 0 to 8; n is an integer from 0 to 8; and p is an integer from 0 to 10. In the embodiment, L is -CH2CH(CH2OH)(CH2). m NH-, , or , where m is an integer from 0 to 8. In the implementation, L is Where n is an integer from 0 to 8. In the implementation, L is -CH=CHC(O)NH(CH2). p NH-, where p is an integer from 0 to 10. In the implementation, L is .

[0225] In the embodiments, L is -CH2CH(CH2OH)(CH2)4NH- or -CH=CHC(O)NH(CH2)6NH-. In the embodiments, L is -CH2CH(CH2OH)(CH2)4NH-. In the embodiments, L is -CH=CHC(O)NH(CH2)6NH-.

[0226] In the implementation method, L is , or In the implementation method, L is... In the implementation method, L is... In the implementation method, L is... In the implementation method, L is... .

[0227] In the embodiments, L is a phosphate ester or a modified phosphate ester. Some non-limiting examples of modified phosphate esters include, but are not limited to, phosphorothioate, dithiophosphate, thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphonate, phosphoroselenoate, selenophosphate, aminophosphate, carbophosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boron phosphate, methylphosphonate, and guanidinopropyl aminophosphate.

[0228] In embodiments, the modified phosphate ester may be, for example, a thiophosphate ester, a selenophosphate ester, a boranophosphate ester, a boranophosphate ester, a hydrophosphonate, an aminophosphate ester, a diaminophosphate ester, an alkyl or aryl phosphonate ester, or a triphosphate ester.

[0229] In some embodiments, the purification tag is a hydrophobic group covalently linked to the 3'-stable region of the RNA molecule described herein; such a group may be removable or non-removable. In some embodiments, the purification tag is directly linked to the 3'-stable region via a covalent bond. In some embodiments, the purification tag is covalently linked to the 3'-stable region via a linker (L). In some embodiments, the purification tag is a removable group. In some embodiments, the purification tag is a non-removable group.

[0230] In implementation methods, purification tags include, for example, but not limited to, C6-C. 24 Alkyl, C4-C 24 alkenyl, C4-C 24 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, silyl, triphenylmethyl, lipid, dye, steroid, vinyl ether compounds, modified and unmodified Fmoc compounds, etc. Fluorine substituents or fluorinated groups can be used to increase the hydrophobicity of the hydrophobic group. In embodiments, the purification label includes C6-C. 24 Alkyl group. In embodiments, the purification label includes C4-C... 24 Alkenyl group. In embodiments, the purification label includes C4-C. 24 Alkyne group. In embodiments, the purification label includes C3-C8 cycloalkyl groups. In embodiments, the purification label includes C6-C... 10 Aryl. In an embodiment, the purification label includes a silicon-based compound. In an embodiment, the purification label includes a triphenylmethyl compound. In an embodiment, the purification label includes a lipid. In an embodiment, the purification label includes a steroid. In an embodiment, the purification label includes a vinyl ether compound. In an embodiment, the purification label includes a modified Fmoc compound. In an embodiment, the purification label includes an unmodified Fmoc compound.

[0231] In embodiments, purification labels include, for example, but not limited to, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, and pentadecyl. In embodiments, purification labels include, for example, but not limited to, phenyl, benzyl, (ethyl)carbonyl (azadibenzocyclooctyne) (DBCO), 4-ethylphenol, dibenzohexyltriazoloazacyclooctene, and 1'-O-butyl3',4',6'-triacetylGalNAc.

[0232] In embodiments, purification labels include, for example, but not limited to, -C(O)-propyl, -C(O)-butyl, -C(O)-pentyl, -C(O)-hexyl, -C(O)-heptyl, -C(O)-octyl, -C(O)-nonyl, -C(O)-decyl, -C(O)-undecyl, -C(O)-dodecyl, -C(O)-tridecyl, -C(O)-tetradecyl, and -C(O)-pentadecanyl. In embodiments, purification labels include, for example, but not limited to, -C(O)-phenyl, -C(O)-benzylmethyl, -C(O)-4-ethylphenol, -C(O)-(ethyl)carbonyl (azadibenzocyclooctyne), -C(O)-dibenzohexyltriazoloazacyclooctene, and -C(O)-1'-O-butyl3',4',6'-triacetylGalNAc.

[0233] In this embodiment, the purification label is (ethyl)carbonyl (azadibenzocyclooctyne) (DBCO). In this embodiment, the purification label is 4-ethylphenol. In this embodiment, the purification label is dibenzohexyltriazolazazacyclooctene, two isomers. and A mixture. In this embodiment, the purification label is 1'-O-butyl3',4',6'-triacetylGalNAc. .

[0234] In this implementation, the purification tag includes lipids. In this implementation, it is not desired to be bound by any particular theory that the inclusion of lipid purification tags can lead to increased encapsulation of mRNA molecules carrying lipid purification tags in pharmaceutical carriers, such as lipid nanoparticles (LNPs).

[0235] In embodiments, this document considers RNA molecules comprising a 3'-stabilizing region as described herein and a 5'-cap analog as described herein and in references incorporated herein. In embodiments, this document considers RNA molecules comprising a 5'-cap analog as described herein and in references incorporated herein and / or a modified 5'-UTR as described herein and / or a modified ORF as described herein and / or a modified 3'-UTR as described herein and / or a modified poly-A tail as described herein and / or a 3'-stabilizing region as described herein.

[0236] compound In one respect, this article provides a compound of formula (III) or (IV): Where N is a nucleoside; L is a adapter that can bind to the purification tag; P stands for purification tag; Q-L1 may optionally exist, where L1 is a connector covalently bonded to N and Q; and Q is hydrogen or a chain-terminating nucleoside.

[0237] In this embodiment, the nucleoside is an unmodified nucleoside. In this embodiment, the nucleoside is a modified nucleoside. In this embodiment, the modified nucleoside is as defined herein, including those defined in this embodiment.

[0238] In this implementation, the connector (L) is as defined herein, including those defined in this implementation.

[0239] In this implementation, the purification tag (P) is as defined herein, including those defined in this implementation.

[0240] In an embodiment, L1 is a connector. In an embodiment, connector L1 may be the same as or different from connector (L). In an embodiment, connector L1 is the same as connector (L) as defined herein. In an embodiment, connector L1 is a phosphate ester (-(HO)P(=O)-). In an embodiment, connector L1 is a modified phosphate ester. In an embodiment, the modified phosphate ester is a phosphorothioate, dithiophosphate, thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphonate, phosphoroselenoate, selenophosphate, aminophosphate, carbophosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boron phosphate, methylphosphonate, or guanidinium aminophosphate.

[0241] In embodiments, Q is hydrogen or a chain-terminating nucleoside. Some non-limiting examples of chain-terminating nucleosides include, but are not limited to, ddC, reverse dT, 3'-phosphate nucleoside, 3'-oxime nucleoside, 3'-azidomethyl nucleoside, or 3'-methyl nucleoside. In embodiments, Q is hydrogen. In embodiments, Q is ddC. In embodiments, Q is reverse dT. In embodiments, Q is 3'-phosphate nucleoside. In embodiments, Q is 3'-oxime nucleoside. In embodiments, Q is 3'-azidomethyl nucleoside. In embodiments, Q is 3'-methyl nucleoside.

[0242] In one embodiment, the linker (L) capable of binding the purification tag is linked to the nucleoside via the 3'-carbon or 2'-carbon of the sugar of the nucleoside. In another embodiment, the linker (L) capable of binding the purification tag is linked to the nucleoside via the nucleobase of the nucleoside. In another embodiment, the nucleoside is an unmodified nucleoside. In another embodiment, the nucleoside is a modified nucleoside. Modified nucleosides are included as defined herein, as defined in this embodiment. In another embodiment, the sugar is a modified sugar. In another embodiment, the sugar is an unmodified sugar. Modified sugars are included as defined herein, as defined in this embodiment. In another embodiment, the nucleobase is an unmodified nucleobase. In another embodiment, the nucleobase is a modified nucleobase. Modified nucleobases are included as defined herein, as defined in this embodiment.

[0243] In one embodiment, the adapter (L) capable of binding the purification tag is linked to the nucleoside via the nucleobase of the nucleoside, and the L1 adapter is linked to the nucleoside via the 3'-carbon or 2'-carbon of the sugar of the nucleoside. In another embodiment, the adapter (L) capable of binding the purification tag is linked to the nucleoside via the 3'-carbon or 2'-carbon of the sugar of the nucleoside, and the L1 adapter is linked to the nucleoside via the nucleobase of the nucleoside.

[0244] In one embodiment, the purification tag is linked to the nucleoside via the 3'-carbon or 2'-carbon of the sugar. In another embodiment, the purification tag is linked to the nucleoside via a nucleobase. In another embodiment, the nucleoside is an unmodified nucleoside. In another embodiment, the nucleoside is a modified nucleoside. Modified nucleosides are included as defined herein. In another embodiment, the sugar is a modified sugar. In another embodiment, the sugar is an unmodified sugar. Modified sugars are included as defined herein. In another embodiment, the nucleobase is an unmodified nucleobase. In another embodiment, the nucleobase is a modified nucleobase. Modified nucleobases are included as defined herein.

[0245] Some non-limiting examples of compounds of formula (III) include, but are not limited to, for example: and Among them, the wavy line ( The symbol indicates a connection to a hydroxyl group (OH), a phosphate ester, a diphosphate ester, a triphosphate ester, or a modified phosphate ester. In embodiments, the modified phosphate ester is a phosphorothioate, a dithiophosphate ester, a thiophosphate ester, a 5'-O-methylphosphonate, a 3'-O-methylphosphonate, a 5'-hydroxyphosphonate, a hydroxyphosphonate, a phosphoroselenoate, a selenophosphate ester, an aminophosphate ester, a carbophosphonate, a phenylphosphonate, an ethylphosphonate, an H-phosphonate, a guanidinium ring, a triazole ring, a boron phosphate ester, a methylphosphonate, or a guanidinium aminophosphate propyl ester.

[0246] In the embodiments, the compound of formula (III) contains, for example... or .

[0247] Some non-limiting examples of compounds of formula (IV) include, but are not limited to, for example: and Among them, the wavy line ( The symbol indicates a connection to a hydroxyl group (OH), a phosphate ester, a diphosphate ester, a triphosphate ester, or a modified phosphate ester. In embodiments, the modified phosphate ester is a phosphorothioate, a dithiophosphate ester, a thiophosphate ester, a 5'-O-methylphosphonate, a 3'-O-methylphosphonate, a 5'-hydroxyphosphonate, a hydroxyphosphonate, a phosphoroselenoate, a selenophosphate ester, an aminophosphate ester, a carbophosphonate, a phenylphosphonate, an ethylphosphonate, an H-phosphonate, a guanidinium ring, a triazole ring, a boron phosphate ester, a methylphosphonate, or a guanidinium aminophosphate propyl ester.

[0248] In embodiments, the compound of formula (IV) comprises, for example... or .

[0249] In one aspect, this document provides a method for increasing the expression of a target protein or peptide in cells, comprising contacting cells with an RNA molecule comprising any of the compounds of formula (III) or (IV) described herein, wherein the RNA molecule encodes the target protein or peptide, wherein the expression is increased compared to the expression of RNA molecules not comprising any of the compounds of formula (III) or (IV) described herein, optionally wherein the cells are isolated in vitro. In another aspect, this document provides a method for increasing the expression of a target protein or peptide in cells, comprising contacting cells with an RNA molecule comprising any of the compounds of formula (III) or (IV) described herein, wherein the RNA molecule encodes the target protein or peptide, wherein the expression is increased compared to the expression of RNA molecules not comprising any of the compounds of formula (III) or (IV) described herein, optionally wherein the cells are isolated in vitro. In one aspect, this article provides a method for increasing the expression of a target protein or peptide in a cell, comprising contacting the cell with an RNA molecule comprising any one of the compounds of formula (III) or (IV) described herein, wherein the RNA molecule encodes the target protein or peptide, wherein the expression is increased compared to the expression of an RNA molecule not comprising any one of the compounds of formula (III) or (IV) described herein, optionally wherein the cell is isolated in vitro.

[0250] In one aspect, this document provides a method for prolonging the half-life of RNA molecules in cells, comprising contacting cells with RNA molecules comprising any one of the compounds of formula (III) or (IV) described herein, wherein the half-life is prolonged compared to the half-life of RNA molecules not comprising any one of the compounds of formula (III) or (IV) described herein, optionally wherein the cells are isolated in vitro or ex vivo. In one aspect, this article provides a method for prolonging the half-life of RNA molecules in cells, comprising contacting cells with RNA molecules comprising any one of the compounds of formula (III) or formula (IV) described herein, wherein the half-life is prolonged compared to the half-life of RNA molecules not comprising any one of the compounds of formula (III) or formula (IV) described herein, optionally wherein the cells are isolated in vitro.

[0251] RNA synthesis In one aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises one or more purification tags or one or more adapters capable of binding purification tags. In another aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises one or more purification tags. In one aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises a purification tag. In another aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises two purification tags. In one aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises three purification tags. In another aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises four purification tags.In one aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises five purification tags. In another aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises more than five purification tags.

[0252] In one aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises one or more adapters capable of binding a purification tag. In another aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises an adapter capable of binding a purification tag. In one aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises two adapters capable of binding purification tags. In another aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises three adapters capable of binding purification tags. In one aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises four adapters capable of binding purification tags. In another aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, a protein-encoding ORF, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises five adapters capable of binding purification tags.In one aspect, this document provides a method for preparing any of the RNA molecules described herein, comprising covalently linking a stable region to a precursor RNA comprising a 5'-cap, optionally a 5' UTR, an ORF encoding a protein, optionally a 3' UTR, and a poly-A region located at the 3' of the ORF, wherein the stable region is added to the 3' of the poly-A region and the stable region comprises more than five adapters capable of binding purification tags.

[0253] In one embodiment, the 3'-stable region is added to the precursor RNA by ligation. In another embodiment, ligation is either enzymatic or chemical. In yet another embodiment, the 3'-stable region is added to the precursor RNA using a polymerase. In yet another embodiment, the 3'-stable region is added to the precursor RNA by ligation followed by polymerase. In yet another embodiment, the 3'-stable region is added to the precursor RNA by using a polymerase followed by ligation.

[0254] In one aspect, this document provides an in vitro method for synthesizing capped RNA transcripts, including capped messenger RNA (mRNA) transcripts. The method described herein comprises (a) forming a reaction mixture containing a cap analog, a DNA template, and an RNA polymerase; and (b) cultivating the reaction mixture under conditions that allow the DNA template to be transcribed to produce capped mRNA transcripts. In the method described herein, the reaction mixture contains NTPs, including ATP, CTP, GTP, and UTP. One or more NTPs in the in vitro transcription reaction mixture may be modified NTPs. Exemplary modified nucleosides include, but are not limited to, inosine, 7-denitroguanosine, 7-methylguanosine, dihydrouridine, 2'-O-methylguanosine, 2'-fluoro-2'-deoxycytidine, pseudouridine, N1-methylpseudouridine, and 5-methyluridine. In some methods, one or more uridines in the in vitro transcribed RNA are replaced by modified nucleosides. Optionally, some methods further include mixing a reaction mixture containing a capped mRNA transcript with a mixture containing Ca 2+ Incubate with DNase I buffer to degrade and remove DNA template.

[0255] Optionally, some methods further comprise removing proteins from the in vitro transcription reaction by subjecting the DNase-treated reaction mixture to phosphatase treatment. Optionally, the methods further comprise subjecting the DNase-treated reaction mixture to one or more purification steps. mRNA transcripts generated by the methods described herein can be purified using one or more purification techniques known to those skilled in the art. See Baronti et al., (2018). Anal. Bioanal. Chem. 410(14): 3239-33252.

[0256] For example, mRNA can be purified by: liquid chromatography (e.g., HPLC, reversed-phase ion-paired HPLC (RP-IP-HPLC), anion exchange chromatography, cation exchange chromatography, affinity chromatography, size exclusion chromatography), precipitation, dialysis, tangential flow filtration, oligo dT chromatography, silica membrane purification, and hydrophobic interaction chromatography, to name just a few. Synthetic capped mRNA transcripts can be largely free of impurities such as DNA, proteins, double-stranded RNA, and / or incomplete mRNA transcripts.

[0257] In one embodiment, the 3'-stable region is covalently linked to the 3' end of the precursor RNA via a linker that can be formed by ligation. In another embodiment, the linker is a bioconjugate linker. In yet another embodiment, the linker can be formed by enzyme ligation, splinting, or chemical ligation. In yet another embodiment, the linker can be formed by enzyme ligation or chemical ligation. In yet another embodiment, the linker can be formed by enzyme ligation. In yet another embodiment, the linker can be formed by chemical ligation. In yet another embodiment, the linker can be formed by splinting.

[0258] In embodiments, the linker formed by the connection between the 3'-stable region and the 3'-end of the precursor RNA includes, but is not limited to, reactions involving, for example, thiol, amino, phosphate, and / or hydroxyl groups or any suitable reactive groups. Various cross-linking agents are known for the coupling of the 3'-stable region to the precursor RNA. For example, NHS / EDC allows coupling of the primary amino group to the carboxyl group; sulfonyl-EMCS ([N-ε-cis-butenyliminohexanoic acid] hydrazide (cis-butenylimide and NHS-ester) is reactive to both thiol and amino groups.

[0259] In this embodiment, an accessible amino group located at the 3'-stable region or the 3'-terminus of the precursor RNA can react with the NHS ester. When the NHS ester reacts with a primary amine, an amide bond is formed. In this embodiment, an accessible thiol group located at the 3'-stable region or the 3'-terminus of the precursor RNA can react with a maleic anhydride to form a thioether bond. In this embodiment, an accessible phosphate group located at the 3'-stable region or the 3'-terminus of the precursor RNA can react with an imidazole, triazole, or tetraazole-activated phosphate ester.

[0260] In this embodiment, the linker can be formed via a click chemistry reaction. In this embodiment, one of the reactive groups is attached to the 3'-stable region, and the other reactive group is attached to the 3'-end of the precursor RNA. Reactive group pairs include, but are not limited to, the following: alkynyl and azide groups; dienyl groups (e.g., 1,3-butadiene, cyclopentadiene, cyclohexadiene, or furan) and dienophiles (e.g., any alkenyl or any alkynyl); aldehydes and amino groups; Michael acceptors (e.g., conjugated alkenyl groups, such as α,β-unsaturated ketones, α,β-unsaturated esters, α,β-unsaturated nitriles) and Michael donors (e.g., thiols, amines, enols, enamines). These are merely non-limiting examples of various reactive group pairs that can be used in click chemistry reactions.

[0261] In one embodiment, the 3'-stable region contains one nucleoside. In another embodiment, the 3'-stable region contains two nucleosides. In another embodiment, the 3'-stable region contains three nucleosides. In another embodiment, the 3'-stable region contains four nucleosides. In another embodiment, the 3'-stable region contains five nucleosides. In another embodiment, the 3'-stable region contains six nucleosides. In another embodiment, the 3'-stable region contains seven nucleosides. In another embodiment, the 3'-stable region contains eight nucleosides. In another embodiment, the 3'-stable region contains nine nucleosides. In another embodiment, the 3'-stable region contains ten nucleosides. In another embodiment, the 3'-stable region contains twenty nucleosides. In another embodiment, the 3'-stable region contains thirty nucleosides. In another embodiment, the 3'-stable region contains forty nucleosides. In another embodiment, the 3'-stable region contains fifty nucleosides.

[0262] In one embodiment, the 3'-stable region is covalently linked to the 3' end of the precursor RNA via a linker that can be formed using a polymerase. In another embodiment, the polymerase is adenosine monophosphate, polyuridine monophosphate, or RNA nucleotidyltransferase. In yet another embodiment, the polymerase is adenosine monophosphate. In yet another embodiment, the polymerase is polyuridine monophosphate. In yet another embodiment, the polymerase is an RNA nucleotidyltransferase. In yet another embodiment, the polymerase catalyzes the reaction between a polyphosphate group (e.g., diphosphate, triphosphate, or tetraphosphate) at the 5' end of the 3'-stable region and a nucleophilic reagent (e.g., a hydroxyl group, amine, or thiol) at the 3' end of the precursor RNA.

[0263] In one embodiment, the polymerase catalyzes the reaction of one NTP with the 3' end of the precursor RNA. In another embodiment, the polymerase catalyzes the reaction of two NTPs with the 3' end of the precursor RNA. In yet another embodiment, the polymerase catalyzes the reaction of three NTPs with the 3' end of the precursor RNA. In yet another embodiment, the polymerase catalyzes the reaction of four NTPs with the 3' end of the precursor RNA. In yet another embodiment, the polymerase catalyzes the reaction of five NTPs with the 3' end of the precursor RNA. In yet another embodiment, the polymerase catalyzes the reaction of six NTPs with the 3' end of the precursor RNA. In yet another embodiment, the polymerase catalyzes the reaction of seven NTPs with the 3' end of the precursor RNA. In yet another embodiment, the polymerase catalyzes the reaction of eight NTPs with the 3' end of the precursor RNA. In yet another embodiment, the polymerase catalyzes the reaction of nine NTPs with the 3' end of the precursor RNA. In yet another embodiment, the polymerase catalyzes the reaction of ten NTPs with the 3' end of the precursor RNA.

[0264] In one embodiment, the polymerase catalyzes a reaction between a protected NTP and the 3' end of the precursor RNA, then deprotects the NTP and subsequently reacts with a second protected NTP, then deprotects the NTP and subsequently reacts with a third protected NTP. In another embodiment, the polymerase may catalyze at least one reaction with a protected NTP, then deprotect it. In yet another embodiment, the polymerase may catalyze at least two sequential reactions with a protected NTP, then deprotect it. In yet another embodiment, the polymerase may catalyze at least three sequential reactions with a protected NTP, then deprotect it. In yet another embodiment, the polymerase may catalyze at least four sequential reactions with a protected NTP, then deprotect it. In yet another embodiment, the polymerase may catalyze at least ten sequential reactions with a protected NTP, then deprotect it. In yet another embodiment, the polymerase may catalyze at least twenty sequential reactions with a protected NTP, then deprotect it.

[0265] In this embodiment, the purification tag is connected to the 3'-stable region via a connector (L). In this embodiment, L is a bond, -S(O)2-, -N(R)-, -O-, -S-, -C(O)-, -C(O)N(R)-, -N(R)C(O)-, -N(R)C(O)NH-, -NHC(O)N(R)-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or any combination thereof; and R is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC( O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or any combination thereof.

[0266] In embodiments, L is a substituted alkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is an unsubstituted alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is a substituted heteroalkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene). In embodiments, L is an unsubstituted heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene). In embodiments, L is a substituted cycloalkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene). In embodiments, L is an unsubstituted heterocyclic alkylene (e.g., C3-C8 heterocyclic alkylene, C3-C6 heterocyclic alkylene, or C5-C6 heterocyclic alkylene). In embodiments, L is a substituted heterocyclic alkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heterocyclic alkylene (e.g., 3- to 8-membered heterocyclic alkylene, 3- to 6-membered heterocyclic alkylene, or 5- to 6-membered heterocyclic alkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heterocyclic alkylene (e.g., 3- to 8-membered heterocyclic alkylene, 3- to 6-membered heterocyclic alkylene, or 5- to 6-membered heterocyclic alkylene). In embodiments, L is an unsubstituted heterocyclic alkylene (e.g., 3- to 8-membered heterocyclic alkylene, 3- to 6-membered heterocyclic alkylene, or 5- to 6-membered heterocyclic alkylene). In the implementation, L is a substituted arylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted arylene (e.g., C6-C). 10 Alpha-aryl, C 10(Arylidene or phenylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) arylene (e.g., C6-C). 10 Alpha-aryl, C 10 (Arylidene or phenylene). In embodiments, L is an unsubstituted arylidene (e.g., C6-C). 10 Alpha-aryl, C 10 (Arylidene or phenylene). In embodiments, L is a substituted heteroaryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, L is an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl).

[0267] In the embodiments, R independently represents hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCBr3, -OCF3, -OCI3, -OCH 2Cl, -OCH2Br, -OCH2F, -OCH2I, -OCHCl2, -OCHBr2, -OCHF2, -OCHI2, substituted or unsubstituted alkyl groups (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), substituted or unsubstituted heteroalkyl groups (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), substituted or unsubstituted cycloalkyl groups (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), substituted or unsubstituted heterocycloalkyl groups (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), substituted or unsubstituted aryl groups (e.g., C6-C... 10 Aryl, C 10 Aryl or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl).

[0268] In embodiments, R is a substituted alkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted alkyl group (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) alkyl group (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R is an unsubstituted alkyl group (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R is a substituted heteroalkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In embodiments, R is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In embodiments, R is an unsubstituted heteroalkyl group (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In embodiments, R is a substituted cycloalkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R is an unsubstituted cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R is a substituted heterocyclic alkyl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heterocyclic alkyl group (e.g., 3- to 8-membered heterocyclic alkyl, 3- to 6-membered heterocyclic alkyl, or 5- to 6-membered heterocyclic alkyl). In embodiments, R is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heterocyclic alkyl group (e.g., 3- to 8-membered heterocyclic alkyl, 3- to 6-membered heterocyclic alkyl, or 5- to 6-membered heterocyclic alkyl). In embodiments, R is an unsubstituted heterocyclic alkyl group (e.g., 3- to 8-membered heterocyclic alkyl, 3- to 6-membered heterocyclic alkyl, or 5- to 6-membered heterocyclic alkyl). In embodiments, R is a substituted aryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted aryl group (e.g., C6-C). 10 Aryl, C 10 Aryl or phenyl). In embodiments, R is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) aryl group (e.g., C6-C). 10 Aryl, C 10Aryl or phenyl). In embodiments, R is an unsubstituted aryl group (e.g., C6-C). 10 Aryl, C 10 Aryl or phenyl). In embodiments, R is a substituted heteroaryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, R is a substituted heteroaryl group (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, R is an unsubstituted heteroaryl group (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl).

[0269] In embodiments, L is a substituted or unsubstituted alkylene or a substituted or unsubstituted heteroalkylene. In embodiments, L is a substituted alkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is an unsubstituted alkylene (e.g., C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene). In embodiments, L is a substituted heteroalkylene (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) or an unsubstituted heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene). In embodiments, L is a substituted (e.g., substituted with a substituent, a size-restricted substituent, or a lower substituent) heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene). In embodiments, L is an unsubstituted heteroalkylene (e.g., 2- to 8-membered heteroalkylene, 2- to 6-membered heteroalkylene, or 2- to 4-membered heteroalkylene).

[0270] In this embodiment, L stands for -CH2CH(CH2OH)(CH2). m NH-, -CH=CHC(O)NH(CH2) p NH-, , or Where m is an integer from 0 to 8; n is an integer from 0 to 8; and p is an integer from 0 to 10. In the embodiment, L is -CH2CH(CH2OH)(CH2). m NH-, , or , where m is an integer from 0 to 8. In the implementation, L is Where n is an integer from 0 to 8. In the implementation, L is -CH=CHC(O)NH(CH2). p NH-, where p is an integer from 0 to 10. In the implementation, L is .

[0271] In the embodiments, L is -CH2CH(CH2OH)(CH2)4NH- or -CH=CHC(O)NH(CH2)6NH-. In the embodiments, L is -CH2CH(CH2OH)(CH2)4NH-. In the embodiments, L is -CH=CHC(O)NH(CH2)6NH-.

[0272] In the implementation method, L is , or In the implementation method, L is... In the implementation method, L is... In the implementation method, L is... In the implementation method, L is... .

[0273] In the embodiments, L is a phosphate ester or a modified phosphate ester. Some non-limiting examples of modified phosphate esters include, but are not limited to, phosphorothioate, dithiophosphate, thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphonate, phosphoroselenoate, selenophosphate, aminophosphate, carbophosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boron phosphate, methylphosphonate, and guanidinopropyl aminophosphate.

[0274] In embodiments, the modified L can be, for example, a thiophosphate, a selenophosphate, a boranophosphate, a boranophosphate ester, a hydrophosphonate, an aminophosphate, a diaminophosphate, an alkyl or aryl phosphonate, or a triphosphate.

[0275] In one embodiment, the purification tag is a hydrophobic group covalently linked to the 3'-stable region of the RNA molecule described herein; this group may be removable or non-removable. In another embodiment, the purification tag is directly linked to the 3'-stable region via a covalent bond. In yet another embodiment, the purification tag is covalently linked to the 3'-stable region via a linker (L). In one embodiment, the purification tag is a removable group. In yet another embodiment, the purification tag is a non-removable group.

[0276] In implementation methods, purification tags include, for example, but not limited to, C6-C. 24 Alkyl, C4-C 24 alkenyl, C4-C 24 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, silyl, triphenylmethyl, lipid, dye, steroid, vinyl ether compounds, modified and unmodified Fmoc compounds, etc. Fluorine substituents or fluorinated groups can be used to increase the hydrophobicity of the hydrophobic group. In embodiments, the purification label includes C6-C. 24 Alkyl group. In embodiments, the purification label includes C4-C... 24 Alkenyl group. In embodiments, the purification label includes C4-C. 24 Alkyne group. In embodiments, the purification label includes C3-C8 cycloalkyl groups. In embodiments, the purification label includes C6-C... 10 Aryl. In an embodiment, the purification label includes a silicon-based compound. In an embodiment, the purification label includes a triphenylmethyl compound. In an embodiment, the purification label includes a lipid. In an embodiment, the purification label includes a steroid. In an embodiment, the purification label includes a vinyl ether compound. In an embodiment, the purification label includes a modified Fmoc compound. In an embodiment, the purification label includes an unmodified Fmoc compound.

[0277] In embodiments, purification labels include, for example, but not limited to, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, and pentadecyl. In embodiments, purification labels include, for example, but not limited to, phenyl, benzyl, (ethyl)carbonyl (azadibenzocyclooctyne) (DBCO), 4-ethylphenol, dibenzohexyltriazoloazacyclooctene, and 1'-O-butyl3',4',6'-triacetylGalNAc.

[0278] In embodiments, purification labels include, for example, but not limited to, -C(O)-propyl, -C(O)-butyl, -C(O)-pentyl, -C(O)-hexyl, -C(O)-heptyl, -C(O)-octyl, -C(O)-nonyl, -C(O)-decyl, -C(O)-undecyl, -C(O)-dodecyl, -C(O)-tridecyl, -C(O)-tetradecyl, and -C(O)-pentadecanyl. In embodiments, purification labels include, for example, but not limited to, -C(O)-phenyl, -C(O)-benzylmethyl, -C(O)-4-ethylphenol, -C(O)-(ethyl)carbonyl (azadibenzocyclooctyne), -C(O)-dibenzohexyltriazoloazacyclooctene, and -C(O)-1'-O-butyl3',4',6'-triacetylGalNAc.

[0279] In this embodiment, the purification label is (ethyl)carbonyl (azadibenzocyclooctyne) (DBCO). In this embodiment, the purification label is 4-ethylphenol. In this embodiment, the purification label is two isomers of dibenzohexyltriazolazazacyclooctene. and A mixture. In this embodiment, the purification label is 1'-O-butyl3',4',6'-triacetylGalNAc. .

[0280] In this implementation, the purification tag includes lipids. In this implementation, it is not desired to be bound by any particular theory that the inclusion of lipid purification tags can lead to increased encapsulation of the mRNA molecule carrying the lipid purification tag in a pharmaceutical carrier, such as lipid nanoparticles (LNPs).

[0281] Therapeutic uses In one aspect, this document provides a cell comprising any of the RNA molecules described herein. In one embodiment, the cell is an isolated cell. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell.

[0282] In one aspect, this document provides a cell comprising a protein or peptide translated from any of the RNA molecules described herein. In another aspect, this document provides a cell comprising a protein translated from any of the RNA molecules described herein. In yet another aspect, this document provides a cell comprising a peptide translated from any of the RNA molecules described herein. In an embodiment, the cell is an isolated cell. In an embodiment, the cell is a mammalian cell. In an embodiment, the cell is a human cell.

[0283] In embodiments, the RNA molecules described herein include pharmaceutically acceptable salts of the RNA molecules described herein. As used herein, a "pharmaceutically acceptable salt" refers to a derivative of the disclosed RNA molecule wherein the parent RNA molecule is modified by converting an existing acid or base portion into its salt form (e.g., by reacting the free base group with a suitable organic or inorganic acid). Representative salts include, but are not limited to, hydrobromide, hydrochloride, sulfate, hydrogen sulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthalenedicarboxylate, methanesulfonate, glucono-p-ethyl, lactobionate, methanesulfonate, and laurylsulfonate, etc. Salts can include, for example, alkali metal and alkaline earth metal-based cations such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. (See SM Barge et al., J. Pharm. Sci. (1977) 66, 1; and Remington: The Science and Practice of Pharmacy, 23rd ed., Adejare et al., Academic Press (2020); which is incorporated herein by reference in its entirety.

[0284] In one aspect, this document provides a pharmaceutical composition comprising any of the RNA molecules described herein and a pharmaceutically acceptable carrier. In embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, solvents, dispersion media, diluents, surfactants, isotonic agents, thickeners or emulsifiers, lipids, liposomes, nanoparticles, lipid nanoparticles (LNPs), polymers, lipid complexes, proteins, or mixtures thereof. In embodiments, the pharmaceutical composition comprises cells containing the RNA molecules described herein.

[0285] In embodiments, a pharmaceutically acceptable carrier is a solvent. In embodiments, a pharmaceutically acceptable carrier is a dispersion medium. In embodiments, a pharmaceutically acceptable carrier is a diluent. In embodiments, a pharmaceutically acceptable carrier is a surfactant. In embodiments, a pharmaceutically acceptable carrier is an isotonic agent. In embodiments, a pharmaceutically acceptable carrier is a thickener. In embodiments, a pharmaceutically acceptable carrier is an emulsifier. In embodiments, a pharmaceutically acceptable carrier is a lipid. In embodiments, a pharmaceutically acceptable carrier is a liposome. In embodiments, a pharmaceutically acceptable carrier is a nanoparticle. In embodiments, a pharmaceutically acceptable carrier is a lipid nanoparticle (LNP). In embodiments, a pharmaceutically acceptable carrier is a polymer. In embodiments, a pharmaceutically acceptable carrier is a lipid complex. In embodiments, a pharmaceutically acceptable carrier is a protein. In embodiments, a pharmaceutically acceptable carrier is a mixture of two or more of the following: solvent, dispersion medium, diluent, surfactant, isotonic agent, thickener or emulsifier, lipid, liposome, nanoparticle, lipid nanoparticle (LNP), polymer, lipid complex or protein.

[0286] In practice, the preparation of pharmaceutically acceptable carriers and formulations containing these materials is described, for example, in: Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Loyd et al., Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences (2012).

[0287] Examples of physiologically acceptable carriers include: buffers, such as phosphate buffers, citrate buffers, and buffers containing other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming ions, such as sodium; and / or nonionic surfactants, such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS. TM (BASF; Florham Park, NJ).

[0288] Compositions containing the RNA molecules or derivatives thereof described herein, suitable for parenteral injection, may comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or mediators include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Appropriate flowability may be maintained, for example, by using coatings (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0289] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. Antimicrobial activity can be enhanced by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenols, sorbic acid, etc.). Isotonic agents, such as sugars and sodium chloride, may also be included. Prolonged absorption in injectable pharmaceutical forms can be achieved by using delayed absorption agents (e.g., aluminum monostearate and gelatin).

[0290] The application of the RNA molecules and compositions described herein, or their pharmaceutically acceptable salts, in a therapeutically effective amount for the prevention or treatment of a disease or condition for a period of time. The application of the RNA molecules and compositions described herein, or their pharmaceutically acceptable salts, in a therapeutically effective amount for the prevention of a disease or condition for a period of time. The application of the RNA molecules and compositions described herein, or their pharmaceutically acceptable salts, in a therapeutically effective amount for the treatment of a disease or condition for a period of time. The effective amount of the RNA molecules and compositions described herein, or their pharmaceutically acceptable salts, can be determined by those skilled in the art.

[0291] Those skilled in the art will understand that the specific dose level and frequency of administration for any particular subject can vary and will depend on a variety of factors, including the activity of the specific compound used; the metabolic stability and duration of action of the compound; the subject's species, age, weight, general health condition, sex, and diet; the administration pattern and time; the excretion rate; the combination of drugs; and the severity of the specific condition.

[0292] The precise dose to be used in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be determined based on the physician's judgment and the individual patient's situation. The effective dose can be obtained by extrapolation from dose-response curves derived from in vitro or animal model testing systems. Furthermore, depending on the route of administration, those skilled in the art should know how to determine the plasma concentration that produces the desired level of response in the subject's cells, tissues, and / or organs.

[0293] Any suitable formulation of the RNA molecule described herein can be prepared. See Remington's Pharmaceutical Sciences, (2000) Hoover, JE ed., 20th edition, Lippincott Williams and Wilkins Publishing Company, Easton, Pa., pp. 780-857. Select a formulation suitable for the appropriate route of administration. In one embodiment, the RNA molecule is formulated for oral administration; in other embodiments, the RNA molecule is formulated for parenteral administration, such as injection or infusion.

[0294] When the RNA molecule of consideration is administered in a pharmacological composition, the RNA molecule of consideration may be co-formulated with a pharmaceutically acceptable excipient and / or carrier. For example, the RNA molecule of consideration may be administered orally as a neutral compound or a pharmaceutically acceptable salt, or intravenously in a physiological saline solution. Conventional buffers, such as phosphates, bicarbonates, or citrates, may be used for this purpose. Of course, those skilled in the art can modify the formulations within the teachings of this specification to obtain a number of formulations for a particular route of administration. Specifically, the RNA molecule of consideration may be modified to make it more soluble in water or other media, for example, easily achieved by minor modifications (salt formulations, esterification, etc.) well known to those skilled in the art. Also entirely within the general skill of the art is the modification of the route of administration and dosing regimen of a particular compound to manage the pharmacokinetics of the compounds of the present invention to maximize the beneficial effects in the patient.

[0295] Depending on the intended mode of administration, the pharmaceutical composition may be in solid, semi-solid, or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids, or suspensions; preferably in a unit dosage form suitable for a precise single-dose administration. The composition will comprise a therapeutically effective amount of the RNA molecule described herein or a derivative thereof combined with a pharmaceutically acceptable carrier, and may additionally include other pharmaceutical agents, medicines, carriers / excipients, or diluents. Pharmaceutically acceptable means a material that is not biologically or otherwise undesirable, which can be administered to a subject together with the selected RNA molecule without producing unacceptable biological effects or interacting in an adverse manner with other components of the pharmaceutical composition containing it.

[0296] To practice the methods of the present invention, the RNA molecules and pharmaceutical compositions thereof can be administered orally, parenterally, by inhalation, topically (including percutaneously, buccally, and sublingually), rectally, nasally, vaginally, via implantable receptacles, or by other methods of drug administration. As used herein, the term "parenterectomy" includes subcutaneous, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. The compositions can be prepared by any method well known in the field of pharmaceutical science.

[0297] Such methods include the step of binding the RNA molecule or combination thereof of the present invention with any adjuvant. Adjuvants, also known as auxiliary ingredients, include those conventional in the art, such as excipients (e.g., starch, lactose), fillers, binders (e.g., gelatin, cellulose, astragalus gum), diluents, disintegrants (e.g., alginate, Primogel, and corn starch), lubricants (e.g., magnesium stearate, silica), colorants, flavoring agents (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint), antioxidants, wetting agents, or other materials well known in the art for use in pharmaceutical formulations.

[0298] Preparation of pharmaceutically acceptable carriers and formulations containing these materials is described, for example, in Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Loyd et al., Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences (2012).

[0299] Solid dosage forms for oral administration of the RNA molecules or their derivatives described herein include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the RNA molecule described herein or a derivative thereof is blended with at least one inert conventional excipient (or carrier, such as sodium citrate or dicalcium phosphate) or the following substances: (a) filler or extender, such as starch, lactose, sucrose, glucose, mannitol and silica; (b) binder, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectant, such as glycerin; (d) disintegrant, such as agar, calcium carbonate, potato or cassava starch, alginate, certain complex silicates and sodium carbonate; (e) solution retarder, such as paraffin; (f) absorption promoter, such as quaternary ammonium compounds; (g) wetting agent, such as cetyl alcohol and glyceryl monostearate; (h) adsorbent, such as kaolin and bentonite; and (i) lubricant, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffers.

[0300] Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules that use excipients such as lactose or milk candy, oxy-coated agents, and high molecular weight polyethylene glycol.

[0301] Solid dosage forms, such as tablets, sugar-coated pills, capsules, pellets, and granules, can be formulated with coatings and shells, such as enteric coatings and other coatings and shells known in the art. Solid dosage forms may contain masking agents, and the compositions therein may also allow for the delayed release of the active compound or compound at a site in the intestine. Examples of encapsulation compositions that can be used are polymeric substances and waxes. The active compound may also be in microencapsulated form, if appropriate, together with one or more of the excipients mentioned above.

[0302] Liquid dosage forms for oral administration of the RNA molecules or derivatives thereof described herein include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, and glycerin), tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, or mixtures of these substances.

[0303] In addition to such inert diluents, the composition may also include other agents, such as wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, or aroma agents.

[0304] In addition to the active compound, the suspension may contain additional agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and astragalus gum, or mixtures of these substances.

[0305] Topical application routes include nasal, buccal, transmucosal, rectal, or vaginal administration. Compositions of the RNA molecules or their derivatives described herein for rectal administration are optionally suppositories, which can be prepared by mixing the compound with a suitable non-irritating excipient or carrier (e.g., cocoa butter, polyethylene glycol, or suppository wax), said composition being solid at room temperature but liquid at body temperature, and thus melting in the rectal or vaginal cavity and releasing the active ingredient.

[0306] Dosage forms for the topical application of the RNA molecules or their derivatives described herein include ointments, lotions, creams, gels, pastes, suspensions, drops, powders, sprays, inhalants, and transdermal patches. One or more thickeners, humectants, and stabilizers may be included in the formulation. Examples of such agents include, but are not limited to, polyethylene glycol, sorbitol, xanthan gum, paraffin oil, beeswax or mineral oil, lanolin, squalene, etc. Methods for preparing transdermal patches are disclosed, for example, by Brown et al. (1988). Ann. Rev. Med References to 39:221-229 are incorporated herein by reference. The RNA molecules or derivatives thereof described herein are mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary. Ophthalmic formulations, ointments, powders, and solutions are also considered within the scope of the compositions.

[0307] Optionally, the RNA molecules described herein may be contained in a drug reservoir. The drug reservoir comprises a solid structure to facilitate implantation and retention in the desired site (e.g., synovial joint, intervertebral disc space, spinal canal, abdominal region, patient tissue, etc.). The drug reservoir can provide an optimal concentration gradient of the compound at a distance of up to about 0.1 cm to about 5 cm from the implantation site. As used herein, the reservoir includes, but is not limited to, capsules, microspheres, microparticles, microcapsules, microfiber particles, nanospheres, nanoparticles, coatings, matrices, powders, pills, pellets, emulsions, liposomes, microcells, gels, antibody-compound conjugates, protein-compound conjugates, or other pharmaceutical delivery compositions. Suitable materials for the reservoir include pharmaceutically acceptable biodegradable materials, preferably FDA-approved or GRAS-approved materials. These materials may be polymeric or non-polymeric, synthetic or naturally occurring, or combinations thereof. The reservoir may optionally include a drug pump.

[0308] For transdermal application, gels, patches, or sprays may be considered. Compositions or formulations suitable for pulmonary application (e.g., nasal inhalation) include fine dust or sprays that can be generated by metered-dose pressurized sprays, nebulizers, or inhalers. Nasal aerosols or inhalation compositions can be prepared according to techniques well known in pharmaceutical formulation and can be prepared as solutions, for example, in physiological saline, using suitable preservatives (e.g., benzyl alcohol), absorption enhancers to improve bioavailability, and / or prepared as other solvents or dispersants known in the art.

[0309] The composition may be presented in single-dose or multi-dose container form (e.g., sealed vials and ampoules) and may be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as water, before use.

[0310] Additionally, RNA molecules having Formula I or Formula II, or any exemplary compound disclosed herein, or their enantiomers, mixtures of enantiomers, mixtures of two or more diastereomers, tautomers, mixtures of two or more tautomers, or isotopic variants; or their pharmaceutically acceptable salts, solvates, hydrates, or prodrugs, may be administered alone or in combination with other therapeutic agents. Combination therapies according to the invention comprise at least one exemplary RNA molecule of the present disclosure and at least one other therapeutic agent administered in a pharmaceutical composition. The at least one exemplary RNA molecule of the present disclosure and at least one other therapeutic agent may be administered separately or together as a pharmaceutical composition. The amount and relative timing of administration of the at least one exemplary RNA molecule of the present disclosure and at least one other therapeutic agent will be selected to achieve the desired combination therapeutic effect.

[0311] In one aspect, this invention provides a method for treating a disease in a subject in need, comprising introducing an effective amount of any of the RNA molecules described herein. In another aspect, this invention provides a method for treating a disease in a subject in need, comprising introducing an effective amount of cells containing any of the RNA molecules described herein. In another aspect, this invention provides a method for treating a disease in a subject in need, comprising introducing an effective amount of cells containing a protein or peptide translated from any of the RNA molecules described herein. In another aspect, this invention provides a method for treating a disease in a subject in need, comprising introducing an effective amount of cells containing a peptide translated from any of the RNA molecules described herein. In another aspect, this invention provides a method for treating a disease in a subject in need, comprising introducing an effective amount of cells containing a protein translated from any of the RNA molecules described herein. In an embodiment, the cells are isolated cells. In an embodiment, the cells are mammalian cells. In an embodiment, the cells are human cells.

[0312] In one aspect, this document provides a method for treating a disease in a subject of need, comprising introducing an effective amount of a pharmaceutical composition comprising any of the RNA molecules described herein and a pharmaceutically acceptable carrier. In embodiments, a pharmaceutically acceptable carrier is a solvent, dispersion medium, diluent, surfactant, isotonic agent, thickener or emulsifier, lipid, liposome, nanoparticle, lipid nanoparticle (LNP), polymer, lipid complex, protein, or mixture thereof. In an embodiment, a pharmaceutically acceptable carrier is a lipid nanoparticle (LNP).

[0313] In one aspect, this invention provides a method for preventing disease in a subject in need, comprising introducing an effective amount of any of the RNA molecules described herein. In another aspect, this invention provides a method for preventing disease in a subject in need, comprising introducing an effective amount of cells containing any of the RNA molecules described herein. In another aspect, this invention provides a method for preventing disease in a subject in need, comprising introducing an effective amount of cells containing a protein or peptide translated from any of the RNA molecules described herein. In another aspect, this invention provides a method for preventing disease in a subject in need, comprising introducing an effective amount of cells containing a peptide translated from any of the RNA molecules described herein. In another aspect, this invention provides a method for preventing disease in a subject in need, comprising introducing an effective amount of cells containing a protein translated from any of the RNA molecules described herein. In an embodiment, the cells are isolated cells. In an embodiment, the cells are mammalian cells. In an embodiment, the cells are human cells.

[0314] In one aspect, this document provides a method for preventing disease in a subject of need, comprising introducing an effective amount of a pharmaceutical composition comprising any of the RNA molecules described herein and a pharmaceutically acceptable carrier. In embodiments, a pharmaceutically acceptable carrier is a solvent, dispersion medium, diluent, surfactant, isotonic agent, thickener or emulsifier, lipid, liposome, nanoparticle, lipid nanoparticle (LNP), polymer, lipid complex, protein, or mixture thereof. In embodiments, a pharmaceutically acceptable carrier is a lipid nanoparticle (LNP).

[0315] In one aspect, this document provides a method for increasing the expression of a target protein or peptide in cells, comprising contacting the cells with any of the RNA molecules described herein, wherein the RNA molecule encodes the target protein or peptide, wherein the expression is increased compared to the expression of an RNA molecule lacking a 3'-stabilizing region. In another aspect, this document provides a method for increasing the expression of a target peptide in cells, comprising contacting the cells with any of the RNA molecules described herein, wherein the RNA molecule encodes the target peptide, wherein the expression is increased compared to the expression of an RNA molecule lacking a 3'-stabilizing region. In another aspect, this document provides a method for increasing the expression of a target protein in cells, comprising contacting the cells with any of the RNA molecules described herein, wherein the RNA molecule encodes the target protein, wherein the expression is increased compared to the expression of an RNA molecule lacking a 3'-stabilizing region. In embodiments, the cells are isolated in vitro or ex vivo. In embodiments, the cells are isolated cells. In embodiments, the cells are in vitro cells. In embodiments, the cells are ex vivo cells.

[0316] In one aspect, this document provides a method for expressing a target protein or peptide in cells, comprising contacting the cells with any of the RNA molecules described herein, wherein the RNA molecules encode the target protein or peptide, and the cells translating the target protein or peptide from the RNA molecules. In another aspect, this document provides a method for expressing a target peptide in cells, comprising contacting the cells with any of the RNA molecules described herein, wherein the RNA molecules encode the target peptide, and the cells translating the target peptide from the RNA molecules. In yet another aspect, this document provides a method for expressing a target protein in cells, comprising contacting the cells with any of the RNA molecules described herein, wherein the RNA molecules encode the target protein, and the cells translating the target protein from the RNA molecules. In embodiments, the cells are isolated in vitro or ex vivo. In embodiments, the cells are isolated cells. In embodiments, the cells are in vitro cells. In embodiments, the cells are ex vivo cells.

[0317] In one aspect, this document provides a method for prolonging the half-life of RNA molecules in cells, comprising contacting cells with any of the RNA molecules described herein, wherein the half-life is prolonged compared to the half-life of RNA molecules without a 3'-stable region, optionally wherein the cells are isolated in vitro or ex vivo. In another aspect, this document provides a method for increasing the half-life of RNA molecules in cells, comprising contacting cells with any of the RNA molecules described herein, wherein the half-life is prolonged compared to the half-life of RNA molecules without a 3'-stable region, optionally wherein the cells are isolated in vitro. In one aspect, this document provides a method for prolonging the half-life of RNA molecules in cells, comprising contacting cells with any of the RNA molecules described herein, wherein the half-life is prolonged compared to the half-life of RNA molecules without a 3'-stable region, optionally wherein the cells are isolated in ex vivo. In an embodiment, the cells are isolated in vitro or ex vivo. In an embodiment, the cells are isolated cells. In an embodiment, the cells are cells isolated in vitro. In an embodiment, the cells are cells isolated in ex vivo.

[0318] In implementation, the RNA molecules described herein can be used as guide RNA (gRNA) in gene editing.

[0319] Reagent test kit In one aspect, this document provides a kit comprising any of the RNA molecules described herein. In one embodiment, the kit may contain an amount of the desired components sufficient to allow for multiple treatments of a subject requiring such treatment. In another embodiment, the kit may contain an amount of the desired components sufficient to allow for multiple experiments.

[0320] In this embodiment, a kit for protein production is provided, comprising an RNA molecule of Formula I or Formula II and instructions for using the kit, the molecule comprising a 5'-cap, optionally a 5' UTR, a translatable region, optionally a 3' UTR, a poly-A region, and a 3'-stabilizing region, wherein the RNA molecule expresses reduced exonuclease degradation and can be separated from prematurely terminated RNA transcripts lacking a poly-A tail (using an oligo dT column) or a 3'-stabilizing region (using an HPLC column).

[0321] In embodiments, this document provides a kit for protein production comprising an RNA molecule of Formula I or Formula II, including a 5'-cap, optionally a 5' UTR, a translatable region, optionally a 3' UTR, a poly-A region, and a ligase or polymerase for ligating the 3'-stable region to the RNA molecule. In embodiments, the kit includes a buffer for performing the ligation. In embodiments, the kit further includes instructions for administering any of the pharmaceutical compositions provided herein to a subject.

[0322] Sequence List: 15-meric oligonucleotide SEQ ID NO:1 5'-Monophosphate-AAAAAAAAAAAAA(dTAm)(ddC) 39-meric oligonucleotide SEQ ID NO:2 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA FLuc mRNA SEQ ID NO:20 (All T nucleotides are 5-methoxyuridine) eGFP mRNA SEQ ID NO:21 (All T nucleotides are N1-methylpseuuridine) m6AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA As used in this article, the commonly used organic chemistry abbreviations are defined as follows: Example The following examples are intended to illustrate and can be used to further understand the implementation of this disclosure and should not be construed as limiting the scope of this teaching in any way.

[0323] The chemical reactions described in the examples can be readily adapted to prepare many other compounds of this disclosure, and alternative methods for preparing the compounds of the present invention are considered to be within the scope of this disclosure. For example, compounds not illustrated in this disclosure can be successfully synthesized by modifications obvious to those skilled in the art, such as by using suitable reagents other than those known in the art, or by conventional modifications to the reaction conditions, reagents, and starting materials. Alternatively, other reactions disclosed herein or known in the art are considered suitable for preparing other compounds of this disclosure.

[0324] Synthesis Examples Unless otherwise stated, the amide reagents used in the synthesis were purchased from Chemgene or Glen Research. (The remaining text appears to be incomplete and requires further context.) 3'OMe ppp m6 A 2'OMe pG-capped eGFP mRNA (without tail modification) is used as an m7G with a modified 3'-end. 3'OMe ppp m6 A 2'OMe Control of pG-capped eGFP mRNA (all eGFP mRNAs with modified 3' ends were treated with m7G) 3'OMe ppp m6 A 2'OMe pG capping; all FLuc mRNAs with modified 3' ends are capped with m7GpppA. 2'OMe (pG capped). The retention time of eGFP mRNA without tail modification was 9.335 minutes.

[0325] If instructed, use the following Oligo dT purification procedure: Sample preparation: 50 μg mRNA was diluted to <0.3 mg / mL in high-salt wash buffer (250 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7), and 5 M NaCl was added to bring the final concentration in the sample to 250 mM.

[0326] column: CIMmultus® Oligo dT18 (C6 connector) - 1 mL (2 μm), Supplier: BIA Separations Inc., Catalog No. 311.1218-2 instrument: AKTA Avant 25 HPLC System Method description: Equilibrate the column to ≥4 column volumes (CV) in high-salt wash buffer. Load the sample onto the column. Set the flow rate to 5 mL / min. Example S1: Synthetic sequence 1a A 10 mM solution (2 μL, 20 nmol) of sequence 1 (containing 13 adenosine ribonucleotides, dTAm, and ddC) (SEQ ID NO: 1) in water was added to a 1.5 mL microcentrifuge tube containing 16 μL of 20 mM sodium phosphate buffer (pH 8.5). The solution was cooled in an ice bath for 3 minutes. A 100 mM solution (2 μL, 200 nmol) of freshly prepared compound 3 was added to the cooled solution in the microcentrifuge tube and mixed thoroughly by pipetting. The reaction mixture was stirred at room temperature for 21 hours. An aliquot (1 μL) of the reaction mixture was diluted in water (19 μL) and analyzed by LC-MS. The crude reaction mixture was stored at -20°C and used as is. The yield was 99% as determined by LC-MS.

[0327] MS m / z = 5125.8 [MH].

[0328] Example S2: Synthetic Sequence 1b A 10 mM solution of sequence 1 in water (custom-made from Trilink Biotechnologies, 2 μL, 20 nmol) was added to a 1.5 mL microcentrifuge tube containing 16 μL of 20 mM sodium phosphate buffer (pH 8.5). The solution was cooled in an ice bath for 3 minutes. A 100 mM solution (2 μL, 200 nmol) of freshly prepared compound 5 was added to the cooled solution in the microcentrifuge tube and thoroughly mixed by pipetting. The reaction mixture was stirred at room temperature for 21 hours. An aliquot of the reaction mixture (1 μL) was diluted in water (19 μL) and analyzed by LC-MS. The crude reaction mixture was stored at -20°C and used as is. The yield, as determined by LC-MS, was approximately 20%.

[0329] MS m / z = 5182.3 [MH].

[0330] Example S3: Synthetic sequence 1c A 10 mM solution of sequence 1 in water (custom-made from Trilink Biotechnologies, 2 μL, 20 nmol) was added to a 1.5 mL microcentrifuge tube containing 20 mM sodium phosphate buffer (pH 8.5, 16 μL). The solution was cooled in an ice bath for 3 minutes. A 100 mM solution (2 μL, 200 nmol) of freshly prepared compound 7 was added to the cooled solution in the microcentrifuge tube and thoroughly mixed by pipetting. The reaction mixture was stirred at room temperature for 21 hours. An aliquot of the reaction mixture (1 μL) was diluted in water (19 μL) and analyzed by LC-MS. The crude reaction mixture was stored at -20°C and used as is. The yield, as determined by LC-MS, was 99%.

[0331] MS m / z = 5146.6 [MH].

[0332] Example S4: Synthetic sequence 2a Add RNase-free water (13.6 μL), T4 RNA ligase reaction buffer (purchased from New England Biolabs Inc., catalog number: M0204S, 500 mM Tris-HCl, 100 mM MgCl2, 10 mM DTT, pH 7.5, 3.0 μL), DMSO (3.0 μL), and mouse RNase inhibitor (purchased from New England Biolabs Inc., catalog number: M0314B, 40 U / μL, 0.4 μL) to a 1.5 mL microcentrifuge tube. Adenosine triphosphate (ATP) (purchased from New England Biolabs Inc., catalog number: M0204S, 10 mM, 3.0 μL), sequence 2 (comprising 39 adenosine ribonucleotides; purchased from TriLink Biotechnologies, 1 mM, 3.0 μL), compound 1 (custom-made from TriLink Biotechnologies, 0.1 mM, 3.0 μL), and T4 RNA ligase 1 (purchased from New England Biolabs Inc., catalog number: M0204S, 10 U / μL, 1.0 μL) were added to the resulting solution and mixed thoroughly. The reaction mixture was incubated at room temperature for 21 hours. LC-MS analysis confirmed the acquisition of sequence 2a (yield of 99% as measured by HPLC). Retention time of sequence 2a: 4.771 min.

[0333] MS m / z = 13287.8 [MH].

[0334] Example S5: Synthetic sequence 2b A 10 mM solution of compound 1 in water (custom-made from Trilink Biotechnologies, 10 μL, 100 nmol) was added to a 1.5 mL microcentrifuge tube containing 20 mM sodium phosphate buffer (pH 8.5, 80 μL). The solution was cooled in an ice bath for 3 minutes. A 100 mM solution of freshly prepared compound 3 (purchased from Sigma-Aldrich Inc., catalog number AMBH97F1164B, 10 μL, 1 μmol) was added to the cooled solution in the microcentrifuge tube and mixed thoroughly by pipette. The reaction mixture was stirred at room temperature for 21 hours. An aliquot of the reaction mixture (1 μL) was diluted in water (19 μL) and analyzed by LC-MS. After confirming the desired product 2, the crude reaction mixture was stored at -20°C and used as is.

[0335] MS m / z = 630.2 [MH]. Add RNase-free water (13.6 μL), T4 RNA ligase reaction buffer (purchased from New England Biolabs Inc., catalog number: M0204S, 500 mM Tris-HCl, 100 mM MgCl2, 10 mM DTT, pH 7.5, 3.0 μL), DMSO (3.0 μL), and mouse RNase inhibitor (purchased from New England Biolabs Inc., catalog number: M0314B, 40 U / μL, 0.4 μL) to a 1.5 mL microcentrifuge tube. Adenosine triphosphate (ATP) (purchased from New England Biolabs Inc., catalog number: M0204S, 10 mM, 3.0 μL), sequence 2 (purchased from TriLink Biotechnologies, 1 mM, 3.0 μL), compound 2 (0.1 mM, 3.0 μL), and T4 RNA ligase 1 (purchased from New England Biolabs Inc., catalog number: M0204S, 10 U / μL, 1.0 μL) were added to the resulting solution and mixed thoroughly. The reaction mixture was incubated at room temperature for 21 hours. LC-MS analysis confirmed the acquisition of sequence 2b (yield of 99% as measured by HPLC). Retention time of sequence 2b: 5.477 min.

[0336] MS m / z = 13386.8 [MH].

[0337] Example S6: Synthetic sequence 2c Add RNase-free water (6.8 μL), T4 RNA ligase reaction buffer (purchased from New England Biolabs Inc., catalog number: M0204S, 500 mM Tris-HCl, 100 mM MgCl2, 10 mM MTT, pH 7.5, 2.0 μL), 50% PEG 8000 (purchased from New England Biolabs Inc., catalog number: M0204S, 4.0 μL), and mouse RNase inhibitor (purchased from New England Biolabs Inc., catalog number: M0314B, 40 U / μL, 0.3 μL) to a 1.5 mL microcentrifuge tube. Adenosine triphosphate (ATP) (purchased from New England Biolabs Inc., catalog number: M0204S, 10 mM, 2.0 μL), sequence 2 (purchased from TriLink Biotechnologies, 10 μM, 2.0 μL), sequence 1 (0.1 mM, 2.0 μL), and T4 RNA ligase 1 (purchased from New England Biolabs Inc., catalog number: M0204S, 10 U / μL, 1.0 μL) were added to the resulting solution and mixed thoroughly. The reaction mixture was incubated at room temperature for 21 hours. LC-MS analysis confirmed the acquisition of sequence 2c. Retention time of sequence 2c: 5.135 minutes.

[0338] MS m / z = 17783.0 [MH].

[0339] Example S7: Synthetic sequence 2d Add RNase-free water (6.8 μL), T4 RNA ligase reaction buffer (purchased from New England Biolabs Inc., catalog number: M0204S, 500 mM Tris-HCl, 100 mM MgCl2, 10 mM MTT, pH 7.5, 2.0 μL), 50% PEG 8000 (purchased from New England Biolabs Inc., catalog number: M0204S, 4.0 μL), and mouse RNase inhibitor (purchased from New England Biolabs Inc., catalog number: M0314B, 40 U / μL, 0.3 μL) to a 1.5 mL microcentrifuge tube. Adenosine triphosphate (ATP) (purchased from New England Biolabs Inc., catalog number: M0204S, 10 mM, 2.0 μL), sequence 2 (purchased from TriLink Biotechnologies, 10 μM, 2.0 μL), sequence 1a (0.1 mM, 2.0 μL), and T4 RNA ligase 1 (purchased from New England Biolabs Inc., catalog number: M0204S, 10 U / μL, 1.0 μL) were added to the resulting solution and mixed thoroughly. The reaction mixture was incubated at room temperature for 21 hours. LC-MS analysis confirmed the acquisition of sequence 2d (yield of 99% as measured by HPLC). Retention time of sequence 2d: 6.024 min.

[0340] MS m / z = 17882.8 [MH].

[0341] Example S8: Synthetic Sequence 3 Add RNase-free water (1.6 μL), T4 RNA ligase reaction buffer (purchased from New England Biolabs Inc., catalog number: M0204S, 500 mM Tris-HCl, 100 mM MgCl2, 10 mM MDT, pH 7.5, 8.0 μL), DMSO (8.0 μL), FLuc mRNA (purchased from TriLink Biotechnologies, 1.0 mg / mL, 50.0 μL), and mouse RNase inhibitor (purchased from New England Biolabs Inc., catalog number: M0314B, 40 U / μL, 1.0 μL) to a 1.5 mL microcentrifuge tube. Adenosine triphosphate (ATP) (purchased from New England Biolabs Inc., catalog number: M0204S, 10 mM, 8.0 μL), compound 1 (1 mM, 0.8 μL), and T4 RNA ligase 1 (purchased from New England Biolabs Inc., catalog number: M0204S, 10 U / μL, 2.6 μL) were added to the resulting solution and mixed thoroughly. The reaction mixture was incubated at room temperature for 21 hours, and mRNA was isolated using the RNeasy kit (purchased from Qiagen, catalog number: 74004), eluted with RNase-free water (50 μL). Nanodrop analysis confirmed the acquisition of mRNA (49.9 µg). Purification was performed by IPRP HPLC using a DNAPac RP 4 μm 3.0 × 50 mm column (Thermofisher, catalog number: 088920). Elution was performed using a gradient of 40% buffer B (100 mM TEAA, 1 mM EDTA, 25% ACN, pH 7.3) to 60% buffer B in buffer A over 14.9 min. Retention time for FLuc mRNA+ sequence 3: 11.352 min.

[0342] Example S9: Synthetic Sequence 5 Add RNase-free water (1.6 μL), T4 RNA ligase reaction buffer (purchased from New England Biolabs Inc., catalog number: M0204S, 500 mM Tris-HCl, 100 mM MgCl2, 10 mM MDT, pH 7.5, 8.0 μL), DMSO (8.0 μL), FLuc mRNA (purchased from TriLink Biotechnologies, 1.0 mg / mL, 50.0 μL), and mouse RNase inhibitor (purchased from New England Biolabs Inc., catalog number: M0314B, 40 U / μL, 1.0 μL) to a 1.5 mL microcentrifuge tube. Adenosine triphosphate (ATP) (purchased from New England Biolabs Inc., catalog number: M0204S, 10 mM, 8.0 μL), compound 2 (1 mM, 0.8 μL), and T4 RNA ligase 1 (purchased from New England Biolabs Inc., catalog number: M0204S, 10 U / μL, 2.6 μL) were added to the resulting solution and mixed thoroughly. The reaction mixture was incubated at room temperature for 21 hours, and mRNA was isolated using the RNeasy kit (purchased from Qiagen, catalog number: 74004), eluted with RNase-free water (50 μL). Nanodrop analysis confirmed the acquisition of mRNA (50.5 µg). Purification was performed by IPRP HPLC using a DNAPac RP 4 μm 3.0 × 50 mm column (Thermofisher, catalog number: 088920). Elution was performed using a gradient from 40% buffer B (100 mM TEAA, 1 mM EDTA, 25% ACN, pH 7.3) in buffer A (100 mM TEAA, 1 mM EDTA, pH 7.3) to 60% buffer B in buffer A over 14.9 min, confirming the presence of a novel product corresponding to sequence 5 (yield of 52% as measured by HPLC). Retention times: FLuc mRNA: 11.477 min; Sequence 5: 12.204 min.

[0343] Example S10: Synthetic Sequence 4 Add RNase-free water (1.6 μL), T4 RNA ligase reaction buffer (purchased from New England Biolabs Inc., catalog number: M0204S, 500 mM Tris-HCl, 100 mM MgCl2, 10 mM MDT, pH 7.5, 8.0 μL), DMSO (8.0 μL), FLuc mRNA (purchased from TriLink Biotechnologies, 1.0 mg / mL, 50.0 μL), and mouse RNase inhibitor (purchased from New England Biolabs Inc., catalog number: M0314B, 40 U / μL, 1.0 μL) to a 1.5 mL microcentrifuge tube. Adenosine triphosphate (ATP) (purchased from New England Biolabs Inc., catalog number: M0204S, 10 mM, 8.0 μL), Sequence 1 (1 mM, 0.8 μL), and T4 RNA ligase 1 (purchased from New England Biolabs Inc., catalog number: M0204S, 10 U / μL, 2.6 μL) were added to the resulting solution and mixed thoroughly. The reaction mixture was incubated at room temperature for 21 hours, and mRNA was isolated using an RNeasy kit (purchased from Qiagen, catalog number: 74004), eluted with RNase-free water (50 μL). Nanodrop analysis confirmed the acquisition of mRNA (50.1 µg). Purification was performed by IPRP HPLC using a DNAPac RP 4 μm 3.0 × 50 mm column (Thermofisher, catalog number: 088920). Elution was performed using a gradient of 40% buffer B (100 mM TEAA, 1 mM EDTA, 25% ACN, pH 7.3) to 60% buffer B in buffer A over 14.9 min. Retention time: FLuc mRNA+ sequence 4: 11,400 min.

[0344] Example S11: Synthetic Sequence 6 Add RNase-free water (1.6 μL), T4 RNA ligase reaction buffer (purchased from New England Biolabs Inc., catalog number: M0204S, 500 mM Tris-HCl, 100 mM MgCl2, 10 mM MDT, pH 7.5, 8.0 μL), DMSO (8.0 μL), FLuc mRNA (purchased from TriLink Biotechnologies, 1.0 mg / mL, 50.0 μL), and mouse RNase inhibitor (purchased from New England Biolabs Inc., catalog number: M0314B, 40 U / μL, 1.0 μL) to a 1.5 mL microcentrifuge tube. Adenosine triphosphate (ATP) (purchased from New England Biolabs Inc., catalog number: M0204S, 10 mM, 8.0 μL), sequence 1a (1 mM, 0.8 μL), and T4 RNA ligase 1 (purchased from New England Biolabs Inc., catalog number: M0204S, 10 U / μL, 2.6 μL) were added to the resulting solution and mixed thoroughly. The reaction mixture was incubated at room temperature for 21 hours, and mRNA was isolated using an RNeasy kit (purchased from Qiagen, catalog number: 74004) and eluted with RNase-free water (50 μL). Nanodrop analysis confirmed the acquisition of mRNA (50.8 µg). Purification was performed by IPRP HPLC using a DNAPac RP 4 μm 3.0 × 50 mm column (Thermofisher, catalog number: 088920). Elution was performed using a gradient from 40% buffer B (100 mM TEAA, 1 mM EDTA, 25% ACN, pH 7.3) in buffer A (100 mM TEAA, 1 mM EDTA, pH 7.3) to 60% buffer B in buffer A over 14.9 min. This confirmed the presence of a novel product corresponding to sequence 6 (yield of 30% according to HPLC measurements). Retention time for FLuc mRNA: 11.433 min; retention time for sequence 6: 13.014 min.

[0345] Example S12: Synthetic Sequence 7 A 10 mM solution of compound 1 in water (custom-made from Trilink Biotechnologies, 10 μL, 100 nmol) was added to a 1.5 mL microcentrifuge tube containing 20 mM sodium phosphate buffer (pH 8.5, 80 μL). The solution was cooled in an ice bath for 3 minutes. A 100 mM solution of freshly prepared compound 5 (purchased from Sigma-Aldrich Inc., catalog number AMBH97F116BA, 10 μL, 1 μmol) was added to the cooled solution in the microcentrifuge tube and mixed thoroughly by pipette. The reaction mixture was stirred at room temperature for 21 hours. An aliquot of the reaction mixture (1 μL) was diluted in water (19 μL) and analyzed by LC-MS. After confirming the desired product 4, the crude reaction mixture was stored at -20°C and used as is.

[0346] MS m / z = 686.3 [MH]. Add RNase-free water (1.6 μL), T4 RNA ligase reaction buffer (purchased from New England Biolabs Inc., catalog number: M0204S, 500 mM Tris-HCl, 100 mM MgCl2, 10 mM MDT, pH 7.5, 8.0 μL), DMSO (8.0 μL), FLuc mRNA (purchased from TriLink Biotechnologies, 1.0 mg / mL, 50.0 μL), and mouse RNase inhibitor (purchased from New England Biolabs Inc., catalog number: M0314B, 40 U / μL, 1.0 μL) to a 1.5 mL microcentrifuge tube. Adenosine triphosphate (ATP) (purchased from New England Biolabs Inc., catalog number: M0204S, 10 mM, 8.0 μL), compound 4 (1 mM, 0.8 μL), and T4 RNA ligase 1 (purchased from New England Biolabs Inc., catalog number: M0204S, 10 U / μL, 2.6 μL) were added to the resulting solution and mixed thoroughly. The reaction mixture was incubated at room temperature for 21 hours, and mRNA was isolated using an RNeasy kit (purchased from Qiagen, catalog number: 74004), eluted with RNase-free water (50 μL). Nanodrop analysis confirmed the acquisition of mRNA (51.0 µg). Purification was performed by IPRP HPLC using a DNAPac RP 4 μm 3.0 × 50 mm column (Thermofisher, catalog number: 088920). Elution was performed using a gradient from 40% buffer B (100 mM TEAA, 1 mM EDTA, 25% ACN, pH 7.3) in buffer A (100 mM TEAA, 1 mM EDTA, pH 7.3) to 60% buffer B in buffer A over 14.9 min. The presence of a novel product corresponding to sequence 7 was confirmed (yield of 29% according to HPLC). Retention time for FLuc mRNA: 11.941 min; retention time for sequence 7: 15.422 min.

[0347] Example S13: Synthetic Sequence 8 Add RNase-free water (1.6 μL), T4 RNA ligase reaction buffer (purchased from New England Biolabs Inc., catalog number: M0204S, 500 mM Tris-HCl, 100 mM MgCl2, 10 mM MDT, pH 7.5, 8.0 μL), DMSO (8.0 μL), FLuc mRNA (purchased from TriLink Biotechnologies, 1.0 mg / mL, 50.0 μL), and mouse RNase inhibitor (purchased from New England Biolabs Inc., catalog number: M0314B, 40 U / μL, 1.0 μL) to a 1.5 mL microcentrifuge tube. Adenosine triphosphate (ATP) (purchased from New England Biolabs Inc., catalog number: M0204S, 10 mM, 8.0 μL), sequence 1b (1 mM, 0.8 μL), and T4 RNA ligase 1 (purchased from New England Biolabs Inc., catalog number: M0204S, 10 U / μL, 2.6 μL) were added to the resulting solution and mixed thoroughly. The reaction mixture was incubated at room temperature for 21 hours, and mRNA was isolated using the RNeasy kit (purchased from Qiagen, catalog number: 74004), eluted with RNase-free water (50 μL). Nanodrop analysis confirmed the acquisition of mRNA (50.2 µg). Purification was performed by IPRP HPLC using a DNAPac RP 4 μm 3.0 × 50 mm column (Thermofisher, catalog number: 088920). Elution was performed using a gradient from 40% buffer B (100 mM TEAA, 1 mM EDTA, 25% ACN, pH 7.3) in buffer A (100 mM TEAA, 1 mM EDTA, pH 7.3) to 60% buffer B in buffer A over 14.9 min. This confirmed the presence of a novel product corresponding to sequence 8 (yield of 0.72% as measured by HPLC). Retention time for FLuc mRNA: 11.921 min; retention time for sequence 8: 16.794 min.

[0348] Example S14: Synthetic Sequence 9 A 10 mM solution of compound 1 in water (custom-made from Trilink Biotechnologies, 10 μL, 100 nmol) was added to a 1.5 mL microcentrifuge tube containing 20 mM sodium phosphate buffer (pH 8.5, 80 μL). The solution was cooled in an ice bath for 3 minutes. A 100 mM solution of freshly prepared compound 7 (purchased from Sigma-Aldrich Inc., catalog number ENAH042579A2, 10 μL, 1 μmol) was added to the cooled solution in the microcentrifuge tube and mixed thoroughly by pipette. The reaction mixture was stirred at room temperature for 21 hours. An aliquot of the reaction mixture (1 μL) was diluted in water (19 ...

Claims

1. An RNA molecule comprising the structure of formula I: Where A includes: a) 5'-cap; b) Open reading frames (ORFs) encoding proteins; and c) The poly-A region, wherein the poly-A region is located at 3' of the open reading frame; and B contains a 3'-stable region containing 1 to 50 nucleosides, wherein one or more nucleosides within the 3'-stable region contain one or more purification tags.

2. The RNA molecule of claim 1, wherein the 3'-stabilizing region is covalently linked to the precursor RNA via a linker that can be formed by ligation.

3. The RNA molecule according to claim 2, wherein the adapter is a adapter formed by enzymatic or chemical linkage.

4. The RNA molecule of claim 1, wherein the 3'-stable region is covalently linked to the precursor RNA via a linker that can be formed using a polymerase.

5. The RNA molecule according to any one of claims 1 to 4, wherein the purification tag is linked to the 3'-stabilization region via a adapter (L).

6. The RNA molecule according to any one of claims 1 to 5, wherein the purification tag comprises lipids.

7. An RNA molecule having a structure containing formula II: Where A includes: a) 5'-cap; b) Open reading frames (ORFs) encoding proteins; and c) The poly-A region, wherein the poly-A region is located at 3' of the open reading frame; and B contains a 3'-stable region containing 1 to 50 nucleosides, wherein one or more nucleosides within the 3'-stable region contain one or more adapters (L) capable of binding to the purification tag.

8. The RNA molecule of claim 7, wherein the 3'-stabilizing region is covalently linked to the precursor RNA via a linker that can be formed by ligation.

9. The RNA molecule according to claim 8, wherein the linkage is an enzyme linkage or a chemical linkage.

10. The RNA molecule of claim 7, wherein the 3'-stabilizing region is covalently linked to the precursor RNA using a polymerase.

11. The RNA molecule according to any one of claims 7 to 10, wherein one or more purification tags are covalently linked to one or more nucleosides or one or more adapters within the 3'-stabilizing region.

12. The RNA molecule of claim 11, wherein the purification tag comprises lipids.

13. The RNA molecule according to any one of claims 1 to 12, wherein the 3'-stabilizing region forms a secondary structure.

14. The RNA molecule according to any one of claims 1 to 13, wherein the secondary structure is a hairpin loop.

15. The RNA molecule according to any one of claims 1 to 14, wherein the 3'-stable region comprises one or more unmodified nucleosides and one or more unmodified nucleotide inter-bonds.

16. The RNA molecule according to any one of claims 1 to 14, wherein the 3'-stable region comprises one or more modified nucleosides and / or one or more modified nucleotide inter-bonds.

17. The RNA molecule of claim 16, wherein the modified nucleoside comprises modified nucleobases and / or modified sugars.

18. The RNA molecule according to claim 16 or 17, wherein the modified nucleoside comprises modified nucleobases.

19. The RNA molecule of claim 18, wherein the modified nucleobase is modified uracil, modified cytosine, modified guanine, or modified adenine.

20. The RNA molecule according to claim 18 or 19, wherein the modified nucleobase is pseudouracil (ψ), 2-thiouracil, 4-thiouracil, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuracil, 5-halouracil, 3-methyluracil, 5-azauracil, or 2-thio-5-azauracil.

21. The RNA molecule according to any one of claims 17 to 20, wherein the modified nucleobase is 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methylcytosine, 5-methylcytosine, 5-halo-cytosine, 2-thio-cytosine, or 2-thio-5-methylcytosine.

22. The RNA molecule according to any one of claims 17 to 21, wherein the modified nucleobase is 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenine, 7-deadenine, N6-methyl-adenine, or 2-methylthio-N6-methyl-adenine.

23. The RNA molecule according to any one of claims 17 to 22, wherein the modified nucleobase is inosine, 1-methyl-inosine, 7-cyano-7-denitro-guanine, 7-aminomethyl-7-denitro-guanine, 6-thio-guanine, 6-thio-7-denitro-guanine, or 6-methoxy-guanine.

24. The RNA molecule according to any one of claims 16 to 23, wherein the modified nucleoside comprises a modified sugar.

25. The RNA molecule of claim 24, wherein the modified sugar has a 5-membered ring or a 6-membered ring, or is a modified ribose.

26. The RNA molecule of claim 25, wherein the modified ribose is 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, or 3'-amino-2',3'-dideoxyribose.

27. The RNA molecule according to any one of claims 16 to 23, wherein the modified nucleoside comprises a morpholino ring.

28. The RNA molecule according to any one of claims 16 to 27, wherein the internucleotide bond comprises a modified phosphate ester.

29. The RNA molecule of claim 28, wherein the modified phosphate ester is a phosphorothioate, a dithiothioate, a thiophosphate, a 5'-O-methylphosphonate, a 3'-O-methylphosphonate, a 5'-hydroxyphosphonate, a hydroxyphosphonate, a phosphoroselenoate, a selenophosphate, an aminophosphate, a carbophosphonate, a phenylphosphonate, an ethylphosphonate, an H-phosphonate, a guanidinium ring, a triazole ring, a boranophosphate, a methylphosphonate, or guanidinopropyl aminophosphate.

30. The RNA molecule according to any one of claims 1 to 29, wherein the last nucleotide of the 3'-stable region does not contain a 3'-hydroxyl group.

31. The RNA molecule of claim 30, wherein the last nucleoside in the 3'-stable region is ddC, reverse dT, 3'-phosphate nucleoside, 3'-oxime nucleoside, 3'-azidomethyl nucleoside, or 3'-methyl nucleoside.

32. The RNA molecule according to any one of claims 1 to 31, wherein the poly-A region is 10 or more nucleotides in length.

33. The RNA molecule according to any one of claims 1 to 31, wherein the length of the poly-A region is 30 or more nucleotides.

34. The RNA molecule according to any one of claims 1 to 31, wherein the poly-A region is 70 or more nucleotides in length.

35. The RNA molecule according to any one of claims 1 to 31, wherein the length of the poly-A region is 100 or more nucleotides.

36. The RNA molecule according to any one of claims 1 to 31, wherein the length of the poly-A region is 2 to 500 nucleotides.

37. The RNA molecule according to any one of claims 1 to 31, wherein the length of the poly-A region is 5 to 250 nucleotides.

38. The RNA molecule according to any one of claims 1 to 31, wherein the length of the poly-A region is 10 to 200 nucleotides.

39. The RNA molecule according to any one of claims 1 to 31, wherein the length of the poly-A region is 15 to 150 nucleotides.

40. The RNA molecule according to any one of claims 1 to 39, wherein the RNA molecule is messenger RNA (mRNA).

41. A cell comprising an RNA molecule according to any one of claims 1 to 40, wherein optionally the cell is isolated.

42. A cell comprising a protein or peptide translated from an RNA molecule according to any one of claims 1 to 40, wherein optionally the cell is isolated.

43. A pharmaceutical composition comprising an RNA molecule according to any one of claims 1 to 40 and a pharmaceutically acceptable carrier.

44. The pharmaceutical composition of claim 43, wherein the pharmaceutically acceptable carrier is a solvent, dispersion medium, diluent, surfactant, isotonic agent, thickener or emulsifier, lipid, liposome, nanoparticle, lipid nanoparticle (LNP), polymer, lipoplex, protein, or mixture thereof.

45. The pharmaceutical composition of claim 44, wherein the pharmaceutically acceptable carrier is an LNP.

46. ​​A pharmaceutical composition comprising cells, said cells comprising an RNA molecule according to any one of claims 1 to 40.

47. A method for increasing the expression of a target protein or peptide in a cell, comprising contacting the cell with an RNA molecule according to any one of claims 1 to 40, wherein the RNA molecule encodes the target protein or peptide, wherein the expression is increased compared to the expression of an RNA molecule that does not contain the 3'-stabilizing region, optionally wherein the cell is isolated in vitro or ex vivo.

48. A method for expressing a target protein or peptide in a cell, comprising contacting the cell with an RNA molecule according to any one of claims 1 to 40, wherein the RNA molecule encodes the target protein or peptide, and wherein the cell is translated from the RNA molecule into the target protein or peptide, optionally wherein the cell is isolated in vitro or ex vivo.

49. A method for prolonging the half-life of an RNA molecule in a cell, comprising contacting the cell with an RNA molecule according to any one of claims 1 to 40, wherein the half-life is prolonged compared to the half-life of an RNA molecule not having the 3'-stable region, optionally wherein the cell is isolated in vitro or ex vivo.

50. A method for preparing an RNA molecule according to any one of claims 1 to 40, comprising covalently linking a stable region to a precursor RNA, said precursor RNA comprising a 5'-cap, an ORF encoding a protein, and a poly-A region located at the 3' of said ORF, wherein said stable region is added to the 3' of said poly-A region and said stable region comprises one or more purification tags and / or one or more adapters capable of binding purification tags.

51. The method of claim 50, wherein the 3'-stabilized region is connected by a link.

52. The method of claim 51, wherein the 3'-stable region is connected by chemical or enzymatic linking.

53. The method of claim 50, wherein the 3'-stabilizing region is ligated using a polymerase.

54. A method of treating a disease in a subject in need, comprising introducing an effective amount of an RNA molecule according to any one of claims 1 to 40, a cell according to claim 41 or 42, or a pharmaceutical composition according to any one of claims 43 to 46.

55. A method for preventing disease in a subject in need, comprising introducing an effective amount of an RNA molecule according to any one of claims 1 to 40, a cell according to claim 41 or 42, or a pharmaceutical composition according to any one of claims 43 to 46.

56. The RNA molecule according to any one of claims 1 to 40, the cell according to claim 41 or 42, or the pharmaceutical composition according to any one of claims 43 to 46, for use in a therapy.

57. Use of an RNA molecule according to any one of claims 1 to 40, a cell according to claim 41 or 42, or a pharmaceutical composition according to any one of claims 43 to 46, for the manufacture of a pharmaceutical agent.

58. A compound of formula (III) or (IV), Where N is a nucleoside; L is a adapter that can bind to the purification tag; P stands for purification tag; Q-L1 may optionally exist, where L1 is a connector covalently bonded to N and Q; and Q is hydrogen or a chain-terminating nucleoside.

59. The compound according to claim 58, wherein the nucleoside is an unmodified nucleoside.

60. The compound according to claim 58, wherein the nucleoside is a modified nucleoside.

61. The compound according to any one of claims 58 to 60, wherein the purification label comprises lipids.

62. The compound according to any one of claims 58 to 61, wherein L1 is a phosphate ester or a modified phosphate ester.

63. The compound according to any one of claims 58 to 62, wherein Q is hydrogen, ddC, reverse dT, 3'-nucleotide phosphate, 3'-oxime nucleotide, 3'-azidomethyl nucleotide, or 3'-methyl nucleotide.

64. The compound according to any one of claims 58 to 63, wherein the linker capable of binding the purification tag is linked to the nucleoside via the 3'-carbon or 2'-carbon of the sugar of the nucleoside.

65. The compound according to any one of claims 58 to 63, wherein the adapter capable of binding the purification tag is linked to the nucleoside via a nucleobase of the nucleoside, and the L1 adapter is linked to the nucleoside via a 3'-carbon or 2'-carbon of the sugar of the nucleoside.

66. The compound according to any one of claims 58 to 63, wherein the purification tag is linked to the nucleoside via the 3'-carbon or 2'-carbon of the sugar of the nucleoside.

67. The compound according to any one of claims 58 to 63, wherein the purification tag is linked to the nucleoside via a nucleobase of the nucleoside.

68. A method for increasing the expression of a target protein or peptide in a cell, comprising contacting the cell with an RNA molecule comprising a compound of formula (III) or (IV) according to any one of claims 58 to 67, wherein the RNA molecule encodes the target protein or peptide, wherein the expression is increased compared to the expression of an RNA molecule not containing the compound of formula (III) or (IV), optionally wherein the cell is isolated in vitro or ex vivo.

69. A method for prolonging the half-life of an RNA molecule in a cell, comprising contacting the cell with an RNA molecule comprising a compound of formula (III) or (IV) according to any one of claims 58 to 67, wherein the half-life is prolonged compared to the half-life of an RNA molecule not containing the compound of formula (III) or (IV), optionally wherein the cell is isolated in vitro or ex vivo.

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