Compositions and methods for capping RNA

Abstract

Provided herein is a composition of 5'-end region modified mRNA and a method for preparing the same.Specifically, the present disclosure relates to novel mRNA 5'-end region motifs and sequence initiators, and thus to these motifs and sequence initiators together with assays that can measure the functional aspects of these motifs and sequence initiators.Further provided herein are compositions and methods for treating conditions related to coronary artery disease.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 315,323, filed March 1, 2022, and U.S. Provisional Application No. 63 / 485,753, filed February 17, 2023, the contents of which are incorporated by reference in their entireties.

[0002] The present disclosure relates to RNA, specifically the 5' end region of an mRNA molecule, including, for example, a novel mRNA 5' end region motif (or mRNA cap) and its initiator. [Background technology]

[0003] The 5'-terminal region of an mRNA may be an important structural and / or functional feature of eukaryotic mRNA molecules because it can confer stability to the mRNA (e.g., by providing protection against 5' exonucleases) and may be involved in other activities that support RNA splicing, mRNA transport, and protein translation. The structural element of a conventional mRNA Cap is an inverted 7-methylguanosine (m) residue linked to a triphosphate (ppp) bridge at the 5' end. 7 The 5'-terminal region mRNA motifs and their sequence initiators described herein differ in several respects from conventional mRNA Caps, including the inclusion of a chemically modified inverted 7-methylguanosine (m7G) nucleoside structure and / or a modified triphosphate (ppp) linkage. Conventional Caps are often one of the most expensive components in the production of mRNA molecules. Therefore, alternatives to conventional Caps that maintain or improve capping efficiency and / or mRNA yield are each independently important aspects of the field. Summary of the Invention

[0004] Thus, novel mRNA 5' end region motifs and initiators are described herein.

[0005] In one aspect there is provided an in vitro transcription (IVT) mRNA sequence initiator comprising a compound of formula (I) or a salt or solvate thereof, [ka] During the ceremony, B1 is, [ka] and each B2, B3, and Bn is independently a natural, modified, or non-natural nucleobase; each Z1 and Z2 is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3, or NHC(=O)CH3; each Z3, Z4, and Zn is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3, or -OCH2CH2OCH3; each Q1 and Q4 is independently -CH2-, -CH=CH-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; each Q2 and Q3 is independently O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X1, X2, X3, X4, and Xn is independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; each Y, Y, Y, Y, and Y is independently =O, =S, =NH, or =NCH; each A, A1, and A2 is independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are in vitro transcription (IVT) mRNA sequence initiators wherein p is 0, 1, 2, 3, 4, 5, or 6.

[0006] In another aspect, compounds of formula (I) are described herein that satisfy one or more of the following conditions (i)-(iii): (i) at least one of X1, X2, X3, X4, and Xn is -SH or -S-; (ii) at least one of Y1, Y2, Y3, Y4, and Yn is =S; and (iii) at least one of A, A1, and A2 is -S-.

[0007] In another embodiment, the IVT mRNA sequence initiator satisfies at least one of Xi, X2, X3, X4, and Xn is -SH or -S-; at least one of Yl, Y2, Y3, Y4, and Yn is =S; or at least one of A, Al, and A2 is -S-.

[0008] In one aspect, there is provided an in vitro transcription (IVT) mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof, [ka] During the ceremony, B1 is, [ka] and each B2, B3, and Bn is independently a natural, modified, or non-natural nucleobase; each Z1 and Z' is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), and -SCH3; each Z2 and Z" is independently fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3, or NHC(=O)CH3; Z"' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3, or -OCH2CH3; each Z3, Z4, and Zn is independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3; each Q1 and Q4 is independently -CH=CH-, -CH2-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; each Q2 and Q3 is independently O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X1, X2, X3, X4, and Xn is independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; each Y, Y, Y, Y, and Y is independently =O, =S, =NH, or =NCH; each A, A1, and A2 is independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are in vitro transcription (IVT) mRNA sequence initiators wherein p is 0, 1, 2, 3, 4, 5, or 6.

[0009] In one aspect, there is provided an in vitro transcription (IVT) mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof, [ka] During the ceremony, B1 is, [ka] and each B2, B3, and Bn is independently a natural, modified, or non-natural nucleobase; each Z' and Z" is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), or -SCH3; Z"' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3, or -OCH2CH3; each Z1 and Z2 is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3, or NHC(=O)CH3; each Z3, Z4, and Zn is independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3; each Q1 and Q4 is independently -CH=CH-, -CH2-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; each Q2 and Q3 is independently O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X1, X2, X3, X4, and Xn is independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; each Y, Y, Y, Y, and Y is independently =O, =S, =NH, or =NCH; each A, A1, and A2 is independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are in vitro transcription (IVT) mRNA sequence initiators wherein p is 0, 1, 2, 3, 4, 5, or 6.

[0010] In one aspect, there is provided an in vitro transcription (IVT) mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof, [ka] During the ceremony, B 1 but, [ka] and Each B 2 , B 3 , and B n is independently a natural, modified, or non-natural nucleobase; each Z' and Z" is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), or -SCH3; Z"' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3, or -OCH2CH3; each Z 1 and Z 2 are independently hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —SCH, —OCHCH, —NH, NHCH, or NHC(═O)CH; each Z 3 , Z 4 , and Z n are independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3; Each Q 1 and Q4 are independently -CH=CH-, -CH2-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; Each Q 2 and Q 3 are independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X 1 , X 2 , X 3 , X 4 , and X n are independently -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; Each Y 1 , Y 2 , Y 3 , Y 4 , and Y n are independently =O, =S, =NH, or =NCH3; Each A, A 1 , and A 2 are independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are in vitro transcription (IVT) mRNA sequence initiators wherein p is 0, 1, 2, 3, 4, 5, or 6.

[0011] In one aspect, there is provided an in vitro transcription (IVT) mRNA sequence initiator comprising a compound of formula (III) or a salt or solvate thereof, [ka] During the ceremony, B1 is, [ka] and B2 is a modified or non-natural nucleobase; each B3 and Bn is independently a natural, modified, or non-natural nucleobase; each Z1 and Z2 is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3, or NHC(=O)CH3; each Z3, Z4, and Zn is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3, or -OCH2CH2OCH3; each Q1 and Q4 is independently -CH2-, -CH=CH-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; each Q2 and Q3 is independently O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X1, X2, X3, X4, and Xn is independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; each Y, Y, Y, Y, and Y is independently =O, =S, =NH, or =NCH; each A, A1, and A2 is independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are in vitro transcription (IVT) mRNA sequence initiators wherein p is 0, 1, 2, 3, 4, 5, or 6.

[0012] In another embodiment, Z3 is hydrogen, fluorine, —OH, —OCH3, or —OCH2CH3. In another embodiment, Z3 is —OCH3. In another embodiment, Z4 and Zn are independently —OH or —OCH3. In another embodiment, each Z3, Z4, and Zn is independently —OH or —OCH3. In another embodiment, Y2, Y4, and Yn are independently ═O or ═S. In another embodiment, X2 and X3 are independently —O— or —S—.

[0013] In some embodiments, the compound of Formula (III) is a salt. In some embodiments, the salt is an alkali metal salt. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is an ammonium salt.

[0014] In another embodiment, the IVT mRNA sequence initiator comprises a phosphorothioate. In another embodiment, the phosphorothioate comprises a chiral phosphorus center. In another embodiment, a composition is described herein that comprises an IVT mRNA sequence initiator, wherein the composition comprises a racemic mixture of R and S diastereomers of the IVT mRNA sequence initiator. In another embodiment, the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 60%. In another embodiment, the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 80%. In another embodiment, the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 90%. In another embodiment, the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 95%. In another embodiment, the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 98%.

[0015] In one embodiment, an mRNA sequence having a 5' end region motif (I'), [ka] During the ceremony, B1 is, [ka] and each B2, B3, and Bn is independently a natural, modified, or non-natural nucleobase; each Z1 and Z2 is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3, or NHC(=O)CH3; each Z3, Z4, and Zn is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3, or -OCH2CH2OCH3; each Q1 and Q4 is independently -CH2-, -CH=CH-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; each Q2 and Q3 is independently O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X1, X2, X3, X4, and Xn is independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; each Y, Y, Y, Y, and Y is independently =O, =S, =NH, or =NCH; each A, A1, and A2 is independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are mRNA sequences having a 5' end region motif (I') where p is 0, 1, 2, 3, 4, 5, or 6.

[0016] In another embodiment, formula (I) satisfies one or more of the following conditions (i) to (iii): (i) at least one of X1, X2, X3, X4, and Xn is -SH or -S-, (ii) at least one of Y1, Y2, Y3, Y4, and Yn is =S, and (iii) at least one of A, A1, and A2 is -S-. In another embodiment, at least one of X1, X2, X3, X4, and Xn is -SH or -S-, or at least one of Y1, Y2, Y3, Y4, and Yn is =S, or at least one of A, A1, and A2 is -S-.

[0017] In one embodiment, an mRNA sequence having a 5' end region motif (II'): [ka] During the ceremony, B1 is, [ka] and each B2, B3, and Bn is independently a natural, modified, or non-natural nucleobase; each Z1 and Z' is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), and -SCH3; each Z2 and Z" is independently fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3, or NHC(=O)CH3; Z"' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3, or -OCH2CH3; each Z3, Z4, and Zn is independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3; each Q1 and Q4 is independently -CH=CH-, -CH2-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; each Q2 and Q3 is independently O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X1, X2, X3, X4, and Xn is independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; each Y, Y, Y, Y, and Y is independently =O, =S, =NH, or =NCH; each A, A1, and A2 is independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are mRNA sequences having a 5' end region motif (II') where p is 0, 1, 2, 3, 4, 5, or 6.

[0018] In one embodiment, an mRNA sequence having a 5' end region motif (II'): [ka] During the ceremony, B1 is, [ka] and each B2, B3, and Bn is independently a natural, modified, or non-natural nucleobase; each Z' and Z" is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), or -SCH3; Z"' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3, or -OCH2CH3; each Z1 and Z2 is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3, or NHC(=O)CH3; each Z3, Z4, and Zn is independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3; each Q1 and Q4 is independently -CH=CH-, -CH2-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; each Q2 and Q3 is independently O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X1, X2, X3, X4, and Xn is independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; each Y, Y, Y, Y, and Y is independently =O, =S, =NH, or =NCH; each A, A1, and A2 is independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are mRNA sequences having a 5' end region motif (II") where p is 0, 1, 2, 3, 4, 5, or 6.

[0019] In one embodiment, an mRNA sequence having a 5' end region motif (II'): [ka] During the ceremony, B 1 but, [ka] and Each B 2 , B 3 , and B n is independently a natural, modified, or non-natural nucleobase; each Z' and Z" is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), or -SCH3; Z"' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3, or -OCH2CH3; each Z 1 and Z 2 are independently hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —SCH, —OCHCH, —NH, NHCH, or NHC(═O)CH; each Z 3 , Z 4 , and Z n are independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3; Each Q 1 and Q 4 are independently -CH=CH-, -CH2-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; Each Q 2 and Q 3 are independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X 1 , X 2 , X 3 , X 4 , and X n are independently -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; Each Y 1 , Y 2 , Y 3 , Y 4 , and Y n are independently =O, =S, =NH, or =NCH3; Each A, A 1 , and A 2 are independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are mRNA sequences having a 5' end region motif (II') where p is 0, 1, 2, 3, 4, 5, or 6.

[0020] In one embodiment, an mRNA sequence having a 5' end region motif (III'): [ka] During the ceremony, B1 is, [ka] and B2 is a modified or non-natural nucleobase; each B3 and Bn is independently a natural, modified, or non-natural nucleobase; each Z1 and Z2 is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3, or NHC(=O)CH3; each Z3, Z4, and Zn is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3, or -OCH2CH2OCH3; each Q1 and Q4 is independently -CH2-, -CH=CH-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; each Q2 and Q3 is independently O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X1, X2, X3, X4, and Xn is independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; each Y, Y, Y, Y, and Y is independently =O, =S, =NH, or =NCH; each A, A1, and A2 is independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are mRNA sequences having a 5' end region motif (III') where p is 0, 1, 2, 3, 4, 5, or 6.

[0021] In one aspect, described herein is an mRNA sequence having a 5'-end region motif, wherein the 5'-end region motif is a compound of Table 1, or a salt or solvate thereof.

[0022] In another aspect, described herein are mRNA sequences having a 5'-end region motif, wherein the 5'-end region motif is a compound in Table 2, or a salt or solvate thereof.

[0023] In another aspect, described herein are mRNA sequences having a 5'-end region motif, wherein the 5'-end region motif is a compound in Table 3, or a salt or solvate thereof.

[0024] In another aspect, the mRNA sequence initiator is a salt. In some embodiments, the mRNA sequence initiator is a sodium salt.

[0025] In another embodiment of the mRNA sequence having a 5' end region motif, Z 3 is hydrogen, fluorine, —OH, —OCH3, or —OCH2CH3.

[0026] In another embodiment of the mRNA sequence having a 5' end region, Z 3 is -OCH3.

[0027] In another embodiment of the mRNA sequence having a 5' end region motif, each Z 4 and Z n are independently —OH or —OCH3.

[0028] In another embodiment of the mRNA sequence having a 5' end region motif, each Z 3 , Z 4 , and Z n are independently —OH or —OCH3.

[0029] In another embodiment of the mRNA sequence having the 5' end region motif, B 1 teeth, [ka] is.

[0030] In another embodiment of the mRNA sequence having the 5' end region motif, B 1 teeth, [ka] is.

[0031] In another embodiment of the mRNA sequence having the 5' end region motif, B 1 teeth, [ka] is.

[0032] In another embodiment of the mRNA sequence having the 5' end region motif, B 1 teeth, [ka] is.

[0033] In another embodiment of the mRNA sequence having the 5' end region motif, B 2 teeth, [ka] is.

[0034] In another embodiment of the mRNA sequence having a 5' end region motif, Z 1 is fluorine, —OH, or —OCH3.

[0035] In another embodiment of the mRNA sequence having a 5' end region motif, Z 1 is fluorine.

[0036] In another embodiment of the mRNA sequence having a 5' end region motif, Z 1 is -OH.

[0037] In another embodiment of the mRNA sequence having a 5' end region motif, Z 1 is -OCH3.

[0038] In another embodiment of the mRNA sequence having a 5' end region motif, Z 2 is fluorine, —OH, or —OCH3.

[0039] In another embodiment of the mRNA sequence having a 5' end region motif, Z 2 is fluorine.

[0040] In another embodiment of the mRNA sequence having a 5' end region motif, Z 2 is -OH.

[0041] In another embodiment of the mRNA sequence having the 5' end region motif, Z 2 is -OCH3.

[0042] In another embodiment of the mRNA sequence having the 5' end region motif, Q 1 and Q 4 is -CH2O-.

[0043] In another embodiment of the mRNA sequence having the 5' end region motif, each Q 2 and Q 3 is -O-.

[0044] In another embodiment of the mRNA sequence having the 5' end region motif, each Y 1 and Y 3 is =O.

[0045] In another embodiment of the mRNA sequence having the 5' end region motif, each Y 2 , Y 4 , and Y n are independently =O or =S.

[0046] In another embodiment of the mRNA sequence having the 5' end region motif, Y 1 , Y 2 , Y 3 , Y 4 , and Y n At least one of is =S.

[0047] In another embodiment of the mRNA sequence having the 5' end region motif, Y 2 is =S.

[0048] In another embodiment of the mRNA sequence having the 5' end region motif, Y 4 is =S.

[0049] In another embodiment of the mRNA sequence having the 5' end region motif, each Y 1 , Y 2 , Y 3 , Y 4 , and Y n is =O.

[0050] In another embodiment of the mRNA sequence having a 5' end region motif, each X 1 , X 4 , and X n -O - is.

[0051] In another embodiment of the mRNA sequence having a 5' end region motif, each X 2 and X 3 independently, -O - or -S - is.

[0052] In another embodiment of the mRNA sequence having the 5' end region motif, X 3 -O - is.

[0053] In another embodiment of the mRNA sequence having the 5' end region motif, X 1 , X 2 , X 3 , X 4 , and X n At least one of the is -S - is.

[0054] In another embodiment of the mRNA sequence having the 5' end region motif, X 2 -S - is.

[0055] In another embodiment of the mRNA sequence having the 5' end region motif, X 4 -S - is.

[0056] In another embodiment of the mRNA sequence having a 5' end region motif, each X 1 , X 2 , X 3 , X 4 , and X n -O - is.

[0057] In another embodiment of the mRNA sequence having the 5' end region motif, each A, A 1, and A 2 is -O-.

[0058] In another embodiment of the mRNA sequence having the 5' end region motif, A, A 1 , and A 2 At least one of is -S-.

[0059] In another embodiment of the mRNA sequence having the 5' end region motif, A is -S- and A 1 and A 2 is -O-.

[0060] In another embodiment of the mRNA sequence having the 5' end region motif, A 2 is -S-, and A and A 1 is -O-.

[0061] In another embodiment of the mRNA sequence having the 5' end region motif, A, A 1 , and A 2 is -O-.

[0062] In another embodiment of the mRNA sequence having the 5' end region motif, p is 0.

[0063] In another embodiment of the mRNA sequence having the 5' end region motif, p is 1.

[0064] In another embodiment of the mRNA sequence having the 5' end region motif, p is 2.

[0065] In another embodiment of the mRNA sequence having the 5' end region motif, each B 2 , B 3 , and B n are independently adenine, cytosine, guanine, uracil, thymine, hypoxanthine, or purine.

[0066] In another embodiment of the mRNA sequence having the 5' end region motif, B 2 is adenine, and B 3 is guanine.

[0067] In another embodiment of the mRNA sequence having the 5' end region motif, B 2 is guanine, and B 3 is adenine.

[0068] Another embodiment of the mRNA sequence having the 5' end region motif increases protein expression.

[0069] In another embodiment of the mRNA sequence having the 5' end region motif, Q 1 and Q 4 is -CH2O-, and Q 2 and Q 3 is -O-, and each X n are independently -OH, -SH, O - , or S - and each Y n are independently =O or =S, and B 1 teeth [ka] is.

[0070] In another embodiment of the mRNA sequence having the 5' end region motif, Q 1 and Q 4 is -CH2O-, and Q 2 and Q 3 is -O-, and each X n are independently -OH, -SH, O - , or S - and each Y n are independently =O or =S, and B 1 teeth [ka] is.

[0071] In another embodiment of the mRNA sequence having the 5' end region motif, Q 1 and Q 4 is -CH2O-, and Q 2 and Q 3 is -O-, and each X nare independently -OH, -SH, O - , or S - and each Y n are independently =O or =S, and B 1 teeth [ka] is.

[0072] In another aspect, described herein is a complex comprising an mRNA sequence having a 5'-end region motif and a DNA template, wherein the mRNA sequence having a 5'-end region motif comprises a compound described herein, the DNA template comprises a promoter region including a transcription start site having a first nucleotide at nucleotide position +1, a second nucleotide at nucleotide position +2, and a third nucleotide at nucleotide position +3, and the mRNA sequence having the 5'-end region motif hybridizes to the DNA template at at least nucleotide positions +1, +2, and +3.

[0073] In another aspect, described herein is a complex comprising an mRNA sequence having a 5'-end region motif and a DNA template, wherein the mRNA sequence having the 5'-end region motif comprises a compound described herein, the DNA template comprises a promoter region including a transcription initiation site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2, and the mRNA sequence having the 5'-end region motif hybridizes to the DNA template at at least nucleotide positions +1 and +2.

[0074] In another aspect, described herein is an RNA molecule comprising an mRNA sequence having a 5' end region motif. In another aspect, the RNA comprises a guide RNA or a nuclease mRNA. In another aspect, the RNA comprises an mRNA.

[0075] In another embodiment, described herein is a method for expressing mRNA, comprising introducing an mRNA described herein into a cell lysate and expressing the mRNA. In another embodiment, the method further comprises measuring the expression level of the mRNA. In another embodiment of the method described herein, the expression level of the mRNA is at least 2-fold, 3-fold, 4-fold, 6-fold, 8-fold, or 20-fold higher than the corresponding mRNA that does not contain the mRNA sequence having the 5'-terminal region motif. In another embodiment described herein, the method requires HeLa cells. In another embodiment described herein, the method comprises producing up to 0.1% by weight of dsRNA. In another embodiment described herein, the method comprises producing up to 0.09% by weight of dsRNA. In another embodiment described herein, the method comprises producing up to 0.06% by weight of dsRNA. In another embodiment described herein, the method comprises producing up to 0.04% by weight of dsRNA.

[0076] In another aspect, described herein is a method for detecting cellular immune stimulation resulting from mRNA, the method comprising: (a) contacting a formulation containing mRNA capped with a compound described herein with a cellular reporter line; and (b) measuring RIG-I activation in the cellular reporter line. In another aspect, the reporter line is a HEK-Lucia RIG-I model. In another aspect described herein, cellular immune stimulation is reduced by at least 20%, 50%, 70%, 100%, and 150% compared to uncapped mRNA.

[0077] In another aspect, described herein is a method for producing an mRNA sequence having a 5'-terminal region motif described herein using an IVT reaction, the method comprising: (a) mixing a DNA template, a polymerase enzyme, an mRNA sequence motif containing a phosphorothioate group (PS), and nucleoside triphosphates (NTPs) at a specific molar ratio of the mRNA sequence motif to the NTPs to produce a mixture; (b) incubating the mixture at a specific temperature and for a specific period of time; and (c) recovering and purifying the mRNA sequence having the 5'-terminal region motif from the mixture. In another aspect, the molar ratio is 1:5, and the method can achieve a yield of at least 80% with a capping efficiency of at least 80%. In another aspect, the molar ratio is 1:2.5, and the method can achieve a yield of at least 80% with a capping efficiency of at least 85%. In another aspect, the molar ratio is 1:1.67, and the method can achieve a yield of at least 80% with a capping efficiency of at least 90%. In another embodiment, the molar ratio is 1:1.25, and the method can achieve a yield of at least 80% with a capping efficiency of at least 90%. In another embodiment, the molar ratio is 1.0:1.0, and the method can achieve a yield of at least 80% with a capping efficiency of at least 80%. In another embodiment, the molar ratio is 1:5, and the method can achieve a yield of at least 3 mg of mRNA per milliliter (mL) of IVT reaction with a capping efficiency of at least 80%. In another embodiment, the molar ratio is 1:2.5, and the method can achieve a yield of at least 3 mg of mRNA per milliliter (mL) of IVT reaction with a capping efficiency of at least 85%. In another embodiment, the molar ratio is 1:1.67, and the method can achieve a yield of at least 3 mg of mRNA per milliliter (mL) of IVT reaction with a capping efficiency of at least 90%. In another embodiment, the molar ratio is 1:1.25, and the method can obtain a yield of at least 3 mg of mRNA per milliliter (mL) of IVT reaction with a capping efficiency of at least 90%.In another embodiment, the molar ratio is 1.0:1.0, and the method can achieve a yield of at least 3 mg of mRNA per milliliter (mL) of IVT reaction with a capping efficiency of at least 80%. In another embodiment, the NTP is GTP, ATP, CTP, UTP, a modified NTP, or a combination thereof. In another embodiment, the modified NTP is N1-methylpseudouridine.

[0078] In another aspect, described herein are cells containing RNA molecules that include mRNA sequences having a 5' end region motif described herein.

[0079] In another aspect, described herein are cells containing polypeptides translated from RNA molecules that include mRNA sequences having a 5' end region motif described herein.

[0080] In another aspect, described herein is a pharmaceutical composition comprising an RNA molecule comprising an mRNA sequence having a 5'-terminal region motif described herein and one or more pharmaceutically acceptable excipients. In another aspect, the pharmaceutical comprises a lipid nanoparticle. In another aspect, the pharmaceutical composition is encapsulated in a lipid nanoparticle. In another aspect, the pharmaceutical composition further comprises one or more single guide RNAs designed to target one or more specific locations of one or more genes of interest to induce a pharmacological effect upon administration to a mammal.

[0081] In another aspect, described herein is a method for synthesizing an RNA molecule, the method comprising introducing an mRNA sequence having a 5'-terminal region motif described herein into a mixture containing an RNA polymerase and incubating the mixture for a sufficient time to allow transcription of the RNA molecule. In another aspect, the mixture further comprises a DNA template and a nucleoside triphosphate.

[0082] In another aspect, described herein are methods of gene editing comprising introducing an RNA molecule or pharmaceutical composition into a cell, wherein the RNA molecule comprises a guide RNA or a nuclease mRNA, and wherein the RNA molecule is translated in the cell.

[0083] In another aspect, described herein is a method for reducing the risk of coronary artery disease in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition described herein.

[0084] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0085] The novel features of the invention are set forth with particularity in the appended claims and are embodied in the 5'-terminal region structures of mRNA described herein. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments of 5'-terminal region structures or compounds (sometimes referred to herein as motifs) in which the principles of the invention are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0086] [Figure 1a] Shown is the corresponding in vitro transcribed mRNA with a phosphorothioate modified 5' end initiator and a 5' end region motif identified herein as 1007a. [Figure 1b] Shown is the corresponding in vitro transcribed mRNA with a phosphorothioate modified 5' end initiator and a 5' end region motif identified herein as 1107a. [Figure 2] 1 illustrates a comparison of the production yield of mRNA prepared using different 5'-end initiators. [Figure 3]1 illustrates a comparison of full-length mRNA purity between mRNAs prepared using different 5'-end initiators. [Figure 4] 1 illustrates a comparison of mRNA capping efficiency between mRNAs prepared using different 5'-end initiators. [Figure 5] 1 illustrates protein expression of mRNAs containing different 5'-end region motifs in HeLa cell lysates. [Figure 6] 1 illustrates the protein expression of mRNAs containing different 5'-terminal region motifs in primary human hepatocytes. [Figure 7] 1 illustrates a comparison of ABE base editing in vivo in mice using proteins encoded by mRNAs containing different 5'-end region motifs. [Figure 8] 1 illustrates a comparison of ABE base editing using proteins encoded by two ABE mRNAs containing different 5'-end region motifs in vivo in NHPs. [Figure 9] 1 illustrates the immune stimulation of mRNAs containing different 5'-terminal region motifs in HEK293 cells. [Figure 10] Illustrating the chirality of the phosphorothioate in 5' initiator 1007a, 1007a_d1 represents the S diastereomer and 1007a_d2 represents the R diastereomer. [Figure 11a] Illustrates the efficiency of analog incorporation of 5' initiator 5232 compared to analog 1002a as determined by ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) at doses ranging from 0.125 mM to 2 mM. [Figure 11b] Figure 1 shows the efficiency of analog incorporation of 5' initiator 5232 compared to analog 1002a as determined by IP-RP-HPLC-mass spectrometry at doses of 0.5, 2, and 4 mM. [Figure 12] 1 shows the yield and quality of mRNA produced using various 5' initiator analogs. [Figure 13]The percent dsRNA detected in mRNA samples generated using 5' initiator analogs is shown. [Figure 14] 1 illustrates the in vitro RIG-I response to mRNAs with various 5' initiator caps. [Figure 15] 1 illustrates the in vitro stability as a measure of relative pyrophosphatase resistance of various 5' initiator caps. [Figure 16] 1 illustrates in vitro 5' initiator cap affinity for protein biogenesis factors by measuring relative ribosome affinity. [Figure 17a] Illustrates the temporal and dose-dependent expression of luciferase from mRNAs synthesized with various 5' initiator caps, with cells harvested 6 hours after exposure to the mRNAs. [Figure 17b] Illustrates the temporal and dose-dependent expression of luciferase from RNA synthesized with various 5' initiator caps, with cells harvested 24 hours after exposure to the mRNA. [Figure 18a] Temporal and dose-dependent expression of Cas9 from mRNA synthesized with various 5' initiator caps is illustrated, where cells are harvested 6 hours after exposure to the mRNA. [Figure 18b] Temporal and dose-dependent expression of Cas9 from mRNA synthesized with various 5' initiator caps is illustrated, and cells are harvested 24 hours after exposure to the mRNA. [Figure 19] 1 illustrates a schematic diagram of MA004 and MA079. [Figure 20] 1 illustrates the in vivo editing efficiency of ABEs synthesized using 5′ initiator analogs in mice. [Figure 21a] 1 illustrates the in vivo immunogenicity of ABE synthesized using a 5' initiator analog as a measure of TNF-α levels over time in mice. [Figure 21b] 1 illustrates the in vivo immunogenicity of ABE synthesized using a 5' initiator analog as a measure of IFN-γ levels over time in mice. [Figure 21c] 1 illustrates the in vivo immunogenicity of ABE synthesized using a 5' initiator analog as a measure of IFN-α levels over time in mice. [Figure 21d] 1 illustrates the in vivo immunogenicity of ABE synthesized using a 5' initiator analog as a measure of IFN-β levels over time in mice. [Figure 21e] 1 illustrates the in vivo immunogenicity of ABE synthesized using a 5' initiator analog as a measure of MIP-1B levels over time in mice. [Figure 21f] 1 illustrates the in vivo immunogenicity of ABE synthesized using a 5' initiator analog as a measure of IP-10 levels over time in mice. [Figure 21g] 1 illustrates the in vivo immunogenicity of ABE synthesized using a 5' initiator analog as a measure of IL-10 levels over time in mice. [Figure 21h] 1 illustrates the in vivo immunogenicity of ABE synthesized using a 5' initiator analog as a measure of IL-6 levels over time in mice. DETAILED DESCRIPTION OF THE INVENTION

[0087] Certain specific details in this description are set forth to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures and / or methods have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context otherwise requires, throughout the following specification and claims, words such as "comprise" and variations thereof, such as "comprises" and "comprising," should be interpreted in an open and inclusive sense, i.e., as "including, but not limited to." Furthermore, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0088] Messenger RNA (mRNA), which encodes physiologically important proteins for therapeutic applications, has shown significant advantages over DNA-based plasmid and viral vectors for delivering genetic material. These include (i) a potential improved level of safety compared with the potential for genomic damage that can result from viral or plasmid integration, (ii) more immediate protein expression upon mRNA delivery (as opposed to the delayed response typically observed with plasmids), (iii) robust dose-dependent regulation of protein expression, and (iv) the facilitated simplification of large-scale mRNA synthesis compared with the production of plasmid and viral vectors.

[0089] Messenger RNA can code for virtually any known protein and can be delivered to specific cells, tissues, and organs by a variety of methods well known to those of skill in the art. Once delivered, such mRNA can induce ribosomal protein expression within the target cell or tissue, resulting in the production of potentially hundreds of copies of the encoded protein from a single mRNA molecule.

[0090] Several structural elements present in active mRNA molecules are utilized for efficient translation of the encoded protein. One of these elements is the 5'-terminal region of the mRNA. In naturally occurring mRNAs, the 5'-terminal region contains a Cap structure, which is an important feature of eukaryotic mRNA molecules (and some viruses). Such Cap structures are well known to be involved in protein translation, 5'-exonuclease protection, splicing, and mRNA transport. The consistent structural element of the naturally occurring 5' Cap is an inverted 7-methylguanosine (m) residue linked to the 5' end of the mRNA through a triphosphate (ppp) bridge. 7 G), which phosphate bridge is linked to the first nucleotide (N1) of the mRNA transcript. 7 This 5'Cap portion, represented as G(ppp)N1, is called cap-0. Methylation of the 2'-hydroxyl on the N1 ribose ring (i.e., cap-1) is known to provide an identifier for self-RNA, thereby helping to protect mRNA from the innate immune system and subsequently improving protein expression. This m 7 The G(ppp)N1m cap-1 structure is a well-known conventional cap moiety used in in vitro transcription (IVT) of mRNA.

[0091] Herein, we present novel mRNA 5'-terminal region motifs and initiators. These novel designs are 7These motifs include modifications of purine bases in the G moiety; phosphorothioate (PS) substitution in triphosphate bridges and chemical modifications of phosphodiester bonds; substitution of 5'-terminal nucleotides with non-standard bases; elongation of 5'-terminal nucleotide oligomers; and chemical modifications of the ribose ring. Specifically, these mRNA 5'-terminal region structures act as the 5'-terminal region at the end of the mRNA and provide stability to the mRNA. The chemical structure of the motif can promote and / or regulate mRNA activity and the rate of translation initiation and elongation; protect mRNA by creating a barrier that prevents or interferes with mRNA cap removal by 5' exonuclease activity; improve mRNA safety by affecting capping efficiency and reducing the formation of immunostimulatory by-products; and promote mRNA manufacturability by modulating binding affinity to the DNA template during the IVT reaction.

[0092] 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 disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All references cited herein are incorporated by reference in their entirety as if fully set forth. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, NY 2001), and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) provide those of ordinary skill in the art with a general guide to many of the terms used in this application.

[0093] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense to include "and / or" unless the content clearly dictates otherwise.

[0094] When referring to the number of substituents, the term "one or more" refers to a range from one substituent to the maximum number of substitutions possible, e.g., replacement of one hydrogen to replacement of all hydrogens by substituents.

[0095] The term "optional" or "optionally" means that the subsequently described event or circumstance does not have to occur, but that the description covers cases where the event or circumstance occurs and cases where it does not occur.

[0096] As used herein, the term "nucleic acid" generally refers to one or more nucleic acid bases, nucleosides, or nucleotides, and includes polynucleobases, polynucleosides, and polynucleotides. Nucleic acids can include polynucleotides, mononucleotides, and oligonucleotides. Nucleic acids may include DNA, RNA, or mixtures thereof, and may be single-stranded, double-stranded, or partially single- or double-stranded, and may form secondary structures. In some embodiments, nucleic acids have multiple double-stranded and single-stranded segments. For example, nucleic acids may include polynucleotides, such as mRNA, having multiple double-stranded segments therein.

[0097] The terms "mRNA sequence initiator," "IVT mRNA sequence initiator," and "initiator" are used interchangeably herein to generally refer to a single-stranded ribo- or deoxyribo- or chimeric ribo / deoxyribo-oligonucleotide, which may be naturally occurring or synthetic and typically comprises a sequence of about 2 to about 10 nucleotides, about 3 to about 8 nucleotides, or about 3 to about 5 nucleotides. The mRNA sequence initiator may comprise one or more modifying groups. The mRNA sequence initiator may be a primer, e.g., an oligonucleotide primer. The mRNA sequence initiator, e.g., an oligonucleotide primer, may comprise RNA, DNA, and / or other modified nucleosides. One skilled in the art can design and prepare an mRNA sequence initiator suitable for transcription of a DNA template sequence.

[0098] The mRNA sequence initiator may be a capped primer or a capped oligonucleotide analog. For example, the capped mRNA sequence initiator may contain an initiating capped oligonucleotide analog or an initiating capped oligonucleotide having a Cap0, Cap1, Cap2, or TMG-Cap structure at the 5'-end. In some cases, the capped initiator, e.g., a capped primer or a capped oligonucleotide analog, has an unmodified or open 3'-OH group and can be extended by RNA polymerase through the incorporation of NTPs at the 3'-end. In some cases, the initiators described herein can initiate in vitro transcription under the control of a promoter in a transcription system containing the necessary components: a DNA template (e.g., a DNA plasmid), RNA polymerase, nucleoside 5'-triphosphates, and an appropriate buffer. The initiator may be an oligonucleotide bearing a terminal 3'-OH group that is an effective substrate for RNA polymerase. In certain embodiments, the initiator is a substrate for RNA polymerase and can be extended by the incorporation of NTPs at the 3'-end. In some embodiments, the initiator is complementary to the DNA template at the initiation site.

[0099] As used herein, the terms "unsubstituted" or "unmodified," in the context of mRNA sequence initiators and nucleoside triphosphates (NTPs), generally refer to unmodified initial capped initiators and NTPs.

[0100] As used herein, the term "modified initiation capped initiator" generally refers to an initiation capped mRNA sequence initiator that contains one or more additional modifying groups or moieties within the sequence initiator.

[0101] As used herein, the term "modifying group(s) or moiety" generally refers to any chemical moiety that can be attached to or substituted into an mRNA sequence initiator, e.g., the starting primer at that position, including, but not limited to, a sugar, a nucleoside base, a triphosphate bridge, and / or an internucleotide phosphate (e.g., U.S. Patent Application Publication No. 2007 / 0281308). The modifying group of a capped initiator may be of any nature that is compatible with the process of transcription.

[0102] The term "internucleotide bond" as used herein generally refers to one or more bonds joining two nucleosides of an initiator, e.g., an oligonucleotide primer or a nucleic acid, and may be a natural phosphodiester bond or a chemically modified nucleic acid backbone bond.

[0103] As used herein, the term "polynucleotide" generally refers to a molecule containing two or more linked nucleic acid subunits, e.g., nucleotides, and may be used interchangeably with "oligonucleotide." For example, a polynucleotide may contain one or more nucleotides selected from the corresponding nucleosides containing the nucleobases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants and combinations thereof. A nucleotide generally contains a nucleoside and at least one, two, three, four, five, six, seven, eight, nine, ten, or more phosphate (PO3) groups. A nucleotide includes a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides include nucleotides in which the sugar is ribose. Deoxyribonucleotides include nucleotides in which the sugar is deoxyribose. A nucleotide may be a nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, or nucleoside polyphosphate. For example, the nucleotide may be a deoxyribonucleoside polyphosphate, such as a deoxyribonucleoside triphosphate (dNTP), and exemplary dNTPs include deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP). The dNTP may also include a detectable tag, such as a luminescent tag or a marker (e.g., a fluorophore). For example, the nucleotide may be a purine (e.g., A or G, or a variant thereof) or a pyrimidine (e.g., C, T, or U, or a variant thereof). In some embodiments, the polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a derivative or variant thereof.Exemplary polynucleotides include, but are not limited to, short interfering RNA (siRNA), microRNA (miRNA), plasmid DNA (pDNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), and heteronuclear RNA (hnRNA), including both the nucleotide sequence and any structural embodiment thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, stem-loop, bulge, etc. In some cases, the polynucleotide is circular. The polynucleotide may have a variety of lengths. For example, a polynucleotide can have a length of at least about 7 bases, 8 bases, 9 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. Polynucleotides can be isolated from cells or tissues. For example, polynucleotide sequences can include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and / or synthetic DNA / RNA analogs.

[0104] A polynucleotide may comprise one or more nucleotide variants, including non-standard nucleotide(s), non-natural nucleotide(s), nucleotide analog(s), and / or modified nucleotides, including acyclic and carbocyclic nucleotides. Examples of modified nucleotides include diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, Examples of suitable nucleotides include, but are not limited to, cuosine, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, and the like. In some cases, a nucleotide may contain a modification in its phosphate moiety, including a modification to the triphosphate moiety. Non-limiting examples of such modifications include longer length phosphate chains (e.g., phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphate).Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms typically available to form hydrogen bonds with a complementary nucleotide and / or at one or more atoms typically incapable of forming hydrogen bonds with a complementary nucleotide), at the sugar moiety, or at the phosphate backbone. Nucleic acid molecules may also contain amine-modifying groups, such as aminoallyl-dUTP (aa-dUTP) and aminohexyl acrylamide-dCTP (aha-dCTP), which allow for the covalent attachment of amine-reactive moieties, such as N-hydroxysuccinimide ester (NHS). Substitution of standard DNA or RNA base pairs in the disclosed oligonucleotides may result in higher bit density per cubic mm, greater safety (resistance to accidental or intentional synthesis of natural toxins), easier recognition in light-programmed polymerases, or lower secondary structure. Such alternative base pairs compatible with natural and mutant polymerases for de novo and / or amplification synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012, 8(7):612-4, which is incorporated herein by reference for all purposes.

[0105] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably and refer to a polymer of amino acid residues linked via peptide bonds and may consist of two or more polypeptide chains. The terms "polypeptide," "protein," and "peptide" refer to a polymer of at least two amino acid monomers linked together through amide bonds. The amino acids may be the L- or D-enantiomers. More specifically, the terms "polypeptide," "protein," and "peptide" refer to a molecule composed of two or more amino acids in a specific order, e.g., an order determined by the nucleotide base sequence of a gene or RNA encoding the protein. Proteins are essential for the structure, function, and regulation of cells, tissues, and organs of the body, and each protein has a unique function. Examples are hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein may be a portion of a protein, e.g., a protein domain, subdomain, or motif. In some cases, a protein may be a variant (or mutation) of a protein in which one or more amino acid residues have been inserted, deleted, and / or substituted in the amino acid sequence of a naturally occurring (or at least known) protein. The protein or variant thereof may be naturally occurring or recombinant.

[0106] As used herein, "hybridizing" refers to the process in which a capped mRNA sequence initiator begins to anneal to a DNA template according to Watson-Crick base pairing rules under appropriately stringent conditions during a transcription reaction. Nucleic acid hybridization techniques are well known in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY. Those skilled in the art will understand how to determine the appropriate stringency of hybridization / washing conditions so that sequences with at least the desired level of complementarity will stably hybridize, while sequences with lower complementarity will not hybridize. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al. 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, NJ, all of which are incorporated by reference in their entireties. In certain embodiments, hybridization can occur between nucleic acid molecules 20 to 100 nucleotides in length.In some embodiments, hybridization occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 consecutive nucleotides. In some embodiments, hybridizing nucleic acid molecules may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mismatches, which are tolerated.

[0107] As used herein, "complement," "complementary," or "complementarity" refers to standard Watson / Crick base pairing rules in the context of a complex, e.g., an initiating capped oligonucleotide primer and a DNA template. For example, the sequence "5'-AGTC-3'" is complementary to the sequence "3'-TCAG-5'." Certain non-natural or synthetic nucleotides may be included in the nucleic acids described herein; these include, but are not limited to, base- and sugar-modified nucleosides, nucleotides, and nucleic acids, such as inosine, 7-deazaguanosine, 2'-O-methylguanosine, 2'-fluoro-2'-deoxycytidine, pseudouridine, locked nucleic acids (LNA), and peptide nucleic acids (PNA). Complementarity need not be perfect; a duplex may contain mismatched base pairs, degenerative, or non-matching nucleotides. One of skill in the art can empirically determine duplex stability by considering numerous variables, including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, the frequency of mismatched base pairs, ionic strength, hybridization buffer components, and reaction conditions.

[0108] Complementarity can be "perfect" or "complete" when all nucleotide bases of the two nucleic acid strands match according to accepted base-pairing rules; "partial" when only some nucleotide bases of the capped mRNA sequence initiator and the DNA target match according to accepted base-pairing rules; or "absent" when none of the nucleotide bases of the two nucleic acid strands match according to accepted base-pairing rules. The degree of complementarity between a capped mRNA sequence initiator, such as a capped primer, and a DNA template can have a significant effect on the strength of hybridization between the initial capped oligonucleotide and the DNA template and the efficiency of the corresponding reaction. The term "complementarity" can also be used in reference to individual nucleotides. For example, a particular nucleotide within an oligonucleotide can be referred to in terms of its complementarity to a nucleotide in another strand, or, in contrast, its lack of complementarity, or in terms of the complementarity between the remainder of the capped mRNA sequence initiator and the DNA strand.

[0109] As used herein, the terms "complete," "total," or "perfectly" complementary mean that the nucleotide bases of the capped mRNA sequence initiator and the DNA target, respectively, exactly match according to accepted base-pairing rules.

[0110] As used herein, the term "substantially complementary" refers to two sequences that hybridize under stringent hybridization conditions. Those skilled in the art will understand that substantially complementary sequences need not hybridize along their entire length. Specifically, a substantially complementary sequence may contain a contiguous sequence of bases that does not hybridize to the target sequence, and may be located 3' or 5' to a contiguous sequence of bases that hybridizes to the target sequence under stringent hybridization conditions.

[0111] As used herein, the term "nucleoside" includes all naturally occurring nucleosides, including all forms of nucleoside bases and furanosides found in nature. The base rings most commonly found in naturally occurring nucleosides are purine and pyrimidine rings. Naturally occurring purine rings include, for example, adenine, guanine, and N 6 2'-O-methyladenine. Naturally occurring pyrimidine rings include, for example, cytosine, thymine, 5-methylcytosine, and pseudouracil. Naturally occurring nucleosides include, but are not limited to, ribo, 2'-O-methyl, or 2'-deoxyribo derivatives of adenosine, guanosine, cytidine, thymidine, uridine, inosine, 7-methylguanosine, or pseudouridine.

[0112] As used herein, the terms "nucleoside analog," "modified nucleoside," or "nucleoside derivative" include synthetic nucleosides described herein. Nucleoside derivatives also include nucleosides having modified base and / or sugar moieties, with or without protecting groups, such as 2'-deoxy-2'-fluorouridine, 5-fluorouridine, and the like. The compounds and methods provided herein include such base rings and their synthetic analogs, as well as non-natural heterocyclic substituted base sugars and acyclic substituted base sugars. Other nucleoside derivatives that may be utilized in the present disclosure include, for example, LNA nucleosides, halogen-substituted purines (e.g., 6-fluoropurine), halogen-substituted pyrimidines, N-substituted nucleosides, and the like. 6 -Ethyladenine, N 4 -(alkyl)-cytosine, 5-ethylcytosine, and the like (U.S. Pat. No. 6,762,298).

[0113] As used herein, the terms "universal base," "degenerate base," "universal base analog," and "degenerate base analog" include, for example, nucleoside analogs containing artificial bases that, in certain embodiments, can be recognized by an RNA polymerase as a substitute for one of the natural NTPs (e.g., ATP, UTP, CTP, and GTP) or other specific NTPs. Universal bases or degenerate bases are described 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, F. P., Oliver, J. S., J. Comput. Biol. 753-765 (2004), and Hill, F. et al., Proc. Natl. Acad. Sci. USA, 95:4258-4263 (1998)).

[0114] As used herein, the term "modified NTP" refers to a nucleoside 5'-triphosphate having a chemical moiety group attached at any position, including the sugar, the base, the triphosphate chain, or any combination of these three positions. Examples of such NTPs can be found, for example, in "Nucleoside Triphosphates and Their Analogs: Chemistry, Biotechnology and Biological Applications," Vaghefi, M. ed., Taylor and Francis, Boca Raton (2005).

[0115] As used herein, the term "specific," when used in reference to a 5'-capped mRNA sequence initiator sequence and its ability to hybridize to a DNA template, is a sequence that has at least 50% sequence identity to a portion of the DNA template when the capped mRNA sequence initiator and DNA strand are aligned. Higher levels of sequence identity that may be preferred include at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, and most preferably 100% sequence identity.

[0116] In addition to "unmodified" or "natural" nucleobases, such as the purine nucleobases adenine (A) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art are suitable for use in the compounds described herein. Unmodified or natural nucleobases may be modified or replaced to provide oligonucleotides with improved properties. For example, nuclease-resistant oligonucleotides may be prepared using these bases, or using synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin) and any one of the oligomeric modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" may be used. When a natural base is replaced with an unnatural and / or universal base, the nucleotide is said herein to contain a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural, unnatural, and universal bases, including conjugate moieties, e.g., ligands, as described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups, which can be conjugated to the nucleobase via a linker having a suitable alkyl, alkenyl, or amide bond.

[0117] As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Exemplary modified nucleobases include other synthetic and natural nucleobases, such as inosine, xanthine, hypoxanthine, nuvalalin, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 9-(methylthio)adenine, 10-(methylthio)adenine, 11-(methylthio)adenine, 12-(methylthio)adenine, 13-(methylthio)adenine, 14-(methylthio)adenine, 15-(methylthio)adenine, 16-(methylthio)adenine, 17-(methylthio)adenine, 18-(methylthio)adenine, 19-(methylthio)adenine, 20-(methylthio)adenine, 21-(methylthio)adenine, 22-(methylthio)adenine, 23-(methylthio)adenine, 24-(methylthio)adenine, 25-(methylthio)adenine, 26-(methylthio)adenine, 27-(methylthio)adenine, 28-(methylthio)adenine, 29-(methylthio)adenine, 30-(methylthio) N6-(isopentyl)adenine, N6-(methyl)adenine, N6,N6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(methyl)guanine, 7-(isopentyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine, N6,N6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(isopentyl)adenine, 8-(isopentyl)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, 8-(isopentyl)adenine, 8-(methyl)adenine, 8-(isopentyl ... (deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine N-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(Dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkoxy)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(di (methylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil , 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-methyl Thilpseudouracil (N1-methylpseudouracil), 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-( 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guani 7-(guanidiniumaralkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumaralkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumaralkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumaralkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(Dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)iso Carbostyryl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N2-substituted purine, N6-substituted purine, O6-substituted purine, substituted 1,2,4-triazole, pyrrolo-pyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ori / zo-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ori / zo-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, Examples of suitable nucleobases include, but are not limited to, ori / zo-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ori / zo-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" may be used. A universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of an oligonucleotide duplex. Some exemplary universal nucleobases include 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazadenine, 4-fluoro-6-methylbenzimidazur, 4-methylbenzimidazur, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl- ... Pyridinyl, pyrrolopyridinyl, isocarbostyryl, 7-propynylisocarbostyryl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof (e.g., see Loakes, 2001, which is incorporated by reference in its entirety).Nucleic Acids Research, 29, 2437-2447). Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in International Application No. PCTUS09 / 038425, filed March 26, 2009; those disclosed in the Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990; those disclosed in English et al., Angewandte Chemie, International Edition, 1991, 30,613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P. Ed. Wiley-VCH, 2008; and those disclosed in Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B. Eds., CRC Press, 1993, the contents of all of which are incorporated herein by reference.

[0118] As used herein, the term "biological sample" means any biological material from which polynucleotides, polypeptides, biomarkers, and / or metabolites can be prepared or extracted and tested. Non-limiting examples include whole blood, plasma, saliva, buccal swab, stool specimen, urine specimen, cell mass, or any other bodily fluid or tissue.

[0119] As used herein, the terms "administer," "administering," "administration," and the like refer to methods that can be used to enable delivery of a compound or composition to a desired site of biological effect. These methods include, but are not limited to, oral (po), intraduodenal (id), parenteral injection (e.g., intravenous (iv), subcutaneous (sc), intraperitoneal (ip), intramuscular (im), intravenous or infusion (inf)), topical (top), and rectal (pr) administration. Those of skill in the art are familiar with administration techniques that can be used with the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally.

[0120] As used herein, terms such as "co-administration" are meant to encompass administration of selected therapeutic agents to a single patient and are intended to include therapeutic regimens in which agents are administered to the same patient or by different routes of administration, or at the same time or at different times.

[0121] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient quantity of an agent or compound being administered to relieve to some extent one or more symptoms of the disease or condition being treated (e.g., reduction and / or alleviation of one or more signs, symptoms, or causes of the disease, or any other desired alteration of a biological system). For example, an "effective amount" for therapeutic purposes can be that amount of an agent that produces a clinically significant reduction in one or more disease symptoms. An appropriate "effective" amount may be determined in each individual case using techniques, such as a dose escalation study.

[0122] The terms "enhance" or "enhancing," as used herein, means to increase or prolong either in amount, potency, or duration a desired effect.

[0123] As used herein, "carbohydrate" refers to either a compound that is a carbohydrate itself, composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound that contains a carbohydrate moiety composed of one or more monosaccharide units (which may be linear, branched, or cyclic), each having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4-9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars of C5 or higher (preferably C5-C8), with disaccharides and trisaccharides including sugars having two or three monosaccharide units (preferably C5-C8).

[0124] The term "monosaccharide" encompasses radicals of allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fuculose, galactosamine, D-galactosaminitol, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphate guloseglyceraldehyde, L-glycero-D-mannos-heprose, glycerol, glycerone, guloseidose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinovose, quinovosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, tulose, xylose, and xylulose. Monosaccharides may be in the D or L configuration. Monosaccharides may also be deoxysugars (alcoholic hydroxy group replaced with hydrogen), aminosugars (alcoholic hydroxy group replaced with amino group), thiosugars (alcoholic hydroxy group replaced with thiol, or C=O replaced with C=S, or in cyclic forms, ring oxygen replaced with sulfur), selenosugars, tellurosugars, azasugars (ring carbon replaced with nitrogen), iminosugars (ring oxygen replaced with nitrogen), phosphanosugars (ring oxygen replaced with phosphorus), phosphosugars (ring carbon replaced with phosphorus), C-substituted monosaccharides (hydrogen on a non-terminal carbon atom replaced with carbon), unsaturated monosaccharides, alditols (carbonyl group replaced with CHOH group), aldonic acids (aldehyde group replaced with carboxy group), ketoaldonic acids, uronic acids, aldaric acids, etc. The amino sugars include amino monosaccharides, preferably galactosamine, glusamine, mannosamine, fucosmine, quinabosamine, neuraminic acid, muramic acid, lactosediamine, acosamine, bacillosamine, daunosamine, desosamine, forosamine, galosamine, kanosamine, kanosamine, mycaminose, myosamine, persosamine, pneumosamine, purpurosamine, and rhodosmin. It is understood that the monosaccharides may be further substituted.

[0125] The terms "disaccharide," "trisaccharide," and "polysaccharide" refer to abequose, acrabose, amicetose, amylopectin, amylose, apiose, alkanose, ascarylose, ascorbic acid, boivinose, cellobiose, cellotriose, cellulose, chacotriose, chalcose, chitin, colitose, cyclodextrin, cymarose, dextrin, 2-deoxyribose, 2-deoxyglucose, diginose, digitalose, digitoxose, evolose, ebemitrose, fructooligosaccharides, galtooligosaccharides, gentianose, genitiobiose, glucan, glucogen, glycogen, hamamelose, heparin, inulin, isolevoglucosenone, isomaltose, isomaltotriose, isopanose, kojibiose, lactose, lactosamine, lactose, Examples of sugars that can be used include diamine, laminarabiose, levoglucosan, levoglucosenone, β-maltose, maltotriose, mannanoligosaccharide, amninotriose, melezitose, melibiose, muramic acid, mycarose, mycinose, neuraminic acid, mygelose, nojirimaicon, nobiose, oleandrose, panose, paratose, planteose, primverose, raffinose, rhodon, rutinose, oleandrose, panose, paratose, planteose, primverose, raffinose, rhodinose, rutinose, sarmentose, sedoheptulose, sedoheptulosan, solatriose, sophorose, stachyose, streptose, sucrose, α,α-trehalose, trahalosamine, turanose, tybelose, xylobiose, and umbelliferose. It is further understood that "disaccharides," "trisaccharides," "polysaccharides," and the like, may be further substituted. Disaccharides also include amino sugars and their derivatives, particularly mycaminose derivatized at the C-4' position or 4-deoxy-3-amino-glucose derivatized at the C-6' position.

[0126] The term "subject" or "patient" includes mammals. Examples of mammals include, but are not limited to, any member of the mammalian class; humans, non-human primates, e.g., chimpanzees, and other ape and monkey species; domestic animals, such as cattle, horses, sheep, goats, and pigs; domestic animals, such as rabbits, dogs, and cats; and laboratory animals, including rodents, such as rats, mice, and guinea pigs. In one aspect, the mammal is a human. The term "animal" as used herein includes human and non-human animals. In one embodiment, a "non-human animal" is a mammal, e.g., a rodent, such as a rat or a mouse. In one embodiment, the non-human animal is a mouse.

[0127] As used herein, the terms "treat," "treating," or "treatment" include alleviating, ameliorating, or improving at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, e.g., arresting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating symptoms caused by a disease or condition, or the prophylactic and / or therapeutic cessation of symptoms of a disease or condition. The term "treat" further encompasses the concepts of "prevent," "preventing," and "prophylaxis," as discussed below. Although not excluded, it is understood that treating a disorder or condition does not require that the disorder, condition, or its associated symptoms be completely eliminated.

[0128] The terms "preventing" or "prevention" of a disease state refer to keeping clinical symptoms of the disease state from occurring in a subject who may be exposed to or predisposed to the disease state but who has not yet experienced or exhibited symptoms of the condition.

[0129] The terms "pharmaceutical composition" and "pharmaceutical formulation" (or "formulation") are used interchangeably and refer to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients that is administered to a subject, e.g., a human in need thereof.

[0130] As used herein, the term "pharmaceutical combination" refers to a product obtained by mixing or combining two or more active pharmaceutical ingredients, and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that both active ingredients, e.g., a compound described herein and a co-agent, are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g., a compound described herein and a co-agent, are administered to a patient simultaneously, concomitantly, or sequentially without a specific intervening time limit as separate entities, such that such administration provides effective levels of the two compounds in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0131] The term "pharmaceutically acceptable" generally refers to the attributes of a material that is safe, non-toxic, not biologically or otherwise undesirable, and useful in preparing pharmaceutical compositions that are acceptable for veterinary and human pharmaceutical use. "Pharmaceutically acceptable" can refer to a material, such as a carrier or diluent, that does not abolish the biological activity or properties of a compound and is relatively non-toxic, e.g., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious way with any of the components of the composition in which it is included.

[0132] The terms "pharmaceutically acceptable excipient," "pharmaceutically acceptable carrier," "pharmaceutically acceptable vehicle," and "therapeutically inactive excipient," may be used interchangeably and refer to any pharmaceutically acceptable ingredient in a pharmaceutical composition that has no therapeutic activity and is non-toxic to a subject to which it is administered, such as a disintegrant, binder, filler, solvent, buffer, isotonicity agent, stabilizer, antioxidant, surfactant, carrier, diluent, excipient, preservative, or lubricant used in formulating a pharmaceutical product.

[0133] The terms "base editing" and "base correction" are used interchangeably to refer to a base change or mutation in a target sequence within a target gene that results in a base modification. In certain embodiments, base editing occurs at a single base in the target sequence. In preferred embodiments, base editing does not involve a double-strand break in the target sequence.

[0134] The term "siRNA" as used herein refers to an agent that mediates targeted cleavage of RNA transcripts. These agents are associated with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Agents that are effective in inducing RNA interference are also referred to herein as siRNAs, RNAi agents, or iRNA agents. The term siRNA as used herein includes microRNAs and pre-microRNAs. The terms "siRNA activity" and "RNAi activity" as used herein refer to gene silencing by siRNA.

[0135] 2'-O-Methoxyethyl (2'-MOE, 2'-O(CH2) 2- The term OCH3 and 2'-O-(2-methoxyethyl) refers to an O-methoxy-ethyl modification at the 2' position of the furosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0136] The term "2'-O-methoxyethyl nucleotide" means a nucleotide containing a 2'-O-methoxyethyl modified sugar moiety.

[0137] The term "5-methylcytosine" means a cytosine modified with a methyl group attached to the 5' position. 5-Methylcytosine is a modified nucleobase.

[0138] The term "oxo" refers to a ═O substituent.

[0139] The term "alkyl" refers to a straight or branched hydrocarbon chain group having from 1 to 20 carbon atoms and attached to the rest of the molecule by a single bond. Alkyl groups containing up to 10 carbon atoms include C1-C 10 Similarly, for example, an alkyl containing up to 6 carbon atoms is a C1-C6 alkyl. Alkyl groups containing other numbers of carbon atoms (and other moieties defined herein) are similarly represented. Alkyl groups include C1-C 10 Examples of alkyl groups include, but are not limited to, alkyl, C1-C9 alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl, and C4-C8 alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl, i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethyl-propyl, and the like. In some embodiments, alkyl is methyl or ethyl. In some embodiments, alkyl is —CH(CH3)2 or —C(CH3)3. Unless otherwise specifically stated herein, alkyl groups may be optionally substituted as described below. "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a radical group. In some embodiments, alkylene is -CH-, -CHCH-, or -CHCHCH-. In some embodiments, alkylene is -CH-. In some embodiments, alkylene is -CHCH-. In some embodiments, alkylene is -CHCHCH-. In some embodiments, alkylene is -CHCHCH-.

[0140] The term "alkoxy" refers to a radical of formula -OR, where R is an alkyl group as defined herein. Unless stated otherwise in the specification, an alkoxy group may be optionally substituted as described below. Representative alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, and pentoxy. In some embodiments, the alkoxy is methoxy. In some embodiments, the alkoxy is ethoxy.

[0141] The term "alkylamino" refers to a radical of the formula -NHR or -NRR, where each R is independently an alkyl group as defined above. Unless stated otherwise in the specification, an alkylamino group can be optionally substituted as described below.

[0142] The term "alkenyl" refers to a type of alkyl group in which at least one carbon-carbon double bond is present. In one embodiment, an alkenyl group has the formula -C(R)=CR, where R refers to the remainder of the alkenyl group and can be the same or different. In some embodiments, R is H or alkyl. In some embodiments, alkenyl is selected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, and the like. Non-limiting examples of alkenyl groups include -CH=CH, -C(CH)=CH, -CH=CHCH, -C(CH)=CHCH, and -CHCH=CH. Depending on the structure, alkenyl groups can be monovalent or divalent (i.e., alkenylene groups).

[0143] The term "alkynyl" refers to a type of alkyl group in which at least one carbon-carbon triple bond is present. Thus, "alkynylene" can refer to a divalent alkynyl group. In one embodiment, an alkenyl group has the formula -C≡CR, where R refers to the remainder of the alkynyl group. In some embodiments, R is H or alkyl. In some embodiments, alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Non-limiting examples of alkynyl groups include -C≡CH, -C≡CCH3, -C≡CCH2CH3, and -CH2C≡CH.

[0144] The term "aryl" refers to an aromatic ring in which each of the atoms forming the ring is a carbon atom. An aryl group may be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl and naphthyl. In some embodiments, an aryl is phenyl. Depending on the structure, an aryl group may be monovalent or divalent (i.e., an "arylene" group). Unless stated otherwise in the specification, the term "aryl" or the prefix "ar-" (e.g., "aralkyl") is meant to include aryl radicals that may be optionally substituted. In some embodiments, an aryl group is partially reduced to form a cycloalkyl group, as defined herein. In some embodiments, an aryl group is fully reduced to form a cycloalkyl group, as defined herein. In some embodiments, an aryl group is a C6-C 14 In some embodiments, the aryl group is C-C 10 It is aryl.

[0145] The term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical in which each of the atoms forming the ring (i.e., skeletal atoms) is a carbon atom. In some embodiments, the cycloalkyl is saturated or partially unsaturated. In some embodiments, the cycloalkyl is a spirocyclic or bridged compound. In some embodiments, the cycloalkyl is fused to an aromatic ring (in which case the cycloalkyl is attached through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls having 3 to 10 carbon atoms, 3 to 8 carbon atoms, 3 to 6 carbon atoms, or 3 to 5 carbon atoms. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the monocyclic cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl. Polycyclic groups include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetraynyl, decalinyl, 3,4-dihydronaphthalenyl-1(2H)-one, spiro[2.2]pentyl, norbornyl, and bicyclo[1.1.1]pentyl. Unless otherwise stated specifically in the specification, cycloalkyl groups may be optionally substituted. Depending on the structure, cycloalkyl groups may be monovalent or divalent (i.e., cycloalkylene groups).

[0146] The term "haloalkyl" refers to an alkyl group in which at least one hydrogen atom of the alkyl group is replaced with the same or different halogen atom, in particular a fluoro atom. Examples of haloalkyl include monofluoro-, difluoro- or trifluoro-methyl, -ethyl or -propyl, such as 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, or trifluoromethyl. The term "perhaloalkyl" refers to an alkyl group in which all hydrogen atoms of the alkyl group are replaced with the same or different halogen atoms.

[0147] The term "heteroalkylene" refers to an alkyl group as defined above in which one or more carbon atoms of the alkyl has been replaced with an O, N, or S atom. A "heteroalkylene" or "heteroalkylene chain" refers to a straight or branched divalent heteroalkyl chain that connects the rest of the molecule to a radical group. Unless stated otherwise in the specification, a heteroalkyl or heteroalkylene group may be optionally substituted as described below. Representative heteroalkylene groups include, but are not limited to, -OCHCHO-, -OCHCHOCHCHO-, or -OCHCHOCHCHOCHCHO-.

[0148] The term "heterocycloalkyl" refers to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless stated otherwise in the specification, a heterocycloalkyl group can be a monocyclic or bicyclic ring system, which can include fused (when fused to an aryl or heteroaryl ring, the heterocycloalkyl is attached through a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon, or sulfur atoms in a heterocyclyl group can be optionally oxidized. The nitrogen atom can be optionally quaternized. The heterocycloalkyl group can be partially or fully saturated. Examples of heterocycloalkyl groups include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all ring forms of carbohydrates, including, but not limited to, monosaccharides, disaccharides, and oligosaccharides. Unless otherwise noted, heterocycloalkyls have 2-12 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons and 1 or 2 N atoms in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons and 3 or 4 N atoms in the ring. In some embodiments, heterocycloalkyls have 2-12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring.In some embodiments, a heterocycloalkyl has 2 to 12 carbons, 1 to 3 N atoms, 0 to 1 O atoms, and 0 to 1 S atoms within the ring. When referring to the number of carbon atoms in a heterocycloalkyl, it is understood that the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including heteroatoms) comprising the heterocycloalkyl (i.e., the skeletal atoms of the heterocycloalkyl ring). Unless otherwise specifically stated in the specification, a heterocycloalkyl group can be optionally substituted. As used herein, the term "heterocycloalkylene" may refer to a divalent heterocycloalkyl group.

[0149] The term "heteroaryl" refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. Heteroaryls are monocyclic or bicyclic. Specific examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. Illustrative examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl. Illustrative examples of bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. In some embodiments, heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl, or furyl. In some embodiments, heteroaryls contain 0 to 6 N atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms in the ring. In some embodiments, the heteroaryl contains 4-6 N atoms in the ring. In some embodiments, the heteroaryl contains 0-4 N atoms, 0-1 O atoms, 0-1 P atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl is a C1-C9 heteroaryl.In some embodiments, the monocyclic heteroaryl is a C1-C5 heteroaryl. In some embodiments, the monocyclic heteroaryl is a 5- or 6-membered heteroaryl. In some embodiments, the bicyclic heteroaryl is a C6-C9 heteroaryl. In some embodiments, the heteroaryl group is partially reduced to form a heterocycloalkyl group, as defined herein. In some embodiments, the heteroaryl group is fully reduced to form a heterocycloalkyl group, as defined herein. Depending on the structure, heteroaryl groups can be monovalent or divalent (i.e., "heteroarylene" groups).

[0150] The terms "substituted," "substituent," and the like, unless otherwise indicated, may individually and independently refer to the replacement of one or more hydrogen radicals in a given structure with the radical of the specified substituent, including, but not limited to, D, halogen, -CN, -NH, -NH(alkyl), -N(alkyl), -OH, -COH, -COalkyl, -C(=O)NH, -C(=O)NH(alkyl), -C(=O)N(alkyl), -S(=O)NH, -S(=O)NH(alkyl), -S(=O)N(alkyl), alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone. In some other embodiments, the optional substituents are independently selected from D, halogen, -CN, -NH, -NH(CH), -N(CH), -OH, -COH, -CO(C-C alkyl), -C(=O)NH, -C(=O)NH(C-C alkyl), -C(=O)N(C-C alkyl), -S(=O)NH, -S(=O)NH(C-C alkyl), -S(=O)N(C-C alkyl), C-C alkyl, C-C cycloalkyl, C-C fluoroalkyl, C-C heteroalkyl, C-C alkoxy, C-C fluoroalkoxy, -SC-C alkyl, -S(=O)C-C alkyl, and -S(=O)(C-C alkyl). In some embodiments, optional substituents are independently selected from D, halogen, -CN, -NH, -OH, -NH(CH), -N(CH), -NH(cyclopropyl), -CH, -CHCH, -CF, -OCH, and -OCF. In some embodiments, substituents are substituted with one or two of the foregoing groups. In some embodiments, optional substituents on aliphatic carbon atoms (acyclic or cyclic) include oxo (=O).

[0151] The term "unsubstituted" means that a particular group has no substituents. The term "optionally substituted" means that a particular group is unsubstituted or substituted with one or more substituents independently selected from a group of possible substituents. When indicating the number of substituents, the term "one or more" means from one substituent to as many substitutions as possible, i.e., from replacement of one hydrogen to replacement of all hydrogens by substituents.

[0152] "About" means within ±10% of the value. For example, a statement that "a marker can be increased by about 50%" means that the marker can be increased by 45% to 55%.

[0153] "Active agent" means one or more substances in a pharmaceutical composition that provide a therapeutic effect when administered to an individual.

[0154] "Dosage unit" means the form in which a pharmaceutical agent is provided, such as a pill, tablet, or other dosage form known in the art. In certain embodiments, the dosage form is a vial containing lyophilized antisense oligonucleotide. In certain embodiments, the dosage form is a vial containing reconstituted antisense oligonucleotide.

[0155] "Dose" refers to a specific amount of pharmaceutical agent provided in a single administration or over a specific period of time. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume that cannot be easily accommodated in a single injection, so two or more injections may be used to achieve the desired dose. In certain embodiments, a pharmaceutical agent is administered by infusion over an extended period of time or continuously. A dose may be expressed as the amount of pharmaceutical agent per hour, day, week, or month. A dose may also be expressed as the mass of pharmaceutical agent or drug substance per mass of target tissue (e.g., mg / kg or g / kg).

[0156] A "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage. For example, a phosphorothioate linkage is a modified internucleoside linkage.

[0157] "Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. For example, 5-methylcytosine is a modified nucleobase. "Unmodified nucleobase" refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0158] "Modified nucleoside" means a nucleoside having at least one modified sugar moiety and / or modified nucleobase.

[0159] "Modified nucleotide" means a nucleotide having at least one modified sugar moiety, modified internucleoside linkage, and / or modified nucleobase.

[0160] "Modified oligonucleotide" means an oligonucleotide containing at least one modified nucleotide.

[0161] A "modified sugar" refers to a substitution or variation from a natural sugar. For example, a 2'-O-methoxyethyl modified sugar is a modified sugar.

[0162] By "motif" is meant a pattern of chemically distinct regions in an antisense compound.

[0163] "Statin" refers to a drug that inhibits the activity of HMG-CoA reductase.

[0164] "Symptoms of a cardiovascular disease or disorder" means phenomena that are caused by or accompany a cardiovascular disease or disorder and serve as indicators thereof. For example, angina, chest pain, dyspnea, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling of the legs, cyanosis, fatigue, fainting, numbness in the face, numbness in the hands and feet, claudication or muscle cramps, abdominal distension, or fever are symptoms of a cardiovascular disease or disorder.

[0165] "Target nucleic acid" and "target sequence" refer to a nucleic acid that can be targeted by a genome editing composition. For example, a target DNA sequence within or adjacent to the ANGPTL3 gene can be targeted by guide nucleotides associated with a Cas9 nuclease.

[0166] Methods for detecting and / or measuring polypeptides in biological materials are well known in the art, including, but not limited to, Western blotting, flow cytometry, ELISA, RIA, and various proteomics techniques. An exemplary method for measuring or detecting polypeptides is an immunoassay such as ELISA. This type of protein quantification can be based on an antibody capable of capturing a specific antigen and a second antibody capable of detecting the captured antigen. An exemplary assay for detecting and / or measuring polypeptides is described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

[0167] Methods for detecting and / or measuring RNA in biological materials are well known in the art, including, but not limited to, Northern blotting, RNA protection assays, and RT-PCR. Suitable methods are described in Molecular Cloning: A Laboratory Manual (Fourth Edition) by Michael R. Green, Joseph Sambrook, and Peter MacCallum, 2012, 2,028 pp, ISBN 978-1-936113-42-2.

[0168] Ribonucleoprotein (RNP) refers to a nuclear protein containing RNA. RNPs may be complexes of ribonucleic acid and RNA-binding proteins. Such combinations are sometimes called protein-RNA complexes. These complexes can function in several biological functions, including, but not limited to, DNA replication, DNA modification, gene expression, RNA metabolism and modification, and pre-mRNA splicing.

[0169] As used herein, the terms "biomarker" or "marker" are used interchangeably to refer to any biochemical, serological, genetic, or other clinical or sonographic feature that can be used to classify a sample from a patient as being associated with a pathological condition, such as a cardiovascular disease or disorder.

[0170] As used herein, the term "antibody" includes, but is not limited to, a population of immunoglobulin molecules, which may be polyclonal or monoclonal, and may be of any class and isotype, or fragments of immunoglobulin molecules. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 (human), IgA2 (human), IgAa (dog), IgAb (dog), IgAc (dog), and IgAd (dog). Such fragments generally contain the portion of an antibody molecule that specifically binds to an antigen. For example, fragments of immunoglobulin molecules known in the art as Fab, Fab', or F(ab')2 are included within the meaning of the term antibody.

[0171] The term "label" as used herein refers to a detectable compound, composition, or solid support that can be directly or indirectly conjugated (e.g., covalently or non-covalently, alone or encapsulated) to a monoclonal antibody or protein. The label may be detectable itself (e.g., a radioisotope label, a chemiluminescent dye, an electrochemical label, a metal chelate, a latex particle, or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a detectable substrate compound or composition (e.g., an enzyme such as horseradish peroxidase or alkaline phosphatase). Labels used in this disclosure may include, but are not limited to, alkaline phosphatase; glucose-6-phosphate dehydrogenase ("G6PDH"); horseradish peroxidase (HRP); chemiluminescent agents such as isoluminol, fluorescent agents such as fluorescein and rhodamine compounds; ribozymes; and dyes. A label may also be a specific binding molecule that is itself detectable (e.g., biotin, avidin, streptavidin, digoxigenin, maltose, oligohistidine, e.g., hexahistidine, 2,4-dinitrobenzene, phenylarsenate, ssDNA, dsDNA, etc.) Use of a label generates a signal that can be detected, and optionally measured, by means such as detection of electromagnetic radiation or direct visualization.

[0172] "Substantial binding" or "substantially binds" refers to the amount of specific binding or recognition between molecules in an assay mixture under particular assay conditions. In its broadest aspect, substantial binding relates to the difference between the inability of a first molecule to bind or recognize a second molecule and the inability of a first molecule to bind or recognize a third molecule, a difference sufficient to enable a meaningful assay to be performed to identify specific binding under a particular set of assay conditions, including the relative concentrations of the molecules and the incubation time and temperature. In another aspect, a molecule is substantially unable to bind or recognize another molecule in the sense of cross-reactivity if the first molecule exhibits less than 25%, e.g., less than 10%, e.g., less than 5%, of the reactivity it exhibits with a third molecule under a particular set of assay conditions, including, e.g., the relative concentrations of the molecules and incubation. Specific binding can be tested using several well-known methods, such as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay.

[0173] As used herein, the term "substantially the same amino acid sequence" includes amino acid sequences that are similar but not identical to a naturally occurring amino acid sequence. For example, an amino acid sequence, e.g., a polypeptide, having substantially the same amino acid sequence as a flagellin protein can have one or more modifications, such as addition, deletion, or substitution of amino acids, compared to the amino acid sequence of a naturally occurring flagellin protein, provided that the modified polypeptide substantially retains at least one biological activity of flagellin, such as immunoreactivity. The "percentage similarity" between two sequences is a function of the number of positions containing matching residues or conserved residues shared by the two sequences, divided by the number of positions compared, multiplied by 100. In this regard, conserved residues in a sequence are residues that are physically or functionally similar to the corresponding reference residues, e.g., have similar size, shape, charge, chemical properties, such as the ability to form covalent or hydrogen bonds.

[0174] The term "targeting moiety" refers to any molecule that provides enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, body region, or compartment, e.g., a cell, tissue, or organ compartment. Some exemplary targeting moieties include, but are not limited to, antibodies, antigens, carbohydrate base moieties, folate, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

[0175] The term "heterologous" refers to any two or more nucleic acid or polypeptide sequences that are not normally found in the same relationship to each other in nature. For example, a heterologous nucleic acid is typically produced recombinantly, having two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous polypeptide often refers to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0176] As used herein, the term "fragment" includes a peptide, polypeptide or protein segment of amino acids of the full-length protein, provided that the fragment retains reactivity with at least one antibody in the serum of a disease patient.

[0177] An "epitope" is an antigenic determinant on a polypeptide that is recognized for binding by an antibody specific for the polypeptide, eg, a paratope on an IBD-associated antibody.

[0178] The term "clinical factor" includes the patient's symptoms related to the disease. Examples of clinical factors for cardiovascular disease include, but are not limited to, angina pectoris; chest pain; shortness of breath; palpitations; weakness; dizziness; nausea; sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling of the lower extremities; cyanosis; fatigue; fainting; facial numbness; numbness of the hands and feet; claudication or muscle cramps; abdominal distension; or fever. In some embodiments, the diagnosis of cardiovascular disease is based on a combination of analyzing the presence or level of one or more markers in a patient using a statistical algorithm and determining whether the patient has one or more clinical factors.

[0179] The term "prognosis" includes a prediction of the likely course and outcome of a pathological condition, e.g., cardiovascular disease, or recovery from disease. In some embodiments, a patient's prognosis for cardiovascular disease is obtained through the use of statistical algorithms. For example, the prognosis may be surgery, the occurrence of one or more clinical factors, the occurrence of cancer of the digestive system, or recovery from disease.

[0180] The term "CRISPR-Cas system" as referred to herein includes a CRISPR-associated protein and a single guide RNA translated from an mRNA encoding the protein. In some embodiments, the CRISPR-associated protein may have inherent endonucleolytic activity. In some embodiments, the CRISPR-Cas system facilitates guide RNA-mediated gene modification. One or more proteins produced from the mRNA encoding the protein can facilitate base or nucleobase and / or gene editing within a gene target segment of interest.

[0181] Provided herein are methods and compositions for targeted delivery of therapeutic agents, such as nucleic acid agents. The therapeutic agents used herein can be linked or associated with a targeting moiety to aid in targeted delivery. For example, the therapeutic agent and the targeting moiety can form a conjugate. The therapeutic agent can include a nucleic acid-guided programmable nuclease system complexed with a nucleic acid, such as a guide RNA. In some embodiments, the guide RNA can be chemically modified. In some embodiments, the modified guide RNA can be used to prepare a medicament for the treatment of any gene-related disease, disorder, or condition in which genes can be altered, manipulated, edited, and modified by DNA insertion or deletion. According to a further aspect of the present disclosure, the modified guide RNA can be used to alter genes by deleting, replacing, repairing, or inserting one or more nucleotides or segments of DNA. This can be done in microorganisms or animals, particularly mammals, and more specifically, humans. Human cells or tissues can be genetically altered or corrected in vitro using the guide RNAs disclosed herein and CRISPR / Cas systems known in the art, and then inoculated into a patient in need thereof. In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising a modified guide RNA according to the present disclosure, a CRISPR-Cas system, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may comprise a vector or a cell harboring the modified guide RNA of the present disclosure.

[0182] compound In one aspect there is provided an IVT mRNA sequence initiator of formula (I) or a salt or solvate thereof, [ka] During the ceremony, B 1 but, [ka] and Each B 2 , B 3 , and B nis independently a natural, modified, or non-natural nucleobase; each Z 1 and Z 2 are independently hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH; each Z 3 , Z 4 , and Z n are independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3, or -OCH2CH2OCH3; Each Q 1 and Q 4 are independently -CH-, -CH=CH-, -CHO-, -CHS-, -CHCH-, -CHCF-, -CHNH-, -CHNH(CH)-, or -CHN(C(=O)CH)-; Each Q 2 and Q 3 are independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X 1 , X 2 , X 3 , X 4 , and X n are independently -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; Each Y 1 , Y 2 , Y 3 , Y 4 , and Y n are independently =O, =S, =NH, or =NCH3; Each A, A 1 , and A 2are independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; p is 0, 1, 2, 3, 4, 5, or 6, However, (i) Z 1 , Z 2 , and Z 3 is hydrogen, or (ii) Q 1 and Q 4 is -CH2O- or (iii) Q 2 and Q 3 is oxygen or (iv) X 1 , X 2 , X 3 , X 4 , and X n At least one of the following is -SH or -S - or (v) Y 1 , Y 2 , Y 3 , Y 4 , and Y n At least one of A, A 1 , A 2 Described herein are IVT mRNA sequence initiators of formula (I) or a salt or solvate thereof, provided that at least one of:

[0183] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (Ia): [ka]

[0184] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (Ib): [ka]

[0185] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (Ic): [ka]

[0186] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (Id): [ka]

[0187] In some embodiments, the IVT mRNA sequence initiator of formula (Id) is [ka] It has the following structure.

[0188] In some embodiments, the IVT mRNA sequence initiator of formula (Id) is [ka] It has the following structure.

[0189] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (Ie): [ka]

[0190] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (If): [ka]

[0191] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (Ig): [ka]

[0192] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (Ih): [ka]

[0193] In some embodiments, a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih) is a salt. In some embodiments, the salt is an alkali metal salt. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is an ammonium salt.

[0194] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Z 1 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH. 1 is hydrogen. In some embodiments, Z 1 is F. In some embodiments, Z 1 is —OH. In some embodiments, Z 1 is -SH. In some embodiments, Z 1 is -CH3. In some embodiments, Z 1 is -CH2CH3. In some embodiments, Z 1 is —OCH. In some embodiments, Z 1 is —OCH2CH3. In some embodiments, Z 1 is -SCH3. In some embodiments, Z 1 is -NH2. In some embodiments, Z 1 is NHCH. In some embodiments, Z 1 is NHC(=O)CH3.

[0195] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ia), (Id), (Ie), (If), (Ig), or (Ih), Z 2 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH. 2 is hydrogen. In some embodiments, Z 2 is F. In some embodiments, Z 2 is —OH. In some embodiments, Z 2 is -SH. In some embodiments, Z 2 is -CH3. In some embodiments, Z 2 is -CH2CH3. In some embodiments, Z 2 is —OCH. In some embodiments, Z 2 is —OCH2CH3. In some embodiments, Z 2 is -SCH3. In some embodiments, Z 2 is -NH2. In some embodiments, Z 2 is NHCH. In some embodiments, Z 2 is NHC(=O)CH3.

[0196] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Z 3 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 3 is hydrogen. In some embodiments, Z 3 is fluorine. In some embodiments, Z 3 is —OH. In some embodiments, Z 3is -SH. In some embodiments, Z 3 is -CH3. In some embodiments, Z 3 is -CH2CH3. In some embodiments, Z 3 is —OCH2OCH3. In some embodiments, Z 3 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 3 is —OCH(CH). In some embodiments, Z 3 is -SCH3. In some embodiments, Z 3 is -OCH2CH2OCH3.

[0197] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Z 4 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 4 is hydrogen. In some embodiments, Z 4 is fluorine. In some embodiments, Z 4 is —OH. In some embodiments, Z 4 is -SH. In some embodiments, Z 4 is -CH3. In some embodiments, Z 4 is -CH2CH3. In some embodiments, Z 4 is —OCH2OCH3. In some embodiments, Z 4 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 4 is —OCH(CH). In some embodiments, Z 4 is -SCH3. In some embodiments, Z 4 is -OCH2CH2OCH3.

[0198] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Z n is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z n is hydrogen. In some embodiments, Z n is fluorine. In some embodiments, Z n is —OH. In some embodiments, Z n is -SH. In some embodiments, Z n is -CH3. In some embodiments, Z n is -CH2CH3. In some embodiments, Z n is —OCH2OCH3. In some embodiments, Z n is —OCH 2 CH 2 CH 3 . In some embodiments, Z n is —OCH(CH). In some embodiments, Z n is -SCH3. In some embodiments, Z n is -OCH2CH2OCH3.

[0199] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), B 2 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 2 is guanine. In some embodiments, B 2 is adenine. In some embodiments, B 2 is cytosine. In some embodiments, B 2 is uracil, and in some embodiments, B 2 is thymine, and in some embodiments, B 2is hypoxanthine. In some embodiments, B 2 is pudding.

[0200] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), B 3 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 3 is guanine. In some embodiments, B 3 is adenine. In some embodiments, B 3 is cytosine. In some embodiments, B 3 is uracil, and in some embodiments, B 3 is thymine, and in some embodiments, B 3 is hypoxanthine. In some embodiments, B 3 is pudding.

[0201] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), B n are independently natural, modified, or non-natural nucleobases. In some embodiments, B n is guanine. In some embodiments, B n is adenine. In some embodiments, B n is cytosine. In some embodiments, B n is uracil, and in some embodiments, B n is thymine, and in some embodiments, B n is hypoxanthine. In some embodiments, B n is pudding.

[0202] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), B 2 , B 3 , and B nAt least one of B is adenine. 2 , B 3 , and B n At least one of B is guanine. 2 is adenine. In some embodiments, B 3 is adenine.

[0203] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Q 1 is —CH—, —CH═CH—, —CHO—, —CHS—, —CHCH—, —CHCF—, —CHNH—, —CHNH(CH)—, or —CHN(C(═O)CH)—. In some embodiments, Q 1 In some embodiments, Q is -CH-. 1 is -CH=CH-. In some embodiments, Q 1 is —CH2O—. In some embodiments, Q 1 is -CHS-. In some embodiments, Q 1 is -CHCH-. In some embodiments, Q 1 is —CHCF—. In some embodiments, Q 1 is —CH 2 NH 2 —. In some embodiments, Q 1 is —CHNH(CH)—. In some embodiments, Q 1 is -CH2N(C(=O)CH3)-.

[0204] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Q 4 is —CH—, —CH═CH—, —CHO—, —CHS—, —CHCH—, —CHCF—, —CHNH—, —CHNH(CH)—, or —CHN(C(═O)CH)—. In some embodiments, Q 4In some embodiments, Q is -CH-. 4 is -CH=CH-. In some embodiments, Q 4 is —CH2O—. In some embodiments, Q 4 is -CHS-. In some embodiments, Q 4 is -CHCH-. In some embodiments, Q 4 is —CHCF—. In some embodiments, Q 4 is —CH 2 NH 2 —. In some embodiments, Q 4 is —CHNH(CH)—. In some embodiments, Q 4 is -CH2N(C(=O)CH3)-.

[0205] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Q 2 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 2 is —O—. In some embodiments, Q 2 is -S-. In some embodiments, Q 2 In some embodiments, Q is -CH-. 2 In some embodiments, Q is -CF-. 2 In some embodiments, Q is -NH-. 2 is —N(CH)—. In some embodiments, Q 2 is -N(C(=O)CH3).

[0206] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Q 3 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 3In some embodiments, Q 3 is -S-. In some embodiments, Q 3 In some embodiments, Q is -CH-. 3 In some embodiments, Q is -CF-. 3 In some embodiments, Q is -NH-. 3 is —N(CH)—. In some embodiments, Q 3 is -N(C(=O)CH3).

[0207] In some embodiments of compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), X 1 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 1 is —OH. In some embodiments, X 1 is -SH. In some embodiments, X 1 -O - In some embodiments, X 1 -S - In some embodiments, X 1 is —NH. In some embodiments, X 1 is -NHCH3. In some embodiments, X 1 is —NH(C(═O)CH). In some embodiments, X 1 is —CH3. In some embodiments, X 1 is -CH2CH3. In some embodiments, X 1 is -CH2CH2CH3. In some embodiments, X 1 is —CH(CH). In some embodiments, X 1 is —OCH3. In some embodiments, X 1is -OCH2CH3.

[0208] In some embodiments of compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), X 2 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 2 is —OH. In some embodiments, X 2 is -SH. In some embodiments, X 2 -O - In some embodiments, X 2 -S - In some embodiments, X 2 is —NH. In some embodiments, X 2 is -NHCH3. In some embodiments, X 2 is —NH(C(═O)CH). In some embodiments, X 2 is —CH3. In some embodiments, X 2 is -CH2CH3. In some embodiments, X 2 is -CH2CH2CH3. In some embodiments, X 2 is —CH(CH). In some embodiments, X 2 is —OCH3. In some embodiments, X 2 is -OCH2CH3.

[0209] In some embodiments of compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), X 3 -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 3 is —OH. In some embodiments, X 3 is -SH. In some embodiments, X 3 -O - In some embodiments, X 3 -S - In some embodiments, X 3 is —NH. In some embodiments, X 3 is -NHCH3. In some embodiments, X 3 is —NH(C(═O)CH). In some embodiments, X 3 is —CH3. In some embodiments, X 3 is -CH2CH3. In some embodiments, X 3 is -CH2CH2CH3. In some embodiments, X 3 is —CH(CH). In some embodiments, X 3 is —OCH3. In some embodiments, X 3 is -OCH2CH3.

[0210] In some embodiments of compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), X 4 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 4 is —OH. In some embodiments, X 4 is -SH. In some embodiments, X 4 -O - In some embodiments, X 4 -S- In some embodiments, X 4 is —NH. In some embodiments, X 4 is -NHCH3. In some embodiments, X 4 is —NH(C(═O)CH). In some embodiments, X 4 is —CH3. In some embodiments, X 4 is -CH2CH3. In some embodiments, X 4 is -CH2CH2CH3. In some embodiments, X 4 is —CH(CH). In some embodiments, X 4 is —OCH3. In some embodiments, X 4 is -OCH2CH3.

[0211] In some embodiments of compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), X n -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X n is —OH. In some embodiments, X n is -SH. In some embodiments, X n -O - In some embodiments, X n -S - In some embodiments, X n is —NH. In some embodiments, X n is -NHCH3. In some embodiments, X n is —NH(C(═O)CH). In some embodiments, X n is —CH3. In some embodiments, X n is -CH2CH3. In some embodiments, X nis -CH2CH2CH3. In some embodiments, X n is —CH(CH). In some embodiments, X n is —OCH3. In some embodiments, X n is -OCH2CH3.

[0212] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Y 1 is =O, =S, =NH, or =NCH3. In some embodiments, Y 1 is ═O. In some embodiments, Y 1 is =S. In some embodiments, Y 1 is ═NH. In some embodiments, Y 1 is =NHCH3.

[0213] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Y 2 is =O, =S, =NH, or =NCH3. In some embodiments, Y 2 is ═O. In some embodiments, Y 2 is =S. In some embodiments, Y 2 is ═NH. In some embodiments, Y 2 is =NHCH3.

[0214] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Y 3 is =O, =S, =NH, or =NCH3. In some embodiments, Y 3 is ═O. In some embodiments, Y 3 is =S. In some embodiments, Y 3 is ═NH. In some embodiments, Y 3 is =NHCH3.

[0215] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Y 4 is =O, =S, =NH, or =NCH3. In some embodiments, Y 4 is ═O. In some embodiments, Y 4 is =S. In some embodiments, Y 4 is ═NH. In some embodiments, Y 4 is =NHCH3.

[0216] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Y n is =O, =S, =NH, or =NCH3. In some embodiments, Y n is ═O. In some embodiments, Y n is =S. In some embodiments, Y n is ═NH. In some embodiments, Y n is =NHCH3.

[0217] In some embodiments of a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), A is -O-, -S-, -CH-, -NH-, -N(CH)-, or -N(C(=O)CH)-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH-. In some embodiments, A is -NH-. In some embodiments, A is -N(CH)-. In some embodiments, A is -N(C(=O)CH)-.

[0218] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), A1 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 1 is —O—. In some embodiments, A 1 is -S-. In some embodiments, A 1 is -CH2-. In some embodiments, A 1 is -NH-. In some embodiments, A 1 is —N(CH)—. In some embodiments, A 1 is -N(C(=O)CH3)-.

[0219] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), A 2 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 2 is —O—. In some embodiments, A 2 is -S-. In some embodiments, A 2 is -CH2-. In some embodiments, A 2 is -NH-. In some embodiments, A 2 is —N(CH)—. In some embodiments, A 2 is -N(C(=O)CH3)-.

[0220] In some embodiments of a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), p is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

[0221] In some embodiments of the compounds of Formula (I), X 1 , X 2 , X 3 , X 4 , and X n At least one of is -SH.

[0222] In some embodiments of the compounds of Formula (I), X 1 , X 2 , X 3 , X 4 , and X n At least one of is -S-.

[0223] In some embodiments of the compounds of Formula (I), Y 1 , Y 2 , Y 3 , Y 4 , and Y n At least one of them is =S.

[0224] In some embodiments of the compounds of Formula (I), A, A 1 , and A 2 At least one of is -S-.

[0225] In some embodiments of the compounds of Formula (I), Z 1 , Z 2 , Z 3 , Z 4 , and Z n At least one of Z is -OCH. 3 is —OCH. In some embodiments, Z 3 and Z 1 is —OCH 3 . In some embodiments of the compounds of Formula (I), Z 1 , Z 2 , Z 3 , Z 4 , and Z n In some embodiments, at least one of Z 1 , Z 2 , and Z 4 is —OH. In some embodiments, Z 2 and Z4 is -OH.

[0226] In some embodiments of the compounds of Formula (I), Q 1 , Q 2 , Q 3 , and Q 4 In some embodiments of the compound of Formula (I), at least one of Q 1 , Q 2 , Q 3 , and Q 4 In some embodiments, at least one of Q 1 and Q 4 is —OCH. In some embodiments, Q 2 and Q 3 is -O-.

[0227] In some embodiments of the compounds of Formula (I), Y 1 , Y 2 , Y 3 , Y 4 , and Y n In some embodiments, at least one of Y 1 , Y 2 , Y 3 , and Y 4 is ═O. In some embodiments, Y 1 , Y 2 , Y 3 , Y 4 , and Y n is =S. In some embodiments, Y 2 is =S. In some embodiments, Y 4 is =S.

[0228] In some embodiments of the compounds of Formula (I), X 1 , X 2 , X 3 , X 4 , and X n At least one of the -O - In some embodiments, X 1 , X 2 , X 3 , and X4 -O - In some embodiments, X 1 -S - In some embodiments, X 2 -S - In some embodiments, X 3 -S - In some embodiments, X 4 -S - is.

[0229] In some embodiments of the compounds of Formula (I), A, A 1 , and A 2 In some embodiments, at least one of A, A 1 , and A 2 is -O-.

[0230] In one embodiment, an mRNA sequence having a 5'-terminal region motif of motif (I'),

[0231] [ka]

[0232] During the ceremony,

[0233] B 1 but, [ka] and

[0234] Each B 2 , B 3 , and B n is independently a natural, modified, or non-natural nucleobase;

[0235] each Z 1 and Z 2are independently hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH;

[0236] each Z 3 , Z 4 , and Z n are independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3, or -OCH2CH2OCH3;

[0237] Each Q 1 and Q 4 are independently -CH-, -CH=CH-, -CHO-, -CHS-, -CHCH-, -CHCF-, -CHNH-, -CHNH(CH)-, or -CHN(C(=O)CH)-;

[0238] Each Q 2 and Q 3 are independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-;

[0239] each X 1 , X 2 , X 3 , X 4 , and X n are independently -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3;

[0240] Each Y 1 , Y 2 , Y 3 , Y 4 , and Y n are independently =O, =S, =NH, or =NCH3;

[0241] Each A, A 1 , and A 2 are independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-;

[0242] p is 0, 1, 2, 3, 4, 5, or 6,

[0243] However, (i) Z 1 , Z 2 , and Z 3 is hydrogen, or (ii) Q 1 and Q 4 is -CH2O- or (iii) Q 2 and Q 3 is oxygen or (iv) X 1 , X 2 , X 3 , X 4 , and X n At least one of the following is -SH or -S - or (v) Y 1 , Y 2 , Y 3 , Y 4 , and Y n At least one of A, A 1 , A 2 Described herein are mRNA sequences having a 5'-end region motif of motif (I'), provided that at least one of: is -S-.

[0244] In some embodiments, the mRNA sequence having the 5' end region motif has the structure of motif (I'-a).

[0245] [ka]

[0246] In some embodiments, the mRNA sequence having the 5'-end region motif has the structure of motif (I'-b).

[0247] [ka]

[0248] In some embodiments, the mRNA sequence having the 5' end region motif has the structure of the motif (I'-c).

[0249] [ka]

[0250] In some embodiments, the mRNA sequence having the 5' end region motif has the structure of motif (I'-d).

[0251] [ka]

[0252] In some embodiments, the mRNA sequence having the 5' end region motif has the structure of the motif (I'-e).

[0253] [ka]

[0254] In some embodiments, the mRNA sequence having the 5' end region motif has the structure of the motif (I'-f).

[0255] [ka]

[0256] In some embodiments, the mRNA sequence having a 5' end region motif has the structure of motif (I'-g).

[0257] [ka]

[0258] In some embodiments, the mRNA sequence having a 5' end region motif has the structure of the motif (I'-h).

[0259] [ka]

[0260] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Z 1 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH. 1 is hydrogen. In some embodiments, Z 1 is F. In some embodiments, Z 1 is —OH. In some embodiments, Z 1 is -SH. In some embodiments, Z 1 is -CH3. In some embodiments, Z 1 is -CH2CH3. In some embodiments, Z 1 is —OCH. In some embodiments, Z 1 is —OCH2CH3. In some embodiments, Z 1 is -SCH3. In some embodiments, Z 1 is -NH2. In some embodiments, Z 1 is NHCH. In some embodiments, Z 1 is NHC(=O)CH3.

[0261] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-a), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Z 2is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH. 2 is hydrogen. In some embodiments, Z 2 is F. In some embodiments, Z 2 is —OH. In some embodiments, Z 2 is -SH. In some embodiments, Z 2 is -CH3. In some embodiments, Z 2 is -CH2CH3. In some embodiments, Z 2 is —OCH. In some embodiments, Z 2 is —OCH2CH3. In some embodiments, Z 2 is -SCH3. In some embodiments, Z 2 is -NH2. In some embodiments, Z 2 is NHCH. In some embodiments, Z 2 is NHC(=O)CH3.

[0262] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Z 3 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 3 is hydrogen. In some embodiments, Z 3 is fluorine. In some embodiments, Z 3 is —OH. In some embodiments, Z 3 is -SH. In some embodiments, Z 3 is -CH3. In some embodiments, Z 3is -CH2CH3. In some embodiments, Z 3 is —OCH2OCH3. In some embodiments, Z 3 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 3 is —OCH(CH). In some embodiments, Z 3 is -SCH3. In some embodiments, Z 3 is -OCH2CH2OCH3.

[0263] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Z 4 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 4 is hydrogen. In some embodiments, Z 4 is fluorine. In some embodiments, Z 4 is —OH. In some embodiments, Z 4 is -SH. In some embodiments, Z 4 is -CH3. In some embodiments, Z 4 is -CH2CH3. In some embodiments, Z 4 is —OCH2OCH3. In some embodiments, Z 4 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 4 is —OCH(CH). In some embodiments, Z 4 is -SCH3. In some embodiments, Z 4 is -OCH2CH2OCH3.

[0264] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Z n is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z n is hydrogen. In some embodiments, Z n is fluorine. In some embodiments, Z n is —OH. In some embodiments, Z n is -SH. In some embodiments, Z n is -CH3. In some embodiments, Z n is -CH2CH3. In some embodiments, Z n is —OCH2OCH3. In some embodiments, Z n is —OCH 2 CH 2 CH 3 . In some embodiments, Z n is —OCH(CH). In some embodiments, Z n is -SCH3. In some embodiments, Z n is -OCH2CH2OCH3.

[0265] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), B 2 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 2 is guanine. In some embodiments, B 2 is adenine. In some embodiments, B 2 is cytosine. In some embodiments, B 2 is uracil, and in some embodiments, B 2 is thymine, and in some embodiments, B2 is hypoxanthine. In some embodiments, B 2 is pudding.

[0266] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), B 3 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 3 is guanine. In some embodiments, B 3 is adenine. In some embodiments, B 3 is cytosine. In some embodiments, B 3 is uracil, and in some embodiments, B 3 is thymine, and in some embodiments, B 3 is hypoxanthine. In some embodiments, B 3 is pudding.

[0267] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), B n are independently natural, modified, or non-natural nucleobases. In some embodiments, B n is guanine. In some embodiments, B n is adenine. In some embodiments, B n is cytosine. In some embodiments, B n is uracil, and in some embodiments, B n is thymine, and in some embodiments, B n is hypoxanthine. In some embodiments, B n is pudding.

[0268] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), B 2 , B 3 , and B n At least one of B is adenine. 2 , B 3 , and B n At least one of B is guanine. 2 is adenine. In some embodiments, B 3 is adenine.

[0269] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Q 1 is —CH—, —CH═CH—, —CHO—, —CHS—, —CHCH—, —CHCF—, —CHNH—, —CHNH(CH)—, or —CHN(C(═O)CH)—. In some embodiments, Q 1 In some embodiments, Q is -CH-. 1 is -CH=CH-. In some embodiments, Q 1 is —CH2O—. In some embodiments, Q 1 is -CHS-. In some embodiments, Q 1 is -CHCH-. In some embodiments, Q 1 is —CHCF—. In some embodiments, Q 1 is —CH 2 NH 2 —. In some embodiments, Q 1 is —CHNH(CH)—. In some embodiments, Q 1 is -CH2N(C(=O)CH3)-.

[0270] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Q 4 is —CH—, —CH═CH—, —CHO—, —CHS—, —CHCH—, —CHCF—, —CHNH—, —CHNH(CH)—, or —CHN(C(═O)CH)—. In some embodiments, Q 4 In some embodiments, Q is -CH-. 4 is -CH=CH-. In some embodiments, Q 4 is —CH2O—. In some embodiments, Q 4 is -CHS-. In some embodiments, Q 4 is -CHCH-. In some embodiments, Q 4 is —CHCF—. In some embodiments, Q 4 is —CH 2 NH 2 —. In some embodiments, Q 4 is —CHNH(CH)—. In some embodiments, Q 4 is -CH2N(C(=O)CH3)-.

[0271] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Q 2 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 2 In some embodiments, Q 2 is -S-. In some embodiments, Q 2 In some embodiments, Q is -CH-. 2 In some embodiments, Q is -CF-. 2 In some embodiments, Q is -NH-. 2 is —N(CH)—. In some embodiments, Q 2is -N(C(=O)CH3).

[0272] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Q 3 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 3 In some embodiments, Q 3 is -S-. In some embodiments, Q 3 In some embodiments, Q is -CH-. 3 In some embodiments, Q is -CF-. 3 In some embodiments, Q is -NH-. 3 is —N(CH)—. In some embodiments, Q 3 is -N(C(=O)CH3).

[0273] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), X 1 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 1 is —OH. In some embodiments, X 1 is -SH. In some embodiments, X 1 -O - In some embodiments, X 1 -S - In some embodiments, X 1 is —NH. In some embodiments, X 1 is -NHCH3. In some embodiments, X 1is —NH(C(═O)CH). In some embodiments, X 1 is —CH3. In some embodiments, X 1 is -CH2CH3. In some embodiments, X 1 is -CH2CH2CH3. In some embodiments, X 1 is —CH(CH). In some embodiments, X 1 is —OCH3. In some embodiments, X 1 is -OCH2CH3.

[0274] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), X 2 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 2 is —OH. In some embodiments, X 2 is -SH. In some embodiments, X 2 -O - In some embodiments, X 2 -S - In some embodiments, X 2 is —NH. In some embodiments, X 2 is -NHCH3. In some embodiments, X 2 is —NH(C(═O)CH). In some embodiments, X 2 is —CH3. In some embodiments, X 2 is -CH2CH3. In some embodiments, X 2 is -CH2CH2CH3. In some embodiments, X 2 is —CH(CH). In some embodiments, X 2is —OCH3. In some embodiments, X 2 is -OCH2CH3.

[0275] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), X 3 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 3 is —OH. In some embodiments, X 3 is -SH. In some embodiments, X 3 -O - In some embodiments, X 3 -S - In some embodiments, X 3 is —NH. In some embodiments, X 3 is -NHCH3. In some embodiments, X 3 is —NH(C(═O)CH). In some embodiments, X 3 is —CH3. In some embodiments, X 3 is -CH2CH3. In some embodiments, X 3 is -CH2CH2CH3. In some embodiments, X 3 is —CH(CH). In some embodiments, X 3 is —OCH3. In some embodiments, X 3 is -OCH2CH3.

[0276] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), X 4 -OH, -SH, -O - , -S- , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 4 is —OH. In some embodiments, X 4 is -SH. In some embodiments, X 4 -O - In some embodiments, X 4 -S - In some embodiments, X 4 is —NH. In some embodiments, X 4 is -NHCH3. In some embodiments, X 4 is —NH(C(═O)CH). In some embodiments, X 4 is —CH3. In some embodiments, X 4 is -CH2CH3. In some embodiments, X 4 is -CH2CH2CH3. In some embodiments, X 4 is —CH(CH). In some embodiments, X 4 is —OCH3. In some embodiments, X 4 is -OCH2CH3.

[0277] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), X n -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X n is —OH. In some embodiments, X n is -SH. In some embodiments, X n -O - In some embodiments, Xn -S - In some embodiments, X n is —NH. In some embodiments, X n is -NHCH3. In some embodiments, X n is —NH(C(═O)CH). In some embodiments, X n is —CH3. In some embodiments, X n is -CH2CH3. In some embodiments, X n is -CH2CH2CH3. In some embodiments, X n is —CH(CH). In some embodiments, X n is —OCH3. In some embodiments, X n is -OCH2CH3.

[0278] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Y 1 is =O, =S, =NH, or =NCH3. In some embodiments, Y 1 is ═O. In some embodiments, Y 1 is =S. In some embodiments, Y 1 is ═NH. In some embodiments, Y 1 is =NHCH3.

[0279] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Y 2 is =O, =S, =NH, or =NCH3. In some embodiments, Y 2 is ═O. In some embodiments, Y 2 is =S. In some embodiments, Y 2 is ═NH. In some embodiments, Y 2 is =NHCH3.

[0280] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Y 3 is =O, =S, =NH, or =NCH3. In some embodiments, Y 3 is ═O. In some embodiments, Y 3 is =S. In some embodiments, Y 3 is ═NH. In some embodiments, Y 3 is =NHCH3.

[0281] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Y 4 is =O, =S, =NH, or =NCH3. In some embodiments, Y 4 is ═O. In some embodiments, Y 4 is =S. In some embodiments, Y 4 is ═NH. In some embodiments, Y 4 is =NHCH3.

[0282] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), Y n is =O, =S, =NH, or =NCH3. In some embodiments, Y n is ═O. In some embodiments, Y n is =S. In some embodiments, Y n is ═NH. In some embodiments, Y n is =NHCH3.

[0283] In some embodiments of a compound of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), A is -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH2-. In some embodiments, A is -NH-. In some embodiments, A is -N(CH3)-. In some embodiments, A is -N(C(=O)CH3)-.

[0284] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), A 1 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 1 is —O—. In some embodiments, A 1 is -S-. In some embodiments, A 1 is -CH2-. In some embodiments, A 1 is -NH-. In some embodiments, A 1 is —N(CH)—. In some embodiments, A 1 is -N(C(=O)CH3)-.

[0285] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), A 2 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 2 is —O—. In some embodiments, A 2 is -S-. In some embodiments, A 2 is -CH2-. In some embodiments, A2 is -NH-. In some embodiments, A 2 is —N(CH)—. In some embodiments, A 2 is -N(C(=O)CH3)-.

[0286] In some embodiments of compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), p is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

[0287] In some embodiments of the compounds of motif (I′), X 1 , X 2 , X 3 , X 4 , and X n At least one of is -SH.

[0288] In some embodiments of the compounds of motif (I′), X 1 , X 2 , X 3 , X 4 , and X n At least one of is -S-.

[0289] In some embodiments of the compounds of motif (I′), Y 1 , Y 2 , Y 3 , Y 4 , and Y n At least one of them is =S.

[0290] In some embodiments of the compounds of motif (I'), A, A 1 , and A 2 At least one of is -S-.

[0291] In some embodiments of compounds of motif (I'), Z 1 , Z 2 , Z 3 , Z 4 , and Z n At least one of Z is -OCH. 3 is —OCH. In some embodiments, Z 3 and Z 1 is —OCH 3 . In some embodiments of compounds of motif (I′), Z 1 , Z 2 , Z 3 , Z 4 , and Z n In some embodiments, at least one of Z 1 , Z 2 , and Z 4 is —OH. In some embodiments, Z 2 and Z 4 is -OH.

[0292] In some embodiments of the compounds of motif (I′), Q 1 , Q 2 , Q 3 , and Q 4 In some embodiments of the compound of Formula (I), at least one of Q 1 , Q 2 , Q 3 , and Q 4 In some embodiments, at least one of Q 1 and Q 4 is —OCH. In some embodiments, Q 2 and Q 3 is -O-.

[0293] In some embodiments of the compounds of motif (I′), Y 1 , Y 2 , Y 3 , Y 4 , and Y n In some embodiments, at least one of Y1 , Y 2 , Y 3 , and Y 4 is ═O. In some embodiments, Y 1 , Y 2 , Y 3 , Y 4 , and Y n is =S. In some embodiments, Y 2 is =S. In some embodiments, Y 4 is =S.

[0294] In some embodiments of the compounds of motif (I′), X 1 , X 2 , X 3 , X 4 , and X n At least one of the -O - In some embodiments, X 1 , X 2 , X 3 , and X 4 -O - In some embodiments, X 1 -S - In some embodiments, X 2 -S - In some embodiments, X 3 -S - In some embodiments, X 4 -S - is.

[0295] In some embodiments of the compounds of motif (I'), A, A 1 , and A 2 In some embodiments, at least one of A, A 1 , and A 2 is -O-.

[0296] In one aspect, there is provided an mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof, [ka] During the ceremony, B 1 but, [ka] and Each B 2 , B 3 , and B n is independently a natural, modified, or non-natural nucleobase; each Z 1 and Z' is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), or -SCH3; each Z 2 and Z" is independently fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3, or NHC(=O)CH3; Z"' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3, or -OCH2CH3; each Z 3 , Z 4 , and Z n are independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3; Each Q 1 and Q 4 are independently -CH=CH-, -CH2-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; Each Q 2 and Q 3 are independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X 1 , X 2 , X 3 , X4 , and X n are independently -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; Each Y 1 , Y 2 , Y 3 , Y 4 , and Y n are independently =O, =S, =NH, or =NCH3; Each A, A 1 , and A 2 are independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are mRNA sequence initiators comprising a compound of formula (II), or a salt or solvate thereof, wherein p is 0, 1, 2, 3, 4, 5, or 6.

[0297] In some embodiments, a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u) is a salt. In some embodiments, the salt is an alkali metal salt. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is an ammonium salt.

[0298] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), B 2are independently natural, modified, or non-natural nucleobases. In some embodiments, B 2 is adenine. In some embodiments, B 2 is guanine. In some embodiments, B 2 is cytosine. In some embodiments, B 2 is uracil, and in some embodiments, B 2 is thymine, and in some embodiments, B 2 is hypoxanthine. In some embodiments, B 2 is pudding.

[0299] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), B 3 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 3 is adenine. In some embodiments, B 3 is guanine. In some embodiments, B 3 is cytosine. In some embodiments, B 3 is uracil, and in some embodiments, B 3 is thymine, and in some embodiments, B 3 is hypoxanthine. In some embodiments, B 3 is pudding.

[0300] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), B nare independently natural, modified, or non-natural nucleobases. In some embodiments, B n is adenine. In some embodiments, B n is guanine. In some embodiments, B n is cytosine. In some embodiments, B n is uracil, and in some embodiments, B n is thymine, and in some embodiments, B n is hypoxanthine. In some embodiments, B n is pudding.

[0301] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Z 1 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH(CH), —NH, —NH(C(═O)CH), or —SCH. In some embodiments, Z 1 is hydrogen. In some embodiments, Z 1 is fluorine. In some embodiments, Z 1 is —OH. In some embodiments, Z 1 is -SH. In some embodiments, Z 1 is -CH3. In some embodiments, Z 1 is -CH2CH3. In some embodiments, Z 1 is —OCH. In some embodiments, Z 1 is —NH(CH), and in some embodiments, Z 1 is -NH-. In some embodiments, Z 1 is —NH(C(═O)CH). In some embodiments, Z 1 is -SCH3.

[0302] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Z' is hydrogen, fluorine, -OH, -SH, -CH, -CHCH, -OCH, -NH(CH), -NH, -NH(C(=O)CH), or -SCH. In some embodiments, Z' is hydrogen. In some embodiments, Z' is fluorine. In some embodiments, Z' is -OH. In some embodiments, Z' is -SH. In some embodiments, Z' is -CH. In some embodiments, Z' is -CHCH. In some embodiments, Z' is -OCH. In some embodiments, Z' is -NH(CH), and in some embodiments, Z' is -NH-. In some embodiments, Z' is -NH(C(=O)CH). In some embodiments, Z' is -SCH.

[0303] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Z 2 is fluorine, —OH, —SH, —CH, —CHCH, —OCH, —SCH, —OCHCH, —NH, NHCH, or NHC(═O)CH. 2 is fluorine. In some embodiments, Z 2 is —OH. In some embodiments, Z 2 is -SH. In some embodiments, Z 2 is -CH3. In some embodiments, Z2 is -CH2CH3. In some embodiments, Z 2 is —OCH. In some embodiments, Z 2 is -SCH3. In some embodiments, Z 2 is —OCH2CH3. In some embodiments, Z 2 is -NH2. In some embodiments, Z 2 is NHCH. In some embodiments, Z 2 is NHC(=O)CH3.

[0304] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Z" is fluorine, -OH, -SH, -CH, -CHCH, -OCH, -SCH, -OCHCH, -NH, NHCH, or NHC(=O)CH. In some embodiments, Z" is fluorine. In some embodiments, Z" is -OH. In some embodiments, Z" is -SH. In some embodiments, Z" is -CH3. In some embodiments, Z" is -CH2CH3. In some embodiments, Z" is -OCH3. In some embodiments, Z" is -SCH3. In some embodiments, Z" is -OCH2CH3. In some embodiments, Z" is -NH2. In some embodiments, Z" is NHCH3. In some embodiments, Z" is NHC(=O)CH3.

[0305] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Z"' is hydrogen, fluorine, -CH, -CHCH, -OCH, or -OCHCH. In some embodiments, Z"' is hydrogen. In some embodiments, Z"' is fluorine. In some embodiments, Z"' is -CH. In some embodiments, Z"' is -CHCH. In some embodiments, Z"' is -OCH. In some embodiments, Z"' is -OCH2CH3.

[0306] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Z 3 is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 3 is hydrogen. In some embodiments, Z 3 is fluorine. In some embodiments, Z 3 is —OH. In some embodiments, Z 3 is -CH3. In some embodiments, Z 3 is -CH2CH3. In some embodiments, Z 3 is —OCH. In some embodiments, Z 3 is -NH2. In some embodiments, Z 3is -NHCH3. In some embodiments, Z 3 is —NH(C(═O)CH). In some embodiments, Z 3 is —OCH2CH3. In some embodiments, Z 3 is —OCH2OCH3. In some embodiments, Z 3 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 3 is —OCH(CH). In some embodiments, Z 3 is -SCH3. In some embodiments, Z 3 is -OCH2CH2OCH3.

[0307] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Z 4 is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 4 is hydrogen. In some embodiments, Z 4 is fluorine. In some embodiments, Z 4 is —OH. In some embodiments, Z 4 is -CH3. In some embodiments, Z 4 is -CH2CH3. In some embodiments, Z 4 is —OCH. In some embodiments, Z 4 is -NH2. In some embodiments, Z 4 is -NHCH3. In some embodiments, Z 4 is —NH(C(═O)CH). In some embodiments, Z4 is —OCH2CH3. In some embodiments, Z 4 is —OCH2OCH3. In some embodiments, Z 4 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 4 is —OCH(CH). In some embodiments, Z 4 is -SCH3. In some embodiments, Z 4 is -OCH2CH2OCH3.

[0308] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Z n is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z n is hydrogen. In some embodiments, Z n is fluorine. In some embodiments, Z n is —OH. In some embodiments, Z n is -CH3. In some embodiments, Z n is -CH2CH3. In some embodiments, Z n is —OCH. In some embodiments, Z n is -NH2. In some embodiments, Z n is -NHCH3. In some embodiments, Z n is —NH(C(═O)CH). In some embodiments, Z n is —OCH2CH3. In some embodiments, Z nis —OCH2OCH3. In some embodiments, Z n is —OCH 2 CH 2 CH 3 . In some embodiments, Z n is —OCH(CH). In some embodiments, Z n is -SCH3. In some embodiments, Z n is -OCH2CH2OCH3.

[0309] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Q 1 is -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 In some embodiments, Q is -CH-. 1 is —CH2O—. In some embodiments, Q 1 is -CHS-. In some embodiments, Q 1 is -CHCH-. In some embodiments, Q 1 is —CHCF—. In some embodiments, Q 1 is —CH 2 NH 2 —. In some embodiments, Q 1 is —CHNH(CH)—. In some embodiments, Q 1 is -CH2N(C(=O)CH3)-.

[0310] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Q 4 is -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 4 In some embodiments, Q is -CH-. 4 is —CH2O—. In some embodiments, Q 4 is -CHS-. In some embodiments, Q 4 is -CHCH-. In some embodiments, Q 4 is —CHCF—. In some embodiments, Q 4 is —CH 2 NH 2 —. In some embodiments, Q 4 is —CHNH(CH)—. In some embodiments, Q 4 is -CH2N(C(=O)CH3)-.

[0311] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Q 2 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 2 In some embodiments, Q 2is -S-. In some embodiments, Q 2 In some embodiments, Q is -CH-. 2 In some embodiments, Q is -CF-. 2 In some embodiments, Q is -NH-. 2 is —N(CH)—. In some embodiments, Q 2 is -N(C(=O)CH3)-.

[0312] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Q 3 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 3 In some embodiments, Q 3 is -S-. In some embodiments, Q 3 In some embodiments, Q is -CH-. 3 In some embodiments, Q is -CF-. 3 In some embodiments, Q is -NH-. 3 is —N(CH)—. In some embodiments, Q 3 is -N(C(=O)CH3)-.

[0313] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), X 1 -OH, -SH, -O - , -S- , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 1 is —OH. In some embodiments, X 1 is -SH. In some embodiments, X 1 -O - In some embodiments, X 1 -S - In some embodiments, X 1 is —NH. In some embodiments, X 1 is -NHCH3. In some embodiments, X 1 is —NH(C(═O)CH). In some embodiments, X 1 is —CH3. In some embodiments, X 1 is -CH2CH3. In some embodiments, X 1 is -CH2CH2CH3. In some embodiments, X 1 is —CH(CH). In some embodiments, X 1 is —OCH3. In some embodiments, X 1 is -OCH2CH3.

[0314] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), X 2 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 2is —OH. In some embodiments, X 2 is -SH. In some embodiments, X 2 -O - In some embodiments, X 2 -S - In some embodiments, X 2 is —NH. In some embodiments, X 2 is -NHCH3. In some embodiments, X 2 is —NH(C(═O)CH). In some embodiments, X 2 is —CH3. In some embodiments, X 2 is -CH2CH3. In some embodiments, X 2 is -CH2CH2CH3. In some embodiments, X 2 is —CH(CH). In some embodiments, X 2 is —OCH3. In some embodiments, X 2 is -OCH2CH3.

[0315] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), X 3 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 3 is —OH. In some embodiments, X 3 is -SH. In some embodiments, X 3 -O - In some embodiments, X 3 -S -In some embodiments, X 3 is —NH. In some embodiments, X 3 is -NHCH3. In some embodiments, X 3 is —NH(C(═O)CH). In some embodiments, X 3 is —CH3. In some embodiments, X 3 is -CH2CH3. In some embodiments, X 3 is -CH2CH2CH3. In some embodiments, X 3 is —CH(CH). In some embodiments, X 3 is —OCH3. In some embodiments, X 3 is -OCH2CH3.

[0316] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), X 4 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 4 is —OH. In some embodiments, X 4 is -SH. In some embodiments, X 4 -O - In some embodiments, X 4 -S - In some embodiments, X 4 is —NH. In some embodiments, X 4 is -NHCH3. In some embodiments, X 4 is —NH(C(═O)CH). In some embodiments, X4 is —CH3. In some embodiments, X 4 is -CH2CH3. In some embodiments, X 4 is -CH2CH2CH3. In some embodiments, X 4 is —CH(CH). In some embodiments, X 4 is —OCH3. In some embodiments, X 4 is -OCH2CH3.

[0317] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), X n -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X n is —OH. In some embodiments, X n is -SH. In some embodiments, X n -O - In some embodiments, X n -S - In some embodiments, X n is —NH. In some embodiments, X n is -NHCH3. In some embodiments, X n is —NH(C(═O)CH). In some embodiments, X n is —CH3. In some embodiments, X n is -CH2CH3. In some embodiments, X n is -CH2CH2CH3. In some embodiments, X nis —CH(CH). In some embodiments, X n is —OCH3. In some embodiments, X n is -OCH2CH3.

[0318] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Y 1 is =O, =S, =NH, or =NCH3. In some embodiments, Y 1 is ═O. In some embodiments, Y 1 is =S. In some embodiments, Y 1 is ═NH. In some embodiments, Y 1 is =NCH3.

[0319] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Y 2 is =O, =S, =NH, or =NCH3. In some embodiments, Y 2 is ═O. In some embodiments, Y 2 is =S. In some embodiments, Y 2 is ═NH. In some embodiments, Y 2 is =NCH3.

[0320] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Y 3 is =O, =S, =NH, or =NCH3. In some embodiments, Y 3 is ═O. In some embodiments, Y 3 is =S. In some embodiments, Y 3 is ═NH. In some embodiments, Y 3 is =NCH3.

[0321] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Y 4 is =O, =S, =NH, or =NCH3. In some embodiments, Y 4 is ═O. In some embodiments, Y 4 is =S. In some embodiments, Y 4 is ═NH. In some embodiments, Y 4 is =NCH3.

[0322] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), Y n is =O, =S, =NH, or =NCH3. In some embodiments, Yn is ═O. In some embodiments, Y n is =S. In some embodiments, Y n is ═NH. In some embodiments, Y n is =NCH3.

[0323] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), A is -O-, -S-, -CH-, -NH-, -N(CH)-, or -N(C(=O)CH)-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH-. In some embodiments, A is -NH-. In some embodiments, A is -N(CH)-. In some embodiments, A is —N(C(═O)CH 3 )—.

[0324] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), A 1 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 1 is —O—. In some embodiments, A 1 is -S-. In some embodiments, A 1 is -CH2-. In some embodiments, A 1 is -NH-. In some embodiments, A 1is —N(CH)—. In some embodiments, A 1 is -N(C(=O)CH3)-.

[0325] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), A 2 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 2 is —O—. In some embodiments, A 2 is -S-. In some embodiments, A 2 is -CH2-. In some embodiments, A 2 is -NH-. In some embodiments, A 2 is —N(CH)—. In some embodiments, A 2 is -N(C(=O)CH3)-.

[0326] In some embodiments of a compound of Formula (II), (II-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-l), (II-m), (II-n), (II-o), (II-p), (II-q), (II-r), (II-s), (II-t), or (II-u), p is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

[0327] In some embodiments, B 1 teeth, [ka] In some embodiments, B 1 teeth, [ka] In some embodiments, each B 2 and B 3 are independently adenine or guanine. In some embodiments, B 2 is adenine. In some embodiments, B 3 is guanine.

[0328] In some embodiments, each Z 1 , Z 2 , Z 3 , and Z 4 is independently —OH or —OCH. In some embodiments, Z 3 is —OCH. In some embodiments, Z 1 is —OCH. In some embodiments, Z 2 is —OH. In some embodiments, Z 1 , Z 2 , and Z 4 is -OH and Z 3 is —OCH. In some embodiments, Z 1 and Z 3 is -OCH3, and Z 2 and Z 4 is —OH. In some embodiments, Z′, Z″, and Z′″ are hydrogen.

[0329] In some embodiments, Q 1 and Q 4 is -OCH2-. In some embodiments, Q 2 and Q 3 is -O-.

[0330] In some embodiments, X 1 , X 2 , X 3 , and X4 is —O—. In some embodiments, X 1 , X 2 , X 3 , and X 4 is -S-. In some embodiments, X 1 , X 3 , X 4 is -O- and X 2 is -S-.

[0331] In some embodiments, Y 1 , Y 2 , Y 3 , and Y 4 is ═O. In some embodiments, Y 1 , Y 2 , Y 3 , and Y 4 is =S. In some embodiments, Y 1 , Y 3 , and Y 4 is = O and Y 2 is =S. In some embodiments, Y 1 , Y 2 , Y 3 is = O and Y 4 is =S.

[0332] In some embodiments, A, A 1 , and A 2 is -O-.

[0333] In some embodiments, p is 0.

[0334] In one embodiment, an mRNA sequence having a 5' end region motif (motif (II'), [ka] During the ceremony, B 1 but, [ka] and Each B 2 , B3 , and B n is independently a natural, modified, or non-natural nucleobase; each Z 1 and Z' is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), or -SCH3; each Z 2 and Z" is independently fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3, or NHC(=O)CH3; Z"' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3, or -OCH2CH3; each Z 3 , Z 4 , and Z n are independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3; Each Q 1 and Q 4 are independently -CH=CH-, -CH2-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; Each Q 2 and Q 3 are independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X 1 , X 2 , X 3 , X 4 , and X n are independently -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; Each Y1 , Y 2 , Y 3 , Y 4 , and Y n are independently =O, =S, =NH, or =NCH3; Each A, A 1 , and A 2 are independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are mRNA sequences having a 5' end region motif (motif (II')) in which p is 0, 1, 2, 3, 4, 5, or 6.

[0335] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), B 2 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 2 is adenine. In some embodiments, B 2 is guanine. In some embodiments, B 2 is cytosine. In some embodiments, B 2 is uracil, and in some embodiments, B 2 is thymine, and in some embodiments, B 2 is hypoxanthine. In some embodiments, B 2 is pudding.

[0336] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), B 3 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 3 is adenine. In some embodiments, B 3 is guanine. In some embodiments, B 3 is cytosine. In some embodiments, B 3 is uracil, and in some embodiments, B 3 is thymine, and in some embodiments, B 3 is hypoxanthine. In some embodiments, B 3 is pudding.

[0337] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), B n are independently natural, modified, or non-natural nucleobases. In some embodiments, B n is adenine. In some embodiments, B n is guanine. In some embodiments, B n is cytosine. In some embodiments, B n is uracil, and in some embodiments, B n is thymine, and in some embodiments, B n is hypoxanthine. In some embodiments, B nis pudding.

[0338] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Z 1 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH(CH), —NH, —NH(C(═O)CH), or —SCH. In some embodiments, Z 1 is hydrogen. In some embodiments, Z 1 is fluorine. In some embodiments, Z 1 is —OH. In some embodiments, Z 1 is -SH. In some embodiments, Z 1 is -CH3. In some embodiments, Z 1 is -CH2CH3. In some embodiments, Z 1 is —OCH. In some embodiments, Z 1 is —NH(CH), and in some embodiments, Z 1 is -NH-. In some embodiments, Z 1 is —NH(C(═O)CH). In some embodiments, Z 1 is -SCH3.

[0339] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Z' is hydrogen, fluorine, -OH, -SH, -CH, -CHCH, -OCH, -NH(CH), -NH, -NH(C(=O)CH), or -SCH. In some embodiments, Z' is hydrogen. In some embodiments, Z' is fluorine. In some embodiments, Z' is -OH. In some embodiments, Z' is -SH. In some embodiments, Z' is -CH. In some embodiments, Z' is -CHCH. In some embodiments, Z' is -OCH. In some embodiments, Z' is -NH(CH), and in some embodiments, Z' is -NH-. In some embodiments, Z' is -NH(C(=O)CH). In some embodiments, Z' is -SCH.

[0340] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Z 2 is fluorine, —OH, —SH, —CH, —CHCH, —OCH, —SCH, —OCHCH, —NH, NHCH, or NHC(═O)CH. 2 is fluorine. In some embodiments, Z 2 is —OH. In some embodiments, Z 2 is -SH. In some embodiments, Z 2is -CH3. In some embodiments, Z 2 is -CH2CH3. In some embodiments, Z 2 is —OCH. In some embodiments, Z 2 is -SCH3. In some embodiments, Z 2 is —OCH2CH3. In some embodiments, Z 2 is -NH2. In some embodiments, Z 2 is NHCH. In some embodiments, Z 2 is NHC(=O)CH3.

[0341] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Z" is fluorine, -OH, -SH, -CH, -CHCH, -OCH, -SCH, -OCHCH, -NH, NHCH, or NHC(=O)CH. In some embodiments, Z" is fluorine. In some embodiments, Z" is -OH. In some embodiments, Z" is -SH. In some embodiments, Z" is -CH3. In some embodiments, Z" is -CH2CH3. In some embodiments, Z" is -OCH3. In some embodiments, Z" is -SCH3. In some embodiments, Z" is -OCH2CH3. In some embodiments, Z" is -NH2. In some embodiments, Z" is NHCH3. In some embodiments, Z" is NHC(=O)CH3.

[0342] In some embodiments of a compound of motif (II′), (II′-a), (II′-b), (II′-c), (II′-d), (II′-e), (II′-f), (II′-g), (II′-h), (II′-i), (II′-j), (II′-k), (II′-l), (II′-m), (II′-n), (II′-o), (II′-p), (II′-q), (II′-r), (II′-s), (II′-t), or (II′-u), Z′″ is hydrogen, fluorine, —CH, —CHCH, —OCH, or —OCHCH. In some embodiments, Z′″ is hydrogen. In some embodiments, Z′″ is fluorine. In some embodiments, Z′″ is —CH. In some embodiments, Z"' is -CH2CH3. In some embodiments, Z"' is -OCH3. In some embodiments, Z"' is -OCH2CH3.

[0343] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Z 3 is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 3 is hydrogen. In some embodiments, Z 3 is fluorine. In some embodiments, Z 3 is —OH. In some embodiments, Z 3 is -CH3. In some embodiments, Z 3 is -CH2CH3. In some embodiments, Z 3 is —OCH. In some embodiments, Z3 is -NH2. In some embodiments, Z 3 is -NHCH3. In some embodiments, Z 3 is —NH(C(═O)CH). In some embodiments, Z 3 is —OCH2CH3. In some embodiments, Z 3 is —OCH2OCH3. In some embodiments, Z 3 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 3 is —OCH(CH). In some embodiments, Z 3 is -SCH3. In some embodiments, Z 3 is -OCH2CH2OCH3.

[0344] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Z 4 is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 4 is hydrogen. In some embodiments, Z 4 is fluorine. In some embodiments, Z 4 is —OH. In some embodiments, Z 4 is -CH3. In some embodiments, Z 4 is -CH2CH3. In some embodiments, Z 4 is —OCH. In some embodiments, Z 4 is -NH2. In some embodiments, Z 4is -NHCH3. In some embodiments, Z 4 is —NH(C(═O)CH). In some embodiments, Z 4 is —OCH2CH3. In some embodiments, Z 4 is —OCH2OCH3. In some embodiments, Z 4 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 4 is —OCH(CH). In some embodiments, Z 4 is -SCH3. In some embodiments, Z 4 is -OCH2CH2OCH3.

[0345] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Z n is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z n is hydrogen. In some embodiments, Z n is fluorine. In some embodiments, Z n is —OH. In some embodiments, Z n is -CH3. In some embodiments, Z n is -CH2CH3. In some embodiments, Z n is —OCH. In some embodiments, Z n is -NH2. In some embodiments, Z n is -NHCH3. In some embodiments, Z nis —NH(C(═O)CH). In some embodiments, Z n is —OCH2CH3. In some embodiments, Z n is —OCH2OCH3. In some embodiments, Z n is —OCH 2 CH 2 CH 3 . In some embodiments, Z n is —OCH(CH). In some embodiments, Z n is -SCH3. In some embodiments, Z n is -OCH2CH2OCH3.

[0346] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Q 1 is -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 In some embodiments, Q is -CH-. 1 is —CH2O—. In some embodiments, Q 1 is -CHS-. In some embodiments, Q 1 is -CHCH-. In some embodiments, Q 1 is —CHCF—. In some embodiments, Q 1 is —CH 2 NH 2 —. In some embodiments, Q 1 is —CHNH(CH)—. In some embodiments, Q 1 is -CH2N(C(=O)CH3)-.

[0347] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Q 4 is -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 4 In some embodiments, Q is -CH-. 4 is —CH2O—. In some embodiments, Q 4 is -CHS-. In some embodiments, Q 4 is -CHCH-. In some embodiments, Q 4 is —CHCF—. In some embodiments, Q 4 is —CH 2 NH 2 —. In some embodiments, Q 4 is —CHNH(CH)—. In some embodiments, Q 4 is -CH2N(C(=O)CH3)-.

[0348] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Q 2 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q2 In some embodiments, Q 2 is -S-. In some embodiments, Q 2 In some embodiments, Q is -CH-. 2 In some embodiments, Q is -CF-. 2 In some embodiments, Q is -NH-. 2 is —N(CH)—. In some embodiments, Q 2 is -N(C(=O)CH3)-.

[0349] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Q 3 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 3 In some embodiments, Q 3 is -S-. In some embodiments, Q 3 In some embodiments, Q is -CH-. 3 In some embodiments, Q is -CF-. 3 In some embodiments, Q is -NH-. 3 is —N(CH)—. In some embodiments, Q 3 is -N(C(=O)CH3)-.

[0350] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), X 1 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 1 is —OH. In some embodiments, X 1 is -SH. In some embodiments, X 1 -O - In some embodiments, X 1 -S - In some embodiments, X 1 is —NH. In some embodiments, X 1 is -NHCH3. In some embodiments, X 1 is —NH(C(═O)CH). In some embodiments, X 1 is —CH3. In some embodiments, X 1 is -CH2CH3. In some embodiments, X 1 is -CH2CH2CH3. In some embodiments, X 1 is —CH(CH). In some embodiments, X 1 is —OCH3. In some embodiments, X 1 is -OCH2CH3.

[0351] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), X 2 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 2 is —OH. In some embodiments, X 2 is -SH. In some embodiments, X 2 -O - In some embodiments, X 2 -S - In some embodiments, X 2 is —NH. In some embodiments, X 2 is -NHCH3. In some embodiments, X 2 is —NH(C(═O)CH). In some embodiments, X 2 is —CH3. In some embodiments, X 2 is -CH2CH3. In some embodiments, X 2 is -CH2CH2CH3. In some embodiments, X 2 is —CH(CH). In some embodiments, X 2 is —OCH3. In some embodiments, X 2 is -OCH2CH3.

[0352] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), X 3 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 3 is —OH. In some embodiments, X 3 is -SH. In some embodiments, X 3 -O - In some embodiments, X 3 -S - In some embodiments, X 3 is —NH. In some embodiments, X 3 is -NHCH3. In some embodiments, X 3 is —NH(C(═O)CH). In some embodiments, X 3 is —CH3. In some embodiments, X 3 is -CH2CH3. In some embodiments, X 3 is -CH2CH2CH3. In some embodiments, X 3 is —CH(CH). In some embodiments, X 3 is —OCH3. In some embodiments, X 3 is -OCH2CH3.

[0353] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), X 4 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 4 is —OH. In some embodiments, X 4 is -SH. In some embodiments, X 4 -O - In some embodiments, X 4 -S - In some embodiments, X 4 is —NH. In some embodiments, X 4 is -NHCH3. In some embodiments, X 4 is —NH(C(═O)CH). In some embodiments, X 4 is —CH3. In some embodiments, X 4 is -CH2CH3. In some embodiments, X 4 is -CH2CH2CH3. In some embodiments, X 4 is —CH(CH). In some embodiments, X 4 is —OCH3. In some embodiments, X 4 is -OCH2CH3.

[0354] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), X n -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X n is —OH. In some embodiments, X n is -SH. In some embodiments, X n -O - In some embodiments, X n -S - In some embodiments, X n is —NH. In some embodiments, X n is -NHCH3. In some embodiments, X n is —NH(C(═O)CH). In some embodiments, X n is —CH3. In some embodiments, X n is -CH2CH3. In some embodiments, X n is -CH2CH2CH3. In some embodiments, X n is —CH(CH). In some embodiments, X n is —OCH3. In some embodiments, X n is -OCH2CH3.

[0355] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Y 1 is =O, =S, =NH, or =NCH3. In some embodiments, Y 1 is ═O. In some embodiments, Y 1 is =S. In some embodiments, Y 1 is ═NH. In some embodiments, Y 1 is =NCH3.

[0356] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Y 2 is =O, =S, =NH, or =NCH3. In some embodiments, Y 2 is ═O. In some embodiments, Y 2 is =S. In some embodiments, Y 2 is ═NH. In some embodiments, Y 2 is =NCH3.

[0357] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Y 3 is =O, =S, =NH, or =NCH3. In some embodiments, Y 3 is ═O. In some embodiments, Y 3 is =S. In some embodiments, Y 3 is ═NH. In some embodiments, Y 3 is =NCH3.

[0358] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Y 4 is =O, =S, =NH, or =NCH3. In some embodiments, Y 4 is ═O. In some embodiments, Y 4 is =S. In some embodiments, Y 4 is ═NH. In some embodiments, Y 4 is =NCH3.

[0359] In some embodiments of compounds of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), Y n is =O, =S, =NH, or =NCH3. In some embodiments, Y n is ═O. In some embodiments, Y n is =S. In some embodiments, Y n is ═NH. In some embodiments, Y n is =NCH3.

[0360] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), A is -O-, -S-, -CH-, -NH-, -N(CH)-, or -N(C(=O)CH)-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH-. In some embodiments, A is -NH-. In some embodiments, A is -N(CH3)-. In some embodiments, A is -N(C(=O)CH3)-.

[0361] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), A 1 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 1 is —O—. In some embodiments, A 1 is -S-. In some embodiments, A 1 is -CH2-. In some embodiments, A 1 is -NH-. In some embodiments, A 1 is —N(CH)—. In some embodiments, A 1 is -N(C(=O)CH3)-.

[0362] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), A 2 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 2 is —O—. In some embodiments, A 2 is -S-. In some embodiments, A 2 is -CH2-. In some embodiments, A 2 is -NH-. In some embodiments, A 2 is —N(CH)—. In some embodiments, A 2is -N(C(=O)CH3)-.

[0363] In some embodiments of a compound of motif (II'), (II'-a), (II'-b), (II'-c), (II'-d), (II'-e), (II'-f), (II'-g), (II'-h), (II'-i), (II'-j), (II'-k), (II'-l), (II'-m), (II'-n), (II'-o), (II'-p), (II'-q), (II'-r), (II'-s), (II'-t), or (II'-u), p is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

[0364] In some embodiments, B 1 teeth, [ka] In some embodiments, each B 2 and B 3 are independently adenine or guanine. In some embodiments, B 2 is adenine. In some embodiments, B 3 is guanine.

[0365] In some embodiments, each Z 1 , Z 2 , Z 3 , and Z 4 is independently —OH or —OCH. In some embodiments, Z 3 is —OCH. In some embodiments, Z 1 is —OCH. In some embodiments, Z 2 is —OH. In some embodiments, Z 1 , Z 2 , and Z 4 is -OH and Z3 is —OCH. In some embodiments, Z 1 and Z 3 is -OCH3, and Z 2 and Z 4 is —OH. In some embodiments, Z′, Z″, and Z′″ are hydrogen.

[0366] In some embodiments, Q 1 and Q 4 is -OCH2-. In some embodiments, Q 2 and Q 3 is -O-.

[0367] In some embodiments, X 1 , X 2 , X 3 , and X 4 is —O—. In some embodiments, X 1 , X 2 , X 3 , and X 4 is -S-. In some embodiments, X 1 , X 3 , X 4 is -O- and X 2 is -S-.

[0368] In some embodiments, Y 1 , Y 2 , Y 3 , and Y 4 is ═O. In some embodiments, Y 1 , Y 2 , Y 3 , and Y 4 is =S. In some embodiments, Y 1 , Y 3 , and Y 4 is = O and Y 2 is =S. In some embodiments, Y 1 , Y 2 , Y 3 is = O and Y 4 is =S.

[0369] In some embodiments, A, A1 , and A 2 is -O-.

[0370] In some embodiments, p is 0.

[0371] In one aspect, there is provided an mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof, [ka] During the ceremony, B 1 but, [ka] and Each B 2 , B 3 , and B n is independently a natural, modified, or non-natural nucleobase; each Z' and Z" is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), or -SCH3; Z"' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3, or -OCH2CH3; each Z 1 and Z 2 are independently hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —SCH, —OCHCH, —NH, NHCH, or NHC(═O)CH; each Z 3 , Z 4 , and Z n are independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3; Each Q 1 and Q 4are independently -CH=CH-, -CH2-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; Each Q 2 and Q 3 are independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X 1 , X 2 , X 3 , X 4 , and X n are independently -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; Each Y 1 , Y 2 , Y 3 , Y 4 , and Y n are independently =O, =S, =NH, or =NCH3; Each A, A 1 , and A 2 are independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are mRNA sequence initiators comprising a compound of formula (II), or a salt or solvate thereof, wherein p is 0, 1, 2, 3, 4, 5, or 6.

[0372] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), B 1 teeth, [ka] In some embodiments, B 1 teeth, [ka] In some embodiments, B 1 teeth, [ka] In some embodiments, B 1 teeth, [ka] is.

[0373] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), B 2 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 2 is adenine. In some embodiments, B 2 is guanine. In some embodiments, B 2 is cytosine. In some embodiments, B 2 is uracil, and in some embodiments, B 2 is thymine, and in some embodiments, B 2 is hypoxanthine. In some embodiments, B 2 is pudding.

[0374] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), B 3 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 3 is adenine. In some embodiments, B 3 is guanine. In some embodiments, B 3 is cytosine. In some embodiments, B 3 is uracil, and in some embodiments, B 3 is thymine, and in some embodiments, B 3 is hypoxanthine. In some embodiments, B 3 is pudding.

[0375] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), B n are independently natural, modified, or non-natural nucleobases. In some embodiments, B n is adenine. In some embodiments, B n is guanine. In some embodiments, B n is cytosine. In some embodiments, B n is uracil, and in some embodiments, B n is thymine, and in some embodiments, B n is hypoxanthine. In some embodiments, B n is pudding.

[0376] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Z' is hydrogen, fluorine, -OH, -SH, -CH, -CHCH, -OCH, -NH(CH), -NH, -NH(C(=O)CH), or -SCH. In some embodiments, Z' is hydrogen. In some embodiments, Z' is fluorine. In some embodiments, Z' is -OH. In some embodiments, Z' is -SH. In some embodiments, Z' is -CH. In some embodiments, Z' is -CHCH. In some embodiments, Z' is -OCH. In some embodiments, Z' is -NH(CH), and in some embodiments, Z' is -NH-. In some embodiments, Z' is -NH(C(=O)CH). In some embodiments, Z' is -SCH.

[0377] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Z" is hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), or -SCH3. In some embodiments, Z" is hydrogen. In some embodiments, Z" is fluorine. In some embodiments, Z" is -OH. In some embodiments, Z" is -SH. In some embodiments, Z" is -CH. In some embodiments, Z" is -CHCH. In some embodiments, Z" is -OCH. In some embodiments, Z" is -NH(CH), and in some embodiments, Z" is -NH-. In some embodiments, Z" is -NH(C(=O)CH). In some embodiments, Z" is -SCH.

[0378] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Z"' is hydrogen, fluorine, -CH, -CHCH, -OCH, or -OCHCH. In some embodiments, Z"' is hydrogen. In some embodiments, Z"' is fluorine. In some embodiments, Z"' is -CH. In some embodiments, Z"' is -CH2CH3. In some embodiments, Z"' is -OCH3. In some embodiments, Z"' is -OCH2CH3.

[0379] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Z 1 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —SCH, —OCHCH, —NH, NHCH, or NHC(═O)CH. 1 is hydrogen. In some embodiments, Z 1 is fluorine. In some embodiments, Z 1 is —OH. In some embodiments, Z 1 is -SH. In some embodiments, Z 1 is -CH3. In some embodiments, Z 1 is -CH2CH3. In some embodiments, Z 1 is —OCH. In some embodiments, Z 1 is -SCH3. In some embodiments, Z 1 is —OCH2CH3. In some embodiments, Z 1 is -NH2. In some embodiments, Z 1 is NHCH. In some embodiments, Z 1 is NHC(=O)CH3.

[0380] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Z 2is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —SCH, —OCHCH, —NH, NHCH, or NHC(═O)CH. 2 is hydrogen. In some embodiments, Z 2 is fluorine. In some embodiments, Z 2 is —OH. In some embodiments, Z 2 is -SH. In some embodiments, Z 2 is -CH3. In some embodiments, Z 2 is -CH2CH3. In some embodiments, Z 2 is —OCH. In some embodiments, Z 2 is -SCH3. In some embodiments, Z 2 is —OCH2CH3. In some embodiments, Z 2 is -NH2. In some embodiments, Z 2 is NHCH. In some embodiments, Z 2 is NHC(=O)CH3.

[0381] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Z 3 is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 3 is hydrogen. In some embodiments, Z 3 is fluorine. In some embodiments, Z 3is —OH. In some embodiments, Z 3 is -CH3. In some embodiments, Z 3 is -CH2CH3. In some embodiments, Z 3 is —OCH. In some embodiments, Z 3 is -NH2. In some embodiments, Z 3 is -NHCH3. In some embodiments, Z 3 is —NH(C(═O)CH). In some embodiments, Z 3 is —OCH2CH3. In some embodiments, Z 3 is —OCH2OCH3. In some embodiments, Z 3 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 3 is —OCH(CH). In some embodiments, Z 3 is -SCH3. In some embodiments, Z 3 is -OCH2CH2OCH3.

[0382] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Z 4 is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 4 is hydrogen. In some embodiments, Z 4 is fluorine. In some embodiments, Z 4 is —OH. In some embodiments, Z 4is -CH3. In some embodiments, Z 4 is -CH2CH3. In some embodiments, Z 4 is —OCH. In some embodiments, Z 4 is -NH2. In some embodiments, Z 4 is -NHCH3. In some embodiments, Z 4 is —NH(C(═O)CH). In some embodiments, Z 4 is —OCH2CH3. In some embodiments, Z 4 is —OCH2OCH3. In some embodiments, Z 4 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 4 is —OCH(CH). In some embodiments, Z 4 is -SCH3. In some embodiments, Z 4 is -OCH2CH2OCH3.

[0383] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Z n is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z n is hydrogen. In some embodiments, Z n is fluorine. In some embodiments, Z n is —OH. In some embodiments, Z n is -CH3. In some embodiments, Z nis -CH2CH3. In some embodiments, Z n is —OCH. In some embodiments, Z n is -NH2. In some embodiments, Z n is -NHCH3. In some embodiments, Z n is —NH(C(═O)CH). In some embodiments, Z n is —OCH2CH3. In some embodiments, Z n is —OCH2OCH3. In some embodiments, Z n is —OCH 2 CH 2 CH 3 . In some embodiments, Z n is —OCH(CH). In some embodiments, Z n is -SCH3. In some embodiments, Z n is -OCH2CH2OCH3.

[0384] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Q 1 is -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 In some embodiments, Q is -CH-. 1 is —CH2O—. In some embodiments, Q 1 is -CHS-. In some embodiments, Q 1 is -CHCH-. In some embodiments, Q 1 is —CHCF—. In some embodiments, Q1 is —CH 2 NH 2 —. In some embodiments, Q 1 is —CHNH(CH)—. In some embodiments, Q 1 is -CH2N(C(=O)CH3)-.

[0385] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Q 4 is -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 4 In some embodiments, Q is -CH-. 4 is —CH2O—. In some embodiments, Q 4 is -CHS-. In some embodiments, Q 4 is -CHCH-. In some embodiments, Q 4 is —CHCF—. In some embodiments, Q 4 is —CH 2 NH 2 —. In some embodiments, Q 4 is —CHNH(CH)—. In some embodiments, Q 4 is -CH2N(C(=O)CH3)-.

[0386] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Q 2 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 2 In some embodiments, Q 2 is -S-. In some embodiments, Q 2 In some embodiments, Q is -CH-. 2 In some embodiments, Q is -CF-. 2 In some embodiments, Q is -NH-. 2 is —N(CH)—. In some embodiments, Q 2 is -N(C(=O)CH3)-.

[0387] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), Q 3 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 3 In some embodiments, Q 3 is -S-. In some embodiments, Q 3 In some embodiments, Q is -CH-. 3 In some embodiments, Q is -CF-. 3In some embodiments, Q is -NH-. 3 is —N(CH)—. In some embodiments, Q 3 is -N(C(=O)CH3)-.

[0388] In some embodiments of a compound of Formula (II), (II-a′), (II-b′), (II-c′), (II-d′), (II-e′), (II-f′), (II-g′), (II-h′), (II-i′), (II-j′), (II-k′), (II-l′), (II-m′), (II-n′), (II-o′), (II-p′), (II-q′), (II-r′), (II-s′), (II-t′), or (II-u′), X 1 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 1 is —OH. In some embodiments, X 1 is -SH. In some embodiments, X 1 -O - In some embodiments, X 1 -S - In some embodiments, X 1 is —NH. In some embodiments, X 1 is -NHCH3. In some embodiments, X 1 is —NH(C(═O)CH). In some embodiments, X 1 is —CH3. In some embodiments, X 1 is -CH2CH3. In some embodiments, X 1 is -CH2CH2CH3. In some embodiments, X 1 is —CH(CH). In some embodiments, X 1 is —OCH3. In some embodiments, X 1 is -OCH2CH3.

[0389] In some embodiments of a compound of Formula (II), (II-a′), (II-b′), (II-c′), (II-d′), (II-e′), (II-f′), (II-g′), (II-h′), (II-i′), (II-j′), (II-k′), (II-l′), (II-m′), (II-n′), (II-o′), (II-p′), (II-q′), (II-r′), (II-s′), (II-t′), or (II-u′), X 2 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 2 is —OH. In some embodiments, X 2 is -SH. In some embodiments, X 2 -O - In some embodiments, X 2 -S - In some embodiments, X 2 is —NH. In some embodiments, X 2 is -NHCH3. In some embodiments, X 2 is —NH(C(═O)CH). In some embodiments, X 2 is —CH3. In some embodiments, X 2 is -CH2CH3. In some embodiments, X 2 is -CH2CH2CH3. In some embodiments, X 2 is —CH(CH). In some embodiments, X 2 is —OCH3. In some embodiments, X 2 is -OCH2CH3.

[0390] In some embodiments of a compound of Formula (II), (II-a′), (II-b′), (II-c′), (II-d′), (II-e′), (II-f′), (II-g′), (II-h′), (II-i′), (II-j′), (II-k′), (II-l′), (II-m′), (II-n′), (II-o′), (II-p′), (II-q′), (II-r′), (II-s′), (II-t′), or (II-u′), X 3 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 3 is —OH. In some embodiments, X 3 is -SH. In some embodiments, X 3 -O - In some embodiments, X 3 -S - In some embodiments, X 3 is —NH. In some embodiments, X 3 is -NHCH3. In some embodiments, X 3 is —NH(C(═O)CH). In some embodiments, X 3 is —CH3. In some embodiments, X 3 is -CH2CH3. In some embodiments, X 3 is -CH2CH2CH3. In some embodiments, X 3 is —CH(CH). In some embodiments, X 3 is —OCH3. In some embodiments, X 3 is -OCH2CH3.

[0391] In some embodiments of a compound of Formula (II), (II-a′), (II-b′), (II-c′), (II-d′), (II-e′), (II-f′), (II-g′), (II-h′), (II-i′), (II-j′), (II-k′), (II-l′), (II-m′), (II-n′), (II-o′), (II-p′), (II-q′), (II-r′), (II-s′), (II-t′), or (II-u′), X 4 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 4 is —OH. In some embodiments, X 4 is -SH. In some embodiments, X 4 -O - In some embodiments, X 4 -S - In some embodiments, X 4 is —NH. In some embodiments, X 4 is -NHCH3. In some embodiments, X 4 is —NH(C(═O)CH). In some embodiments, X 4 is —CH3. In some embodiments, X 4 is -CH2CH3. In some embodiments, X 4 is -CH2CH2CH3. In some embodiments, X 4 is —CH(CH). In some embodiments, X 4 is —OCH3. In some embodiments, X 4 is -OCH2CH3.

[0392] In some embodiments of a compound of Formula (II), (II-a′), (II-b′), (II-c′), (II-d′), (II-e′), (II-f′), (II-g′), (II-h′), (II-i′), (II-j′), (II-k′), (II-l′), (II-m′), (II-n′), (II-o′), (II-p′), (II-q′), (II-r′), (II-s′), (II-t′), or (II-u′), X n -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X n is —OH. In some embodiments, X n is -SH. In some embodiments, X n -O - In some embodiments, X n -S - In some embodiments, X n is —NH. In some embodiments, X n is -NHCH3. In some embodiments, X n is —NH(C(═O)CH). In some embodiments, X n is —CH3. In some embodiments, X n is -CH2CH3. In some embodiments, X n is -CH2CH2CH3. In some embodiments, X n is —CH(CH). In some embodiments, X n is —OCH3. In some embodiments, X n is -OCH2CH3.

[0393] In some embodiments of a compound of Formula (II), (II-a′), (II-b′), (II-c′), (II-d′), (II-e′), (II-f′), (II-g′), (II-h′), (II-i′), (II-j′), (II-k′), (II-l′), (II-m′), (II-n′), (II-o′), (II-p′), (II-q′), (II-r′), (II-s′), (II-t′), or (II-u′), Y 1 is =O, =S, =NH, or =NCH3. In some embodiments, Y 1 is ═O. In some embodiments, Y 1 is =S. In some embodiments, Y 1 is ═NH. In some embodiments, Y 1 is =NCH3.

[0394] In some embodiments of a compound of Formula (II), (II-a′), (II-b′), (II-c′), (II-d′), (II-e′), (II-f′), (II-g′), (II-h′), (II-i′), (II-j′), (II-k′), (II-l′), (II-m′), (II-n′), (II-o′), (II-p′), (II-q′), (II-r′), (II-s′), (II-t′), or (II-u′), Y 2 is =O, =S, =NH, or =NCH3. In some embodiments, Y 2 is ═O. In some embodiments, Y 2 is =S. In some embodiments, Y 2 is ═NH. In some embodiments, Y 2 is =NCH3.

[0395] In some embodiments of a compound of Formula (II), (II-a′), (II-b′), (II-c′), (II-d′), (II-e′), (II-f′), (II-g′), (II-h′), (II-i′), (II-j′), (II-k′), (II-l′), (II-m′), (II-n′), (II-o′), (II-p′), (II-q′), (II-r′), (II-s′), (II-t′), or (II-u′), Y 3 is =O, =S, =NH, or =NCH3. In some embodiments, Y 3 is ═O. In some embodiments, Y 3 is =S. In some embodiments, Y 3 is ═NH. In some embodiments, Y 3 is =NCH3.

[0396] In some embodiments of a compound of Formula (II), (II-a′), (II-b′), (II-c′), (II-d′), (II-e′), (II-f′), (II-g′), (II-h′), (II-i′), (II-j′), (II-k′), (II-l′), (II-m′), (II-n′), (II-o′), (II-p′), (II-q′), (II-r′), (II-s′), (II-t′), or (II-u′), Y 4 is =O, =S, =NH, or =NCH3. In some embodiments, Y 4 is ═O. In some embodiments, Y 4 is =S. In some embodiments, Y 4 is ═NH. In some embodiments, Y 4 is =NCH3.

[0397] In some embodiments of a compound of Formula (II), (II-a′), (II-b′), (II-c′), (II-d′), (II-e′), (II-f′), (II-g′), (II-h′), (II-i′), (II-j′), (II-k′), (II-l′), (II-m′), (II-n′), (II-o′), (II-p′), (II-q′), (II-r′), (II-s′), (II-t′), or (II-u′), Y n is =O, =S, =NH, or =NCH3. In some embodiments, Y n is ═O. In some embodiments, Y n is =S. In some embodiments, Y n is ═NH. In some embodiments, Y n is =NCH3.

[0398] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), A is -O-, -S-, -CH-, -NH-, -N(CH)-, or -N(C(=O)CH)-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH-. In some embodiments, A is -NH-. In some embodiments, A is -N(CH3)-. In some embodiments, A is -N(C(=O)CH3)-.

[0399] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), A 1 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 1 is —O—. In some embodiments, A 1 is -S-. In some embodiments, A 1 is -CH2-. In some embodiments, A 1 is -NH-. In some embodiments, A 1 is —N(CH)—. In some embodiments, A 1 is -N(C(=O)CH3)-.

[0400] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), A 2 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 2 is —O—. In some embodiments, A 2 is -S-. In some embodiments, A 2 is -CH2-. In some embodiments, A 2 is -NH-. In some embodiments, A 2 is —N(CH)—. In some embodiments, A 2is -N(C(=O)CH3)-.

[0401] In some embodiments of a compound of Formula (II), (II-a'), (II-b'), (II-c'), (II-d'), (II-e'), (II-f'), (II-g'), (II-h'), (II-i'), (II-j'), (II-k'), (II-l'), (II-m'), (II-n'), (II-o'), (II-p'), (II-q'), (II-r'), (II-s'), (II-t'), or (II-u'), p is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

[0402] In some embodiments, B 1 teeth, [ka] In some embodiments, each B 2 and B 3 are independently adenine or guanine. In some embodiments, B 2 is adenine. In some embodiments, B 3 is guanine.

[0403] In some embodiments, each Z 1 , Z 2 , Z 3 , and Z 4 is independently —OH or —OCH. In some embodiments, Z 3 is —OCH. In some embodiments, Z 1 is —OCH. In some embodiments, Z 2 is —OH. In some embodiments, Z 1 , Z 2 , and Z 4 is -OH and Z3 is —OCH. In some embodiments, Z 1 and Z 3 is -OCH3, and Z 2 and Z 4 is —OH. In some embodiments, Z′, Z″, and Z′″ are hydrogen.

[0404] In some embodiments, Q 1 and Q 4 is -OCH2-. In some embodiments, Q 2 and Q 3 is -O-.

[0405] In some embodiments, X 1 , X 2 , X 3 , and X 4 is —O—. In some embodiments, X 1 , X 2 , X 3 , and X 4 is -S-. In some embodiments, X 1 , X 3 , X 4 is -O- and X 2 is -S-.

[0406] In some embodiments, Y 1 , Y 2 , Y 3 , and Y 4 is ═O. In some embodiments, Y 1 , Y 2 , Y 3 , and Y 4 is =S. In some embodiments, Y 1 , Y 3 , and Y 4 is = O and Y 2 is =S. In some embodiments, Y 1 , Y 2 , Y 3 is = O and Y 4 is =S.

[0407] In some embodiments, A, A1 , and A 2 is -O-.

[0408] In some embodiments, p is 0.

[0409] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-a): [ka]

[0410] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-a'): [ka]

[0411] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-b): [ka]

[0412] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-b'): [ka]

[0413] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-c): [ka]

[0414] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-c'): [ka]

[0415] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-d): [ka]

[0416] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-d'): [ka]

[0417] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-e): [ka]

[0418] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-e'): [ka]

[0419] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-f): [ka]

[0420] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-g): [ka]

[0421] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-g'): [ka]

[0422] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-h): [ka]

[0423] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-h'): [ka]

[0424] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-i): [ka]

[0425] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-I'): [ka]

[0426] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-j): [ka]

[0427] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-j'): [ka]

[0428] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-k): [ka]

[0429] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-k'): [ka]

[0430] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-1): [ka]

[0431] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-l'): [ka]

[0432] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-m): [ka]

[0433] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-m'): [ka]

[0434] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-n): [ka]

[0435] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-n'): [ka]

[0436] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-o): [ka]

[0437] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-o'): [ka]

[0438] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-p): [ka]

[0439] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-p'): [ka]

[0440] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-q): [ka]

[0441] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-q'): [ka]

[0442] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-r): [ka]

[0443] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-r'): [ka]

[0444] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-s): [ka]

[0445] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-s'): [ka]

[0446] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-t): [ka]

[0447] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-t'): [ka]

[0448] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-u): [ka]

[0449] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-u'): [ka]

[0450] In some embodiments of Formula (II) or Formulas (II-a) through (II-u'), each B is independently a natural nucleobase. In some embodiments of Formula (II) or Formulas (II-a) through (II-u'), each B is independently a modified nucleobase. In some embodiments of Formula (II) or Formulas (II-a) through (II-u'), each B is independently a non-natural nucleobase.

[0451] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), B 1 teeth, [ka] is.

[0452] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), B 1 teeth, is.

[0453] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), B 1 teeth, [ka] is.

[0454] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Z 1 is fluorine.

[0455] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Z 1 is -OH.

[0456] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Z 1 is -OCH3.

[0457] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Z 2 is fluorine.

[0458] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Z 2 is -OH.

[0459] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Z 2 is -OCH3.

[0460] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 1 is -CH2O-.

[0461] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 1 is -O-.

[0462] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 4 is -CH2O-.

[0463] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 4 is -O-.

[0464] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 2 is -O-.

[0465] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 3 is -O-.

[0466] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Y 1 is =O.

[0467] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Y 3 is =O.

[0468] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Y 2 is =O.

[0469] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Y 4 is =O.

[0470] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Y n is =O.

[0471] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Y 1 , Y 2 , Y 3 , Y 4 , and Y n At least one of is =S.

[0472] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Y 2 is =S.

[0473] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Y 4 is =S.

[0474] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), Y n is =S.

[0475] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), each Y 1 , Y 2 , Y 3 , Y 4 , and Y n is =O.

[0476] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), each X 1 , X 4 , and X n -O - is.

[0477] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), X 2 is -O-.

[0478] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), X 3 -O - is.

[0479] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), X 1 , X 2 , X 3 , X 4 , and X n At least one of the is -S - is.

[0480] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), X 2 -S - is.

[0481] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), X 4 -S - is.

[0482] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), X 3 is -S-.

[0483] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), each X 1 , X 2 , X 3 , X 4 , and X n -O - is.

[0484] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), each A, A 1 , and A 2 is -O-.

[0485] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), A, A 1 , and A 2 In some embodiments, one or more of A is -S-. In some embodiments, A is -S-. 1 is —O—. In some embodiments, A 2 is —O—. In some embodiments, A 2 In some embodiments, A is -S-. In some embodiments, A is -O-. In some embodiments, A 1 is -O-.

[0486] In some embodiments of Formula (II) or Formulas (II-a) through (II-u'), p is 0. In some embodiments, p is 1. In some embodiments, p is 2.

[0487] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), B 2is adenine, cytosine, guanine, uracil, thymine, hypoxanthine, or purine.

[0488] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), B 3 is adenine, cytosine, guanine, uracil, thymine, hypoxanthine, or purine.

[0489] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), B n is adenine, cytosine, guanine, uracil, thymine, hypoxanthine, or purine.

[0490] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 1 is —CH2O—. In some embodiments, Q 4 is -CH2O-.

[0491] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 2 In some embodiments, Q 3 is -O-.

[0492] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), each X n are independently -OH, -SH, O - , or S - is.

[0493] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), each Y n are independently ═O or ═S.

[0494] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), B 1 teeth, [ka] is.

[0495] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 1 is -CH2O-, and Q 4 is -CH2O-, and Q 2 is -O- and Q 3 is -O-, and each X n are independently -OH, -SH, O - , or S - and each Y n are independently =O or =S, and B 1 teeth [ka] is.

[0496] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 1 is -CH2O-, and Q 4 is -CH2O-, and Q 2 is -O- and Q 3 is -O-, and each X n are independently -OH, -SH, O - , or S - and each Y n are independently =O or =S, and B 1 teeth [ka] is.

[0497] In some embodiments of Formula (II) or Formulas (II-a) through (II-u′), Q 1 is -CH2O-, and Q 4 is -CH2O-, and Q 2 is -O- and Q 3 is -O-, and each X n are independently -OH, -SH, O - , or S - and each Y n are independently =O or =S, and B 1 teeth [ka] is.

[0498] In some embodiments of Formula (II) or Formulas (II-a) to (II-u′), B 2 is adenine. In some embodiments of Formula (II) or Formulas (II-a) through (II-u'), B 3 is guanine.

[0499] In one embodiment, an mRNA sequence having a 5' end region motif (motif (II') [ka] During the ceremony, B 1 but, [ka] and Each B 2 , B 3 , and B n is independently a natural, modified, or non-natural nucleobase; each Z' and Z" is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3), or -SCH3; Z"' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3, or -OCH2CH3; each Z 1 and Z 2 are independently hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —SCH, —OCHCH, —NH, NHCH, or NHC(═O)CH; each Z 3 , Z 4 , and Z nare independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3; Each Q 1 and Q 4 are independently -CH=CH-, -CH2-, -CHO-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-; Each Q 2 and Q 3 are independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X 1 , X 2 , X 3 , X 4 , and X n are independently -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; Each Y 1 , Y 2 , Y 3 , Y 4 , and Y n are independently =O, =S, =NH, or =NCH3; Each A, A 1 , and A 2 are independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are mRNA sequences having a 5' end region motif (motif (II')) in which p is 0, 1, 2, 3, 4, 5, or 6.

[0500] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), B 1 teeth, [ka] In some embodiments, B 1 teeth, [ka] In some embodiments, B 1 teeth, [ka] In some embodiments, B 1 teeth, [ka] is.

[0501] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), B 2 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 2 is adenine. In some embodiments, B 2 is guanine. In some embodiments, B 2is cytosine. In some embodiments, B 2 is uracil, and in some embodiments, B 2 is thymine, and in some embodiments, B 2 is hypoxanthine. In some embodiments, B 2 is pudding.

[0502] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), B 3 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 3 is adenine. In some embodiments, B 3 is guanine. In some embodiments, B 3 is cytosine. In some embodiments, B 3 is uracil, and in some embodiments, B 3 is thymine, and in some embodiments, B 3 is hypoxanthine. In some embodiments, B 3 is pudding.

[0503] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), B nare independently natural, modified, or non-natural nucleobases. In some embodiments, B n is adenine. In some embodiments, B n is guanine. In some embodiments, B n is cytosine. In some embodiments, B n is uracil, and in some embodiments, B n is thymine, and in some embodiments, B n is hypoxanthine. In some embodiments, B n is pudding.

[0504] In some embodiments of compounds of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Z' is hydrogen, fluorine, -OH, -SH, -CH, -CHCH, -OCH, -NH(CH), -NH, -NH(C(=O)CH), or -SCH. In some embodiments, Z' is hydrogen. In some embodiments, Z' is fluorine. In some embodiments, Z' is -OH. In some embodiments, Z' is -SH. In some embodiments, Z' is -CH. In some embodiments, Z' is -CHCH. In some embodiments, Z' is -OCH. In some embodiments, Z' is -NH(CH), and in some embodiments, Z' is -NH-. In some embodiments, Z' is -NH(C(=O)CH). In some embodiments, Z' is -SCH.

[0505] Motif (II”), (II”-a’), (II”-b’), (II”-c’), (II”-d’), (II”-e’), (II”-f’), (II”-g’), (II”-h '), (II”-i'), (II”-j'), (II”-k'), (II”-l'), (II”-m'), (II”-n'), (II”-o'), (II”-p'), (II” In some embodiments of the compound of (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Z" is hydrogen, fluorine, -OH, -SH, -CH, -CHCH, -OCH, -NH(CH), -NH, -NH(C(=O)CH), or -SCH. In some embodiments, Z" is hydrogen. In some embodiments, Z" is fluorine. In some embodiments, Z" is -OH. In some embodiments, Z" is -SH. In some embodiments, Z" is -CH. In some embodiments, Z" is -CHCH. In some embodiments, Z" is -OCH. In some embodiments, Z" is -NH(CH), and in some embodiments, Z" is -NH-. In some embodiments, Z" is -NH(C(=O)CH). In some embodiments, Z" is -SCH.

[0506] In some embodiments of compounds of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Z"' is hydrogen, fluorine, -CH, -CHCH, -OCH, or -OCHCH. In some embodiments, Z"' is hydrogen. In some embodiments, Z"' is fluorine. In some embodiments, Z"' is -CH3. In some embodiments, Z"' is -CH2CH3. In some embodiments, Z"' is -OCH3. In some embodiments, Z"' is -OCH2CH3.

[0507] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Z 1 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —SCH, —OCHCH, —NH, NHCH, or NHC(═O)CH. 1 is hydrogen. In some embodiments, Z 1 is fluorine. In some embodiments, Z 1 is —OH. In some embodiments, Z 1 is -SH. In some embodiments, Z 1 is -CH3. In some embodiments, Z 1 is -CH2CH3. In some embodiments, Z1 is —OCH. In some embodiments, Z 1 is -SCH3. In some embodiments, Z 1 is —OCH2CH3. In some embodiments, Z 1 is -NH2. In some embodiments, Z 1 is NHCH. In some embodiments, Z 1 is NHC(=O)CH3.

[0508] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Z 2 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —SCH, —OCHCH, —NH, NHCH, or NHC(═O)CH. 2 is hydrogen. In some embodiments, Z 2 is fluorine. In some embodiments, Z 2 is —OH. In some embodiments, Z 2 is -SH. In some embodiments, Z 2 is -CH3. In some embodiments, Z 2 is -CH2CH3. In some embodiments, Z 2 is —OCH. In some embodiments, Z 2 is -SCH3. In some embodiments, Z 2 is —OCH2CH3. In some embodiments, Z 2 is -NH2. In some embodiments, Z 2 is NHCH. In some embodiments, Z 2is NHC(=O)CH3.

[0509] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Z 3 is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 3 is hydrogen. In some embodiments, Z 3 is fluorine. In some embodiments, Z 3 is —OH. In some embodiments, Z 3 is -CH3. In some embodiments, Z 3 is -CH2CH3. In some embodiments, Z 3 is —OCH. In some embodiments, Z 3 is -NH2. In some embodiments, Z 3 is -NHCH3. In some embodiments, Z 3 is —NH(C(═O)CH). In some embodiments, Z 3 is —OCH2CH3. In some embodiments, Z 3 is —OCH2OCH3. In some embodiments, Z 3 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 3 is —OCH(CH). In some embodiments, Z 3 is -SCH3. In some embodiments, Z 3 is -OCH2CH2OCH3.

[0510] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Z 4 is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 4 is hydrogen. In some embodiments, Z 4 is fluorine. In some embodiments, Z 4 is —OH. In some embodiments, Z 4 is -CH3. In some embodiments, Z 4 is -CH2CH3. In some embodiments, Z 4 is —OCH. In some embodiments, Z 4 is -NH2. In some embodiments, Z 4 is -NHCH3. In some embodiments, Z 4 is —NH(C(═O)CH). In some embodiments, Z 4 is —OCH2CH3. In some embodiments, Z 4 is —OCH2OCH3. In some embodiments, Z 4 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 4 is —OCH(CH). In some embodiments, Z 4 is -SCH3. In some embodiments, Z 4 is -OCH2CH2OCH3.

[0511] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Z n is hydrogen, fluorine, —OH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z n is hydrogen. In some embodiments, Z n is fluorine. In some embodiments, Z n is —OH. In some embodiments, Z n is -CH3. In some embodiments, Z n is -CH2CH3. In some embodiments, Z n is —OCH. In some embodiments, Z n is -NH2. In some embodiments, Z n is -NHCH3. In some embodiments, Z n is —NH(C(═O)CH). In some embodiments, Z n is —OCH2CH3. In some embodiments, Z n is —OCH2OCH3. In some embodiments, Z n is —OCH 2 CH 2 CH 3 . In some embodiments, Z n is —OCH(CH). In some embodiments, Z n is -SCH3. In some embodiments, Z n is -OCH2CH2OCH3.

[0512] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Q 1 is -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 In some embodiments, Q is -CH-. 1 is —CH2O—. In some embodiments, Q 1 is -CHS-. In some embodiments, Q 1 is -CHCH-. In some embodiments, Q 1 is —CHCF—. In some embodiments, Q 1 is —CH 2 NH 2 —. In some embodiments, Q 1 is —CHNH(CH)—. In some embodiments, Q 1 is -CH2N(C(=O)CH3)-.

[0513] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Q 4is -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)-, or -CH2N(C(=O)CH3)-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 4 In some embodiments, Q is -CH-. 4 is —CH2O—. In some embodiments, Q 4 is -CHS-. In some embodiments, Q 4 is -CHCH-. In some embodiments, Q 4 is —CHCF—. In some embodiments, Q 4 is —CH 2 NH 2 —. In some embodiments, Q 4 is —CHNH(CH)—. In some embodiments, Q 4 is -CH2N(C(=O)CH3)-.

[0514] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Q 2 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 2 In some embodiments, Q 2 is -S-. In some embodiments, Q 2 In some embodiments, Q is -CH-. 2 In some embodiments, Q is -CF-. 2 In some embodiments, Q is -NH-. 2is —N(CH)—. In some embodiments, Q 2 is -N(C(=O)CH3)-.

[0515] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Q 3 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 3 In some embodiments, Q 3 is -S-. In some embodiments, Q 3 In some embodiments, Q is -CH-. 3 In some embodiments, Q is -CF-. 3 In some embodiments, Q is -NH-. 3 is —N(CH)—. In some embodiments, Q 3 is -N(C(=O)CH3)-.

[0516] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), X 1 -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 1 is —OH. In some embodiments, X 1 is -SH. In some embodiments, X 1 -O - In some embodiments, X 1 -S - In some embodiments, X 1 is —NH. In some embodiments, X 1 is -NHCH3. In some embodiments, X 1 is —NH(C(═O)CH). In some embodiments, X 1 is —CH3. In some embodiments, X 1 is -CH2CH3. In some embodiments, X 1 is -CH2CH2CH3. In some embodiments, X 1 is —CH(CH). In some embodiments, X 1 is —OCH3. In some embodiments, X 1 is -OCH2CH3.

[0517] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), X 2 -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 2 is —OH. In some embodiments, X 2 is -SH. In some embodiments, X 2 -O - In some embodiments, X 2 -S - In some embodiments, X 2 is —NH. In some embodiments, X 2 is -NHCH3. In some embodiments, X 2 is —NH(C(═O)CH). In some embodiments, X 2 is —CH3. In some embodiments, X 2 is -CH2CH3. In some embodiments, X 2 is -CH2CH2CH3. In some embodiments, X 2 is —CH(CH). In some embodiments, X 2 is —OCH3. In some embodiments, X 2 is -OCH2CH3.

[0518] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), X 3 -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 3 is —OH. In some embodiments, X 3 is -SH. In some embodiments, X 3 -O - In some embodiments, X 3 -S - In some embodiments, X 3 is —NH. In some embodiments, X 3 is -NHCH3. In some embodiments, X 3 is —NH(C(═O)CH). In some embodiments, X 3 is —CH3. In some embodiments, X 3 is -CH2CH3. In some embodiments, X 3 is -CH2CH2CH3. In some embodiments, X 3 is —CH(CH). In some embodiments, X 3 is —OCH3. In some embodiments, X 3 is -OCH2CH3.

[0519] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), X 4 -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 4 is —OH. In some embodiments, X 4 is -SH. In some embodiments, X 4 -O - In some embodiments, X 4 -S - In some embodiments, X 4 is —NH. In some embodiments, X 4 is -NHCH3. In some embodiments, X 4 is —NH(C(═O)CH). In some embodiments, X 4 is —CH3. In some embodiments, X 4 is -CH2CH3. In some embodiments, X 4 is -CH2CH2CH3. In some embodiments, X 4 is —CH(CH). In some embodiments, X 4 is —OCH3. In some embodiments, X 4 is -OCH2CH3.

[0520] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), X n -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X n is —OH. In some embodiments, X n is -SH. In some embodiments, X n -O - In some embodiments, X n -S - In some embodiments, X n is —NH. In some embodiments, X n is -NHCH3. In some embodiments, X n is —NH(C(═O)CH). In some embodiments, X n is —CH3. In some embodiments, X n is -CH2CH3. In some embodiments, X n is -CH2CH2CH3. In some embodiments, X n is —CH(CH). In some embodiments, X n is —OCH3. In some embodiments, X n is -OCH2CH3.

[0521] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Y 1 is =O, =S, =NH, or =NCH3. In some embodiments, Y 1 is ═O. In some embodiments, Y 1 is =S. In some embodiments, Y 1is ═NH. In some embodiments, Y 1 is =NCH3.

[0522] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Y 2 is =O, =S, =NH, or =NCH3. In some embodiments, Y 2 is ═O. In some embodiments, Y 2 is =S. In some embodiments, Y 2 is ═NH. In some embodiments, Y 2 is =NCH3.

[0523] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Y 3 is =O, =S, =NH, or =NCH3. In some embodiments, Y 3 is ═O. In some embodiments, Y 3 is =S. In some embodiments, Y 3 is ═NH. In some embodiments, Y 3 is =NCH3.

[0524] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Y 4 is =O, =S, =NH, or =NCH3. In some embodiments, Y 4 is ═O. In some embodiments, Y 4 is =S. In some embodiments, Y 4 is ═NH. In some embodiments, Y 4 is =NCH3.

[0525] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), Y n is =O, =S, =NH, or =NCH3. In some embodiments, Y n is ═O. In some embodiments, Y n is =S. In some embodiments, Y n is ═NH. In some embodiments, Y n is =NCH3.

[0526] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), A is -O-, -S-, -CH-, -NH-, -N(CH)-, or -N(C(=O)CH)-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH2-. In some embodiments, A is -NH-. In some embodiments, A is -N(CH3)-. In some embodiments, A is -N(C(=O)CH3)-.

[0527] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), A 1 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 1 is —O—. In some embodiments, A 1 is -S-. In some embodiments, A 1 is -CH2-. In some embodiments, A 1 is -NH-. In some embodiments, A 1 is —N(CH)—. In some embodiments, A 1 is -N(C(=O)CH3)-.

[0528] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), A 2 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 2 is —O—. In some embodiments, A 2 is -S-. In some embodiments, A 2 is -CH2-. In some embodiments, A 2 is -NH-. In some embodiments, A 2 is —N(CH)—. In some embodiments, A 2 is -N(C(=O)CH3)-.

[0529] In some embodiments of a compound of motif (II"), (II"-a'), (II"-b'), (II"-c'), (II"-d'), (II"-e'), (II"-f'), (II"-g'), (II"-h'), (II"-i'), (II"-j'), (II"-k'), (II"-l'), (II"-m'), (II"-n'), (II"-o'), (II"-p'), (II"-q'), (II"-r'), (II"-s'), (II"-t'), or (II"-u'), p is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

[0530] In some embodiments, B 1 teeth, [ka] In some embodiments, each B 2 and B 3 are independently adenine or guanine. In some embodiments, B 2 is adenine. In some embodiments, B 3 is guanine.

[0531] In some embodiments, B 1 teeth, [ka] In some embodiments, each B 2 and B 3 are independently adenine or guanine. In some embodiments, B 2 is adenine. In some embodiments, B 3 is guanine.

[0532] In some embodiments, each Z 1 , Z 2 , Z 3 , and Z 4 is independently —OH or —OCH. In some embodiments, Z 3 is —OCH. In some embodiments, Z 1 is —OCH. In some embodiments, Z 2 is —OH. In some embodiments, Z 1 , Z 2 , and Z 4 is -OH and Z 3 is —OCH. In some embodiments, Z 1 and Z 3 is -OCH3, and Z 2 and Z 4 is —OH. In some embodiments, Z′, Z″, and Z′″ are hydrogen.

[0533] In some embodiments, Q 1 and Q4 is -OCH2-. In some embodiments, Q 2 and Q 3 is -O-.

[0534] In some embodiments, X 1 , X 2 , X 3 , and X 4 is —O—. In some embodiments, X 1 , X 2 , X 3 , and X 4 is -S-. In some embodiments, X 1 , X 3 , X 4 is -O- and X 2 is -S-.

[0535] In some embodiments, Y 1 , Y 2 , Y 3 , and Y 4 is ═O. In some embodiments, Y 1 , Y 2 , Y 3 , and Y 4 is =S. In some embodiments, Y 1 , Y 3 , and Y 4 is = O and Y 2 is =S. In some embodiments, Y 1 , Y 2 , Y 3 is = O and Y 4 is =S.

[0536] In some embodiments, A, A 1 , and A 2 is -O-.

[0537] In some embodiments, p is 0.

[0538] In one aspect there is provided an IVT mRNA sequence initiator of formula (I) or a salt or solvate thereof, [ka] During the ceremony, B 1 but, [ka] and B 2 is a modified or non-natural nucleobase; Each B 3 and B n is independently a natural, modified, or non-natural nucleobase; each Z 1 and Z 2 are independently hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH; each Z 3 , Z 4 , and Z n are independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3, or -OCH2CH2OCH3; Each Q 1 and Q 4 are independently -CH-, -CH=CH-, -CHO-, -CHS-, -CHCH-, -CHCF-, -CHNH-, -CHNH(CH)-, or -CHN(C(=O)CH)-; Each Q 2 and Q 3 are independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X 1 , X 2 , X 3 , X 4 , and X n are independently -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; Each Y 1 , Y 2 , Y 3 , Y 4 , and Y n are independently =O, =S, =NH, or =NCH3; Each A, A 1 , and A 2 are independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are IVT mRNA sequence initiators of formula (I), or salts or solvates thereof, wherein p is 0, 1, 2, 3, 4, 5, or 6.

[0539] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (III-a): [ka]

[0540] In some embodiments of compounds of Formula (III) or (III-a), Z 1 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH. 1 is hydrogen. In some embodiments, Z 1 is F. In some embodiments, Z 1 is —OH. In some embodiments, Z 1 is -SH. In some embodiments, Z 1 is -CH3. In some embodiments, Z 1 is -CH2CH3. In some embodiments, Z 1 is —OCH. In some embodiments, Z 1 is —OCH2CH3. In some embodiments, Z1 is -SCH3. In some embodiments, Z 1 is -NH2. In some embodiments, Z 1 is NHCH. In some embodiments, Z 1 is NHC(=O)CH3.

[0541] In some embodiments of compounds of Formula (III) or (III-a), Z 2 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH. 2 is hydrogen. In some embodiments, Z 2 is F. In some embodiments, Z 2 is —OH. In some embodiments, Z 2 is -SH. In some embodiments, Z 2 is -CH3. In some embodiments, Z 2 is -CH2CH3. In some embodiments, Z 2 is —OCH. In some embodiments, Z 2 is —OCH2CH3. In some embodiments, Z 2 is -SCH3. In some embodiments, Z 2 is -NH2. In some embodiments, Z 2 is NHCH. In some embodiments, Z 2 is NHC(=O)CH3.

[0542] In some embodiments of compounds of Formula (III) or (III-a), Z 3 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 3 is hydrogen. In some embodiments, Z3 is fluorine. In some embodiments, Z 3 is —OH. In some embodiments, Z 3 is -SH. In some embodiments, Z 3 is -CH3. In some embodiments, Z 3 is -CH2CH3. In some embodiments, Z 3 is —OCH2OCH3. In some embodiments, Z 3 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 3 is —OCH(CH). In some embodiments, Z 3 is -SCH3. In some embodiments, Z 3 is -OCH2CH2OCH3.

[0543] In some embodiments of compounds (III) or (III-a), Z 4 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 4 is hydrogen. In some embodiments, Z 4 is fluorine. In some embodiments, Z 4 is —OH. In some embodiments, Z 4 is -SH. In some embodiments, Z 4 is -CH3. In some embodiments, Z 4 is -CH2CH3. In some embodiments, Z 4 is —OCH2OCH3. In some embodiments, Z 4 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 4 is —OCH(CH). In some embodiments, Z 4 is -SCH3. In some embodiments, Z 4is -OCH2CH2OCH3.

[0544] In some embodiments of compounds of Formula (III) or (III-a), Z n is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z n is hydrogen. In some embodiments, Z n is fluorine. In some embodiments, Z n is —OH. In some embodiments, Z n is -SH. In some embodiments, Z n is -CH3. In some embodiments, Z n is -CH2CH3. In some embodiments, Z n is —OCH2OCH3. In some embodiments, Z n is —OCH 2 CH 2 CH 3 . In some embodiments, Z n is —OCH(CH). In some embodiments, Z n is -SCH3. In some embodiments, Z n is -OCH2CH2OCH3.

[0545] In some embodiments of the compound of Formula (III) or (III-a), B 2 is a modified or non-natural nucleobase. In some embodiments, B 2 is a modified guanine. In some embodiments, B 2 is a modified adenine. In some embodiments, B 2 is a modified cytosine. In some embodiments, B 2 is a modified uracil, and in some embodiments, B 2 is a modified thymine, and in some embodiments, B 2 is a modified hypoxanthine. In some embodiments, B2 is a modified purine. In some embodiments, B 2 is 2-aminoadenine. In some embodiments, B 2 teeth, [ka] is.

[0546] In some embodiments of the compound of Formula (III) or (III-a), B 3 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 3 is guanine. In some embodiments, B 3 is adenine. In some embodiments, B 3 is cytosine. In some embodiments, B 3 is uracil, and in some embodiments, B 3 is thymine, and in some embodiments, B 3 is hypoxanthine. In some embodiments, B 3 is pudding.

[0547] In some embodiments of the compound of Formula (III) or (III-a), B n are independently natural, modified, or non-natural nucleobases. In some embodiments, B n is guanine. In some embodiments, B n is adenine. In some embodiments, B n is cytosine. In some embodiments, B n is uracil, and in some embodiments, B n is thymine, and in some embodiments, B n is hypoxanthine. In some embodiments, B n is pudding.

[0548] In some embodiments of the compound of Formula (III) or (III-a), B 2 is a modified adenine. In some embodiments, B3 is guanine.

[0549] In some embodiments of compounds of Formula (III) or (III-a), Q 1 is —CH—, —CH═CH—, —CHO—, —CHS—, —CHCH—, —CHCF—, —CHNH—, —CHNH(CH)—, or —CHN(C(═O)CH)—. In some embodiments, Q 1 In some embodiments, Q is -CH-. 1 is -CH=CH-. In some embodiments, Q 1 is —CH2O—. In some embodiments, Q 1 is -CHS-. In some embodiments, Q 1 is -CHCH-. In some embodiments, Q 1 is —CHCF—. In some embodiments, Q 1 is —CH 2 NH 2 —. In some embodiments, Q 1 is —CHNH(CH)—. In some embodiments, Q 1 is -CH2N(C(=O)CH3)-.

[0550] In some embodiments of compounds of Formula (III) or (III-a), Q 4 is —CH—, —CH═CH—, —CHO—, —CHS—, —CHCH—, —CHCF—, —CHNH—, —CHNH(CH)—, or —CHN(C(═O)CH)—. In some embodiments, Q 4 In some embodiments, Q is -CH-. 4 is -CH=CH-. In some embodiments, Q 4 is —CH2O—. In some embodiments, Q 4 is -CHS-. In some embodiments, Q 4 is -CHCH-. In some embodiments, Q 4 is —CHCF—. In some embodiments, Q 4is —CH 2 NH 2 —. In some embodiments, Q 4 is —CHNH(CH)—. In some embodiments, Q 4 is -CH2N(C(=O)CH3)-.

[0551] In some embodiments of compounds of Formula (III) or (III-a), Q 2 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 2 is —O—. In some embodiments, Q 2 is -S-. In some embodiments, Q 2 In some embodiments, Q is -CH-. 2 In some embodiments, Q is -CF-. 2 In some embodiments, Q is -NH-. 2 is —N(CH)—. In some embodiments, Q 2 is -N(C(=O)CH3).

[0552] In some embodiments of compounds of Formula (III) or (III-a), Q 3 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 3 is —O—. In some embodiments, Q 3 is -S-. In some embodiments, Q 3 In some embodiments, Q is -CH-. 3 In some embodiments, Q is -CF-. 3 In some embodiments, Q is -NH-. 3 is —N(CH)—. In some embodiments, Q 3 is -N(C(=O)CH3).

[0553] In some embodiments of compounds of Formula (III) or (III-a), X 1-OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 1 is —OH. In some embodiments, X 1 is -SH. In some embodiments, X 1 -O - In some embodiments, X 1 -S - In some embodiments, X 1 is —NH. In some embodiments, X 1 is -NHCH3. In some embodiments, X 1 is —NH(C(═O)CH). In some embodiments, X 1 is —CH3. In some embodiments, X 1 is -CH2CH3. In some embodiments, X 1 is -CH2CH2CH3. In some embodiments, X 1 is —CH(CH). In some embodiments, X 1 is —OCH3. In some embodiments, X 1 is -OCH2CH3.

[0554] In some embodiments of compounds of Formula (III) or (III-a), X 2 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 2 is —OH. In some embodiments, X 2 is -SH. In some embodiments, X 2 -O - In some embodiments, X 2 -S- In some embodiments, X 2 is —NH. In some embodiments, X 2 is -NHCH3. In some embodiments, X 2 is —NH(C(═O)CH). In some embodiments, X 2 is —CH3. In some embodiments, X 2 is -CH2CH3. In some embodiments, X 2 is -CH2CH2CH3. In some embodiments, X 2 is —CH(CH). In some embodiments, X 2 is —OCH3. In some embodiments, X 2 is -OCH2CH3.

[0555] In some embodiments of compounds of Formula (III) or (III-a), X 3 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 3 is —OH. In some embodiments, X 3 is -SH. In some embodiments, X 3 -O - In some embodiments, X 3 -S - In some embodiments, X 3 is —NH. In some embodiments, X 3 is -NHCH3. In some embodiments, X 3 is —NH(C(═O)CH). In some embodiments, X 3 is —CH3. In some embodiments, X 3 is -CH2CH3. In some embodiments, X 3 is -CH2CH2CH3. In some embodiments, X 3is —CH(CH). In some embodiments, X 3 is —OCH3. In some embodiments, X 3 is -OCH2CH3.

[0556] In some embodiments of compounds of Formula (III) or (III-a), X 4 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 4 is —OH. In some embodiments, X 4 is -SH. In some embodiments, X 4 -O - In some embodiments, X 4 -S - In some embodiments, X 4 is —NH. In some embodiments, X 4 is -NHCH3. In some embodiments, X 4 is —NH(C(═O)CH). In some embodiments, X 4 is —CH3. In some embodiments, X 4 is -CH2CH3. In some embodiments, X 4 is -CH2CH2CH3. In some embodiments, X 4 is —CH(CH). In some embodiments, X 4 is —OCH3. In some embodiments, X 4 is -OCH2CH3.

[0557] In some embodiments of compounds of Formula (III) or (III-a), X n -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X n is —OH. In some embodiments, X n is -SH. In some embodiments, X n -O - In some embodiments, X n -S - In some embodiments, X n is —NH. In some embodiments, X n is -NHCH3. In some embodiments, X n is —NH(C(═O)CH). In some embodiments, X n is —CH3. In some embodiments, X n is -CH2CH3. In some embodiments, X n is -CH2CH2CH3. In some embodiments, X n is —CH(CH). In some embodiments, X n is —OCH3. In some embodiments, X n is -OCH2CH3.

[0558] In some embodiments of compounds (III) or (III-a), Y 1 is =O, =S, =NH, or =NCH3. In some embodiments, Y 1 is ═O. In some embodiments, Y 1 is =S. In some embodiments, Y 1 is ═NH. In some embodiments, Y 1 is =NHCH3.

[0559] In some embodiments of compounds of Formula (III) or (III-a), Y 2 is =O, =S, =NH, or =NCH3. In some embodiments, Y 2is ═O. In some embodiments, Y 2 is =S. In some embodiments, Y 2 is ═NH. In some embodiments, Y 2 is =NHCH3.

[0560] In some embodiments of compounds (III) or (III-a), Y 3 is =O, =S, =NH, or =NCH3. In some embodiments, Y 3 is ═O. In some embodiments, Y 3 is =S. In some embodiments, Y 3 is ═NH. In some embodiments, Y 3 is =NHCH3.

[0561] In some embodiments of compounds of Formula (III) or (III-a), Y 4 is =O, =S, =NH, or =NCH3. In some embodiments, Y 4 is ═O. In some embodiments, Y 4 is =S. In some embodiments, Y 4 is ═NH. In some embodiments, Y 4 is =NHCH3.

[0562] In some embodiments of compounds of Formula (III) or (III-a), Y n is =O, =S, =NH, or =NCH3. In some embodiments, Y n is ═O. In some embodiments, Y n is =S. In some embodiments, Y n is ═NH. In some embodiments, Y n is =NHCH3.

[0563] In some embodiments of a compound of Formula (III) or (III-a), A is -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH2-. In some embodiments, A is -NH-. In some embodiments, A is -N(CH3)-. In some embodiments, A is -N(C(=O)CH3)-.

[0564] In some embodiments of compounds (III) or (III-a), A 1 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 1 is —O—. In some embodiments, A 1 is -S-. In some embodiments, A 1 is -CH2-. In some embodiments, A 1 is -NH-. In some embodiments, A 1 is —N(CH)—. In some embodiments, A 1 is -N(C(=O)CH3)-.

[0565] In some embodiments of compounds (III) or (III-a), A 2 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 2 is —O—. In some embodiments, A 2 is -S-. In some embodiments, A 2 is -CH2-. In some embodiments, A 2 is -NH-. In some embodiments, A 2 is —N(CH)—. In some embodiments, A 2 is -N(C(=O)CH3)-.

[0566] In some embodiments of a compound of Formula (III) or (III-a), p is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

[0567] In some embodiments of compounds of Formula (III) or (III-a), Z 1 , Z 2 , Z 3 , Z 4 , and Z n At least one of Z is -OCH. 3 is —OCH. In some embodiments, Z 3 and Z 1 is —OCH 3 . In some embodiments of the compounds of Formula (I), Z 1 , Z 2 , Z 3 , Z 4 , and Z n In some embodiments, at least one of Z 1 , Z 2 , and Z 4 is —OH. In some embodiments, Z 2 and Z 4 is -OH.

[0568] In some embodiments of compounds of Formula (III) or (III-a), Q 1 , Q 2 , Q 3 , and Q 4 In some embodiments of the compound of Formula (I), at least one of Q 1 , Q 2 , Q 3 , and Q 4 In some embodiments, at least one of Q 1 and Q 4is —OCH. In some embodiments, Q 2 and Q 3 is -O-.

[0569] In some embodiments of compounds of Formula (III) or (III-a), Y 1 , Y 2 , Y 3 , Y 4 , and Y n In some embodiments, at least one of Y 1 , Y 2 , Y 3 , and Y 4 is ═O. In some embodiments, Y 1 , Y 2 , Y 3 , Y 4 , and Y n is =S.

[0570] In some embodiments of compounds of Formula (III) or (III-a), X 1 , X 2 , X 3 , X 4 , and X n At least one of the -O - In some embodiments, X 1 , X 2 , X 3 , and X 4 -O - is.

[0571] In some embodiments of the compounds of formula (III) or (III-a), A, A 1 , and A 2 In some embodiments, at least one of A, A 1 , and A 2 is -O-.

[0572] In one embodiment, an mRNA sequence having a 5'-terminal region motif of motif (III'), [ka] During the ceremony, B 1 but, [ka] and B 2 is a modified or non-natural nucleobase; Each B 3 and B n is independently a natural, modified, or non-natural nucleobase; each Z 1 and Z 2 are independently hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH; each Z 3 , Z 4 , and Z n are independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3, or -OCH2CH2OCH3; Each Q 1 and Q 4 are independently -CH-, -CH=CH-, -CHO-, -CHS-, -CHCH-, -CHCF-, -CHNH-, -CHNH(CH)-, or -CHN(C(=O)CH)-; Each Q 2 and Q 3 are independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; each X 1 , X 2 , X 3 , X 4 , and X n are independently -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3; Each Y 1 , Y 2 , Y 3 , Y 4 , and Y n are independently =O, =S, =NH, or =NCH3; Each A, A 1 , and A 2 are independently -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-; Described herein are mRNA sequences having a 5'-terminal region motif of motif (III'), where p is 0, 1, 2, 3, 4, 5, or 6.

[0573] In some embodiments, the mRNA sequence having the 5'-end region motif has the structure of motif (III'-a). [ka]

[0574] In some embodiments of compounds of motif (III') or (III'-a), Z 1 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH. 1 is hydrogen. In some embodiments, Z 1 is F. In some embodiments, Z 1 is —OH. In some embodiments, Z 1 is -SH. In some embodiments, Z 1 is -CH3. In some embodiments, Z 1 is -CH2CH3. In some embodiments, Z 1 is —OCH. In some embodiments, Z 1is —OCH2CH3. In some embodiments, Z 1 is -SCH3. In some embodiments, Z 1 is -NH2. In some embodiments, Z 1 is NHCH. In some embodiments, Z 1 is NHC(=O)CH3.

[0575] In some embodiments of compounds of motif (III') or (III'-a), Z 2 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —OCHCH, —SCH, —NH, NHCH, or NHC(═O)CH. 2 is hydrogen. In some embodiments, Z 2 is F. In some embodiments, Z 2 is —OH. In some embodiments, Z 2 is -SH. In some embodiments, Z 2 is -CH3. In some embodiments, Z 2 is -CH2CH3. In some embodiments, Z 2 is —OCH. In some embodiments, Z 2 is —OCH2CH3. In some embodiments, Z 2 is -SCH3. In some embodiments, Z 2 is -NH2. In some embodiments, Z 2 is NHCH. In some embodiments, Z 2 is NHC(=O)CH3.

[0576] In some embodiments of compounds of motif (III') or (III'-a), Z 3is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 3 is hydrogen. In some embodiments, Z 3 is fluorine. In some embodiments, Z 3 is —OH. In some embodiments, Z 3 is -SH. In some embodiments, Z 3 is -CH3. In some embodiments, Z 3 is -CH2CH3. In some embodiments, Z 3 is —OCH2OCH3. In some embodiments, Z 3 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 3 is —OCH(CH). In some embodiments, Z 3 is -SCH3. In some embodiments, Z 3 is -OCH2CH2OCH3.

[0577] In some embodiments of compounds of motif (III') or (III'-a), Z 4 is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z 4 is hydrogen. In some embodiments, Z 4 is fluorine. In some embodiments, Z 4 is —OH. In some embodiments, Z 4 is -SH. In some embodiments, Z 4 is -CH3. In some embodiments, Z 4 is -CH2CH3. In some embodiments, Z4 is —OCH2OCH3. In some embodiments, Z 4 is —OCH 2 CH 2 CH 3 . In some embodiments, Z 4 is —OCH(CH). In some embodiments, Z 4 is -SCH3. In some embodiments, Z 4 is -OCH2CH2OCH3.

[0578] In some embodiments of compounds of motif (III') or (III'-a), Z n is hydrogen, fluorine, —OH, —SH, —CH, —CHCH, —OCH, —NH, —NHCH, —NH(C(═O)CH), —OCHCH, —OCHOCH, —OCHCHCH, —OCH(CH), —SCH, or —OCHCHOCH. In some embodiments, Z n is hydrogen. In some embodiments, Z n is fluorine. In some embodiments, Z n is —OH. In some embodiments, Z n is -SH. In some embodiments, Z n is -CH3. In some embodiments, Z n is -CH2CH3. In some embodiments, Z n is —OCH2OCH3. In some embodiments, Z n is —OCH 2 CH 2 CH 3 . In some embodiments, Z n is —OCH(CH). In some embodiments, Z n is -SCH3. In some embodiments, Z n is -OCH2CH2OCH3.

[0579] In some embodiments of compounds of motif (III') or (III'-a), B 2 is a modified or non-natural nucleobase. In some embodiments, B 2 is a modified guanine. In some embodiments, B 2is a modified adenine. In some embodiments, B 2 is a modified cytosine. In some embodiments, B 2 is a modified uracil, and in some embodiments, B 2 is a modified thymine, and in some embodiments, B 2 is a modified hypoxanthine. In some embodiments, B 2 is a modified purine. In some embodiments, B 2 is 2-aminoadenine. In some embodiments, B 2 teeth, [ka] is.

[0580] In some embodiments of compounds of motif (III') or (III'-a), B 3 are independently natural, modified, or non-natural nucleobases. In some embodiments, B 3 is guanine. In some embodiments, B 3 is adenine. In some embodiments, B 3 is cytosine. In some embodiments, B 3 is uracil, and in some embodiments, B 3 is thymine, and in some embodiments, B 3 is hypoxanthine. In some embodiments, B 3 is pudding.

[0581] In some embodiments of compounds of motif (III') or (III'-a), B n are independently natural, modified, or non-natural nucleobases. In some embodiments, B n is guanine. In some embodiments, B n is adenine. In some embodiments, B n is cytosine. In some embodiments, B n is uracil, and in some embodiments, B nis thymine, and in some embodiments, B n is hypoxanthine. In some embodiments, B n is pudding.

[0582] In some embodiments of compounds of motif (III') or (III'-a), B 2 , B 3 , and B n At least one of B is adenine. 2 , B 3 , and B n At least one of B is guanine. 2 is a modified adenine. In some embodiments, B 3 is adenine.

[0583] In some embodiments of compounds of motif (III') or (III'-a), Q 1 is —CH—, —CH═CH—, —CHO—, —CHS—, —CHCH—, —CHCF—, —CHNH—, —CHNH(CH)—, or —CHN(C(═O)CH)—. In some embodiments, Q 1 In some embodiments, Q is -CH-. 1 is -CH=CH-. In some embodiments, Q 1 is —CH2O—. In some embodiments, Q 1 is -CHS-. In some embodiments, Q 1 is -CHCH-. In some embodiments, Q 1 is —CHCF—. In some embodiments, Q 1 is —CH 2 NH 2 —. In some embodiments, Q 1 is —CHNH(CH)—. In some embodiments, Q 1 is -CH2N(C(=O)CH3)-.

[0584] In some embodiments of compounds of motif (III') or (III'-a), Q4 is —CH—, —CH═CH—, —CHO—, —CHS—, —CHCH—, —CHCF—, —CHNH—, —CHNH(CH)—, or —CHN(C(═O)CH)—. In some embodiments, Q 4 In some embodiments, Q is -CH-. 4 is -CH=CH-. In some embodiments, Q 4 is —CH2O—. In some embodiments, Q 4 is -CHS-. In some embodiments, Q 4 is -CHCH-. In some embodiments, Q 4 is —CHCF—. In some embodiments, Q 4 is —CH 2 NH 2 —. In some embodiments, Q 4 is —CHNH(CH)—. In some embodiments, Q 4 is -CH2N(C(=O)CH3)-.

[0585] In some embodiments of compounds of motif (III') or (III'-a), Q 2 is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 2 In some embodiments, Q 2 is -S-. In some embodiments, Q 2 In some embodiments, Q is -CH-. 2 In some embodiments, Q is -CF-. 2 In some embodiments, Q is -NH-. 2 is —N(CH)—. In some embodiments, Q 2 is -N(C(=O)CH3).

[0586] In some embodiments of compounds of motif (III') or (III'-a), Q 3is —O—, —S—, —CH—, —CF—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, Q 3 In some embodiments, Q 3 is -S-. In some embodiments, Q 3 In some embodiments, Q is -CH-. 3 In some embodiments, Q is -CF-. 3 In some embodiments, Q is -NH-. 3 is —N(CH)—. In some embodiments, Q 3 is -N(C(=O)CH3).

[0587] In some embodiments of compounds of motif (III') or (III'-a), X 1 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 1 is —OH. In some embodiments, X 1 is -SH. In some embodiments, X 1 -O - In some embodiments, X 1 -S - In some embodiments, X 1 is —NH. In some embodiments, X 1 is -NHCH3. In some embodiments, X 1 is —NH(C(═O)CH). In some embodiments, X 1 is —CH3. In some embodiments, X 1 is -CH2CH3. In some embodiments, X 1 is -CH2CH2CH3. In some embodiments, X 1 is —CH(CH). In some embodiments, X 1is —OCH3. In some embodiments, X 1 is -OCH2CH3.

[0588] In some embodiments of compounds of motif (III') or (III'-a), X 2 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 2 is —OH. In some embodiments, X 2 is -SH. In some embodiments, X 2 -O - In some embodiments, X 2 -S - In some embodiments, X 2 is —NH. In some embodiments, X 2 is -NHCH3. In some embodiments, X 2 is —NH(C(═O)CH). In some embodiments, X 2 is —CH3. In some embodiments, X 2 is -CH2CH3. In some embodiments, X 2 is -CH2CH2CH3. In some embodiments, X 2 is —CH(CH). In some embodiments, X 2 is —OCH3. In some embodiments, X 2 is -OCH2CH3.

[0589] In some embodiments of compounds of motif (III') or (III'-a), X 3 -OH, -SH, -O - , -S -, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 3 is —OH. In some embodiments, X 3 is -SH. In some embodiments, X 3 -O - In some embodiments, X 3 -S - In some embodiments, X 3 is —NH. In some embodiments, X 3 is -NHCH3. In some embodiments, X 3 is —NH(C(═O)CH). In some embodiments, X 3 is —CH3. In some embodiments, X 3 is -CH2CH3. In some embodiments, X 3 is -CH2CH2CH3. In some embodiments, X 3 is —CH(CH). In some embodiments, X 3 is —OCH3. In some embodiments, X 3 is -OCH2CH3.

[0590] In some embodiments of compounds of motif (III') or (III'-a), X 4 -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X 4 is —OH. In some embodiments, X 4 is -SH. In some embodiments, X 4 -O - In some embodiments, X 4 -S - In some embodiments, X 4is —NH. In some embodiments, X 4 is -NHCH3. In some embodiments, X 4 is —NH(C(═O)CH). In some embodiments, X 4 is —CH3. In some embodiments, X 4 is -CH2CH3. In some embodiments, X 4 is -CH2CH2CH3. In some embodiments, X 4 is —CH(CH). In some embodiments, X 4 is —OCH3. In some embodiments, X 4 is -OCH2CH3.

[0591] In some embodiments of compounds of motif (III') or (III'-a), X n -OH, -SH, -O - , -S - , -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3, or -OCH2CH3. In some embodiments, X n is —OH. In some embodiments, X n is -SH. In some embodiments, X n -O - In some embodiments, X n -S - In some embodiments, X n is —NH. In some embodiments, X n is -NHCH3. In some embodiments, X n is —NH(C(═O)CH). In some embodiments, X n is —CH3. In some embodiments, X n is -CH2CH3. In some embodiments, X n is -CH2CH2CH3. In some embodiments, X n is —CH(CH). In some embodiments, Xn is —OCH3. In some embodiments, X n is -OCH2CH3.

[0592] In some embodiments of compounds of motif (III') or (III'-a), Y 1 is =O, =S, =NH, or =NCH3. In some embodiments, Y 1 is ═O. In some embodiments, Y 1 is =S. In some embodiments, Y 1 is ═NH. In some embodiments, Y 1 is =NHCH3.

[0593] In some embodiments of compounds of motif (III') or (III'-a), Y 2 is =O, =S, =NH, or =NCH3. In some embodiments, Y 2 is ═O. In some embodiments, Y 2 is =S. In some embodiments, Y 2 is ═NH. In some embodiments, Y 2 is =NHCH3.

[0594] In some embodiments of compounds of motif (III') or (III'-a), Y 3 is =O, =S, =NH, or =NCH3. In some embodiments, Y 3 is ═O. In some embodiments, Y 3 is =S. In some embodiments, Y 3 is ═NH. In some embodiments, Y 3 is =NHCH3.

[0595] In some embodiments of compounds of motif (III') or (III'-a), Y 4 is =O, =S, =NH, or =NCH3. In some embodiments, Y 4 is ═O. In some embodiments, Y 4is =S. In some embodiments, Y 4 is ═NH. In some embodiments, Y 4 is =NHCH3.

[0596] In some embodiments of compounds of motif (III') or (III'-a), Y n is =O, =S, =NH, or =NCH3. In some embodiments, Y n is ═O. In some embodiments, Y n is =S. In some embodiments, Y n is ═NH. In some embodiments, Y n is =NHCH3.

[0597] In some embodiments of compounds of motif (III') or (III'-a), A is -O-, -S-, -CH2-, -NH-, -N(CH3)-, or -N(C(=O)CH3)-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH2-. In some embodiments, A is -NH-. In some embodiments, A is -N(CH3)-. In some embodiments, A is -N(C(=O)CH3)-.

[0598] In some embodiments of compounds of motif (III') or (III'-a), A 1 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 1 is —O—. In some embodiments, A 1 is -S-. In some embodiments, A 1 is -CH2-. In some embodiments, A 1 is -NH-. In some embodiments, A 1 is —N(CH)—. In some embodiments, A 1 is -N(C(=O)CH3)-.

[0599] In some embodiments of compounds of motif (III') or (III'-a), A 2 is —O—, —S—, —CH—, —NH—, —N(CH)—, or —N(C(═O)CH)—. In some embodiments, A 2 is —O—. In some embodiments, A 2 is -S-. In some embodiments, A 2 is -CH2-. In some embodiments, A 2 is -NH-. In some embodiments, A 2 is —N(CH)—. In some embodiments, A 2 is -N(C(=O)CH3)-.

[0600] In some embodiments of compounds of motif (III') or (III'-a), p is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

[0601] In some embodiments of compounds of motif (III') or (III'-a), Z 1 , Z 2 , Z 3 , Z 4 , and Z n At least one of Z is -OCH. 3 is —OCH. In some embodiments, Z 3 and Z 1 is —OCH 3 . In some embodiments of compounds of motif (I′), Z 1 , Z 2 , Z 3 , Z 4 , and Z n In some embodiments, at least one of Z 1 , Z 2, and Z 4 is —OH. In some embodiments, Z 2 and Z 4 is -OH.

[0602] In some embodiments of compounds of motif (III') or (III'-a), Q 1 , Q 2 , Q 3 , and Q 4 In some embodiments of the compound of Formula (I), at least one of Q 1 , Q 2 , Q 3 , and Q 4 In some embodiments, at least one of Q 1 and Q 4 is —OCH. In some embodiments, Q 2 and Q 3 is -O-.

[0603] In some embodiments of compounds of motif (III') or (III'-a), Y 1 , Y 2 , Y 3 , Y 4 , and Y n In some embodiments, at least one of Y 1 , Y 2 , Y 3 , and Y 4 is ═O. In some embodiments, Y 1 , Y 2 , Y 3 , Y 4 , and Y n is =S. In some embodiments, Y 2 is =S. In some embodiments, Y 4 is =S.

[0604] In some embodiments of compounds of motif (III') or (III'-a), X 1 , X 2 , X 3 , X 4 , and Xn At least one of the -O - In some embodiments, X 1 , X 2 , X 3 , and X 4 -O - In some embodiments, X 1 -S - In some embodiments, X 2 -S - In some embodiments, X 3 -S - In some embodiments, X 4 -S - is.

[0605] In some embodiments of compounds of motif (III') or (III'-a), in some embodiments of compounds of motif (I'), A, A 1 , and A 2 In some embodiments, at least one of A, A 1 , and A 2 is -O-.

[0606] In certain embodiments, the sequence initiator compounds are as set forth in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0607] Disclosed herein, in some embodiments, is a pharmaceutical composition comprising a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate, or one or more mRNAs generated / produced from one or more sequence initiator compounds selected from Table 1, wherein the mRNA encodes one or more pharmaceutically active proteins.

[0608] In certain embodiments, the sequence initiator compounds are as set forth in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0609] Disclosed herein, in some embodiments, is a pharmaceutical composition comprising a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate, or one or more mRNAs generated / produced from one or more sequence initiator compounds selected from Table 2, wherein the mRNA encodes one or more pharmaceutically active proteins.

[0610] In certain embodiments, the sequence initiator compounds are as set forth in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13]

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

Table 3-26

Table 3-27

Table 3-28

Table 3-29

Table 3-30

Table 3-31

Table 3-32

Table 3-33

Table 3-34

Table 3-35

Table 3-36

Table 3-37

Table 3-38

Table 3-39

Table 3-40

Table 3-41

Table 3-42

Table 3-43

Table 3-44

Table 3-45

Table 3-46

Table 3-47

Table 3-48

Table 3-49

Table 3-50

Table 3-51

Table 3-52

Table 3-53

Table 3-54

Table 3-55

Table 3-56

Table 3-57

Table 3-58

Table 3-59

Table 3-60

Table 3-61

Table 3-62

Table 3-63

Table 3-64

Table 3-65

Table 3-66

Table 3-67

Table 3-68

Table 3-69

Table 3-70

Table 3-71

Table 3-72

Table 3-73

Table 3-74

Table 3-75

Table 3-76

Table 3-77

Table 3-78

Table 3-79

Table 3-80

Table 3-81

[0611] In some embodiments, disclosed herein are pharmaceutical compositions comprising a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate, or one or more mRNAs generated / produced from one or more sequence initiator compounds selected from Table 3.

[0612] In another aspect, described herein is a complex comprising an IVT mRNA sequence initiator and a DNA template, wherein the IVT mRNA sequence initiator comprises a compound described herein, and (a) the DNA template comprises a promoter region including a transcription start site having a first nucleotide at nucleotide position +1, a second nucleotide at nucleotide position +2, and a third nucleotide at nucleotide position +3, and (b) the IVT mRNA sequence initiator hybridizes to the DNA template at at least nucleotide positions +1, +2, and +3.

[0613] In another aspect, described herein is a complex comprising an IVT mRNA sequence initiator and a DNA template, wherein the IVT mRNA sequence initiator comprises a compound described herein, and (a) the DNA template comprises a promoter region including a transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2, and (b) the IVT mRNA sequence initiator hybridizes to the DNA template at at least nucleotide positions +1 and +2.

[0614] Pharmaceutical Compositions In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate the processing of the active compound into a pharmaceutically usable preparation. The appropriate formulation depends on the selected route of administration. A summary of the pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Edition (Lippincott Williams & Wilkins 1999), which are incorporated herein by reference for their disclosure.

[0615] A pharmaceutical composition can be a mixture of a sequence initiator compound described herein with one or more other chemical compounds (i.e., pharmaceutically acceptable ingredients), such as carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, humectants, antifoaming agents, antioxidants, preservatives, or one or more combinations thereof. Pharmaceutical compositions facilitate administration of a compound to an organism.

[0616] The compositions described herein can be administered to a subject in a variety of ways, including parenterally, intravenously, intradermally, intramuscularly, intracolonically, intrarectally, or intraperitoneally. In some embodiments, the sequence initiator compounds described herein or pharmaceutically acceptable salts thereof are administered to a subject by intraperitoneal, intramuscular, subcutaneous, or intravenous injection. In some embodiments, the pharmaceutical compositions can be administered parenterally, intravenously, intramuscularly, or orally. Oral formulations containing the sequence initiator compounds described herein can be in any form suitable for oral administration, such as liquids, tablets, capsules, etc. Oral formulations can be further coated or treated to prevent or reduce dissolution in the stomach. The compositions of the present disclosure can be administered to a subject using any suitable method known in the art. Formulations and delivery methods suitable for use in the present disclosure are generally well known in the art. For example, the sequence initiator compounds described herein can be formulated as pharmaceutical compositions containing pharmaceutically acceptable diluents, carriers, or excipients. The composition may contain pharmaceutically acceptable auxiliary substances necessary for approximately physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0617] The pharmaceutical formulations described herein may be administered to a subject in a variety of ways by multiple routes of administration, including, but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intralymphatic, intranasal injection), intranasal, buccal, topical, or transdermal routes of administration. Pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolve formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and combined immediate and controlled release formulations.

[0618] In some embodiments, the pharmaceutical formulation is in the form of a tablet. In other embodiments, the pharmaceutical formulation containing the sequence initiator compound described herein is in the form of a capsule. In one aspect, the liquid pharmaceutical dosage form for oral administration is in the form of an aqueous suspension or solution selected from the group including, but not limited to, aqueous oral dispersion, emulsion, solution, elixir, gel, and syrup.

[0619] For administration by inhalation, the sequence initiator compounds described herein can be formulated for use as an aerosol, mist, or powder. For buccal or sublingual administration, the compositions can take the form of tablets, lozenges, or gels formulated in conventional manner. In some embodiments, the sequence initiator compounds described herein can be prepared as transdermal dosage forms. In some embodiments, the sequence initiator compounds described herein can be formulated into pharmaceutical compositions suitable for intramuscular, subcutaneous, or intravenous injection. In some embodiments, the sequence initiator compounds described herein can be administered topically and can be formulated into various topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, or ointments. In some embodiments, the sequence initiator compounds described herein can be formulated in rectal compositions, such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas.

[0620] In one aspect, the present invention provides a pharmaceutical composition comprising an RNA molecule comprising the IVT mRNA sequence initiator disclosed herein and one or more pharmaceutically acceptable excipients.In some embodiments, the pharmaceutical composition comprises lipid nanoparticles.In some embodiments, the RNA is encapsulated in lipid nanoparticles.

[0621] Transcription In eukaryotes, transcription of messenger RNA (mRNA) is carried out by RNA polymerase II, a complex, multisubunit enzyme with complex regulation. To perform large-scale transcription in vitro, researchers typically use single-subunit phage polymerases derived from T7, T3, SP6, K1-5, K1E, K1F, or K11 bacteriophages. This family of polymerases has a single, minimal promoter sequence of approximately 17 nucleotides, requiring no accessory proteins and with minimal restrictions on the initiation nucleotide sequence. While this application focuses on T7 RNA polymerase (T7 RNAP), those skilled in the art will understand that the present disclosure can be practiced with other RNA polymerases.

[0622] T7 RNAP exists in at least two protein states. The first, called the "abortive complex," is associated with transcription initiation. The second is a highly processive conformation called the "elongation complex." In vitro transcription can be divided into six steps: 1) binding of RNA polymerase to the promoter sequence, 2) initiation of transcription, 3) non-processive elongation, called abortive transcription, during which the polymerase frequently releases the DNA template and short abortive transcripts, 4) conversion of the open complex to a closed complex, 5) processive elongation, and 6) transcription termination. A significant amount of RNA produced during transcription consists of short abortive fragments approximately 2-8 nucleotides in length (Biochemistry 19:3245-3253 (1980); Nucleic Acids Res. 9:31-45 (1981); Nucleic Acids Res. 15:8783-8798 (1987); Biochemistry 27:3966-3974 (1988)). After synthesis of approximately 10-14 bases, RNA polymerase escapes the abortive cycle and simultaneously loses sequence-specific contacts with the promoter DNA to form a processive elongation complex, in which the RNA chain is extended in a sequence-independent manner (J. Mol. Biol. 183:165-177 (1985); Proc. Natl. Acad. Sci. USA 83:3614-3618 (1986); Mol. Cell Biol. 7:3371-3379 (1987)).

[0623] The consensus sequence for the most active class III T7 promoter contains 17 bp of sequence upstream and 6 bp of sequence downstream of the transcription start site (Cell 16:815-25 (1979)). The position of the first transcribed nucleotide is generally referred to as the +1 transcript nucleotide of the RNA, the position of the second transcribed nucleotide as the +2 transcript nucleotide, and so on (Table 2). During transcription, the two strands melt to form a transcription bubble, and the bottom strand of the duplex (3' to 5' shown in Table 4) is the template for transcription. For transcript nucleotides above +3, the template strand defines the identity of the transcribed nucleotide primarily through Watson-Crick base-pairing interactions. Here, the nucleotide encoding the first RNA transcript nucleotide is defined as the +1 nucleotide of the template. In the example shown in Table 4, the +1 transcript nucleotide is G and the +1 template nucleotide is C. Similarly, the +4 transcript nucleotide is A and the +4 template nucleotide is T. [Table 4]

[0624] Unlike DNA polymerase, T7 RNAP initiates RNA synthesis in the absence of a primer. The first step in initiation, called de novo RNA synthesis, involves RNA polymerase recognizing a specific sequence in a DNA template, selecting an initial pair of nucleotide triphosphates complementary to the template residues at positions +1 and +2, and catalyzing the formation of a phosphodiester bond to form a dinucleotide. The initiating nucleotide has a lower affinity for the polymerase than those used during elongation. The Kd values ​​are 2 mM for the first initiating NTP and 80 μM for the second, while the Kd for the elongating NTPs is approximately 5 μM (J. Mol. Biol. (2007) 370, 256-268). De novo synthesis is known to be the rate-limiting step during transcription. T7 RNAP exhibits a strong bias toward GTP as the initiating nucleotide (J. Biol. Chem. 248:2235-2244 (1973)). Of the 17 T7 promoters in the genome, 15 initiate with GTP (and 13 initiate with pppGpG), while there is no clear NTP preference during transcription elongation (J. Mol. Biol. 370:256-268 (2007)). T7 RNA polymerase initiates poorly at promoters encoding A at the +1 position; instead, transcription predominantly initiates when G is encoded at the +2 position (J. Biol. Chem. 278:2819-2823 (2003)).

[0625] During de novo RNA synthesis, binding of the initiating nucleotide is achieved primarily through free energy generated from base stacking, specific interactions between polymerase residues, the guanine moiety of the initiating nucleotide, and base-complementary interactions (J. Mol. Biol. 370:256-268 (2007)).

[0626] It is known that T7 RNAP can also initiate with short oligonucleotide primers. For example, 13 promoters in the T7 genome initiate with pppGpG (J. Mol. Biol. 370:256-268 (2007)). Several groups have shown that T7 RNAP can initiate from dinucleotide primers (Biochemistry 24:5716-5723 (1985)). Axelrod et al. showed that an uncapped GpA dinucleotide can initiate from the +1 and +2 template nucleotides, 2'-deoxycytidine and 2'-deoxythymidine, respectively ("CT" template). Their reaction conditions were 200 micromolar (μM) dimers and 100 μM ATP, CTP, GTP, and UTP. Their reaction mixture also contained 100 μM 3'dATP, 3'dCTP, 3'dUTP, or 50 μM 3'dGTP. They observed only GpA-initiated RNA, not a mixture of GpA-initiated RNA and 5' triphosphate RNA from GTP initiation. This is likely due to the reaction conditions employed. 100 μM GTP is significantly lower than the 2 mM Kd of T7 polymerase for the initial initiating guanosine (J. Mol. Biol. (2007) 370, 256-268). Because GTP competes with the initiator oligonucleotide for initiation, the use of lower GTP concentrations favors GpA initiation but results in lower transcription yields (maximum calculated yields were estimated to be <150 μg / mL of reaction). When initiating transcription on the "CT" template with ApG, CpG, UpG, or GpG, they observed the formation of RNA transcripts with an additional non-template 5' nucleotide (A, C, U, or G, respectively).

[0627] Methods and compositions provided herein for the preparation of 5'-capped RNA include, but are not limited to, mRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and small Cajal body-specific RNA (scaRNA). These methods involve the use of an oligonucleotide primer containing an mRNA sequence initiator, e.g., Cap, along with nucleoside 5'-triphosphates (NTPs) and RNA polymerase for promoter-controlled RNA synthesis from a DNA template. In certain embodiments, the methods use an initiating capped oligonucleotide primer, which provides utility in RNA synthesis, particularly in the synthesis of capped mRNA. Exemplary initiating oligonucleotide primers have a structure similar to Cap0, Cap1, Cap2, or TMG-Cap of natural RNA molecules, and contain 2'-O-methylated nucleoside units at the 5'-position of the RNA (penultimate Cap1 and adjacent penultimate Cap2). The native Cap0 structure has no 2'-O-methylated nucleotide units.

[0628] Methods and compositions for the preparation of RNA include, but are not limited to, mRNA, snRNA, snoRNA, scaRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), and transfer-messenger RNA (tmRNA) with modifications at or near the 5' end of the molecule. These methods involve the use of initiators, such as a starting oligonucleotide primer with or without a cap, nucleoside 5'-triphosphates (NTPs), and RNA polymerase for DNA-templated, promoter-controlled synthesis of RNA. In certain embodiments, the methods use modified starting oligonucleotide primers with structural modifications that provide utility in RNA synthesis, particularly the synthesis of 5'-modified RNAs.

[0629] An exemplary initiation capped oligonucleotide primer has an open-chain 3'-OH group, which allows for the initiation of RNA ...

Claims

1. An in vitro transcription (IVT) mRNA sequence initiator comprising a compound of formula (I) or a salt or solvate thereof, 【Chemistry 1】 During the ceremony, B １ but, 【Chemistry 2】 and Each B ２ , B ３ , and B ｎ is independently a natural, modified, or non-natural nucleobase; Each Z １ and Z ２ are independently hydrogen, fluorine, —OH, —SH, —CH ３ , -CH ２ CH ３ , -OCH ３ , -OCH ２ CH ３ , -SCH ３ , -NH ２ , NHCH ３ , or NHC(═O)CH ３ and Z 3 is —OCH 3 ; Each Z 4 and Z ｎ are independently hydrogen, fluorine, —OH, —SH, —CH ３ , -CH ２ CH ３ , -OCH ３ , -NH ２ , -NHCH ３ , -NH(C(=O)CH ３ ), -OCH ２ CH ３ , -OCH ２ OCH ３ , -OCH ２ CH ２ CH ３ , -OCH(CH ３ ) ２ , -SCH ３ , or -OCH ２ CH ２ OCH ３ and Each Q １ and Q ４ are independently -CH ２ -, -CH=CH-, -CH ２ O-, -CH ２ S-, -CH ２ CH ２ -, -CH ２ CF ２ -, -CH ２ NH ２ -, -CH ２ NH (CH ３ ) - or -CH ２ N(C(=O)CH ３ ) - and Each Q ２ and Q ３ are independently —O—, —S—, or —CH ２ -, -CF ２ -, -NH-, -N(CH ３ ) - or -N(C(=O)CH ３ ) - and X 4 is —OH or —O − ; Each X １ , X ２ , X 3 , and X ｎ are independently —OH, —SH, —O － , -S － , -NH ２ , -NHCH ３ , -NH(C(=O)CH ３ ), -CH ３ , -CH ２ CH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -OCH ３ , or -OCH ２ CH ３ and Y 4 is =S; Each Y １ , Y ２ , Y ３ , and Y ｎ are independently ═O, ═S, ═NH, or ═NCH ３ and Each A, A １ , and A ２ are independently —O—, —S—, or —CH ２ -, -NH-, -N(CH ３ ) - or -N(C(=O)CH ３ ) - and p is 0, 1, 2, 3, 4, 5, or 6; The compound comprises a phosphorothioate, the phosphorothioate comprising a chiral phosphorus center. In vitro transcription (IVT) mRNA sequence initiator.

2. 2. The IVT mRNA sequence initiator of claim 1, wherein the IVT mRNA sequence initiator has the structure of formula (Ic): 【Transformation 3】

3. 2. The IVT mRNA sequence initiator of claim 1, wherein the IVT mRNA sequence initiator has the structure of formula (Id): 【Chemistry 4】

4. B 2 is adenine and B 3 is guanine; each of Q 1 and Q 4 is —CH 2 O—; each of Q 2 and Q 3 is —O—; Each of X 1 , X 2 , and X 3 is —OH or —O − ; each of Y 1 , Y 2 , and Y 3 is ═O; and 3. The IVT mRNA sequence initiator of claim 2, wherein each of A 1 and A is -O-.

5. B 2 is adenine and B 3 is guanine; each of Q 1 and Q 4 is —CH 2 O—; each of Q 2 and Q 3 is —O—; Each of X 1 , X 2 , and X 3 is —OH or —O − ; each of Y 1 , Y 2 , and Y 3 is ═O; and 4. The IVT mRNA sequence initiator of claim 3, wherein each of A 1 and A is -O-.

6. Each Z ４ and Z ｎ are independently —OH or —OCH ３ 2. The IVT mRNA sequence initiator of claim 1,

7. Each of Y 1 , Y 2 , Y 3 , and Y ｎ 7. The IVT mRNA sequence initiator of any one of claims 1 to 6, wherein:

8. Each of X 1 , X 2 , X 3 , and X n independently represents —O － or -S － 7. The IVT mRNA sequence initiator of claim 1, wherein

9. The compound 【Transformation 5】 7. The IVT mRNA sequence initiator of any one of claims 1 and 3 to 6,

10. The compound 【Transformation 6】 7. The IVT mRNA sequence initiator of any one of claims 1 and 3 to 6,

11. An mRNA comprising a sequence comprising an IVT mRNA sequence initiator according to any one of claims 1 and 3 to 6.

12. The array is (a) the 5' untranslated region; (b) a first region encoding a deaminase; (c) a second region encoding a programmable nuclease; (d) a third region encoding a nuclear localization sequence; (e) the 3' untranslated region; (f) a polyadenylic acid region; The mRNA of claim 11, comprising:

13. the deaminase is an adenine base editor, and 13. The mRNA of claim 12, wherein the programmable nuclease is a Cas9 protein.

14. 12. The RNA of claim 11, wherein the sequence comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 3 or 4.

15. An mRNA comprising a sequence having a 5'-terminal region motif (I'), 【Transformation 7】 During the ceremony, B １ but, 【Transformation 8】 and Each B ２ , B ３ , and B ｎ is independently a natural, modified, or non-natural nucleobase; Each Z １ and Z ２ are independently hydrogen, fluorine, —OH, —SH, —CH ３ , -CH ２ CH ３ , -OCH ３ , -OCH ２ CH ３ , -SCH ３ , -NH ２ , NHCH ３ , or NHC(═O)CH ３ and Each Z ３ , Z ４ , and Z ｎ are independently hydrogen, fluorine, —OH, —SH, —CH ３ , -CH ２ CH ３ , -OCH ３ , -NH ２ , -NHCH ３ , -NH(C(=O)CH ３ ), -OCH ２ CH ３ , -OCH ２ OCH ３ , -OCH ２ CH ２ CH ３ , -OCH(CH ３ ) ２ , -SCH ３ , or -OCH ２ CH ２ OCH ３ and Each Q １ and Q ４ are independently -CH ２ -, -CH=CH-, -CH ２ O-, -CH ２ S-, -CH ２ CH ２ -, -CH ２ CF ２ -, -CH ２ NH-, -CH ２ NH (CH ３ ) - or -CH ２ N(C(=O)CH ３ ) - and Each Q ２ and Q ３ are independently —O—, —S—, or —CH ２ -, -CF ２ -, -NH-, -N(CH ３ ) - or -N(C(=O)CH ３ ) - and Each X １ , X ２ , X ３ , X ４ , and X ｎ are independently —OH, —SH, —O － , -S － , -NH ２ , -NHCH ３ , -NH(C(=O)CH ３ ), -CH ３ , -CH ２ CH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -OCH ３ , or -OCH ２ CH ３ and Each Y １ , Y ２ , Y ３ , Y ４ , and Y ｎ are independently ═O, ═S, ═NH, or ═NCH ３ and Each A, A １ , and A ２ are independently —O—, —S—, or —CH ２ -, -NH-, -N(CH ３ ) - or -N(C(=O)CH ３ ) - and p is 0, 1, 2, 3, 4, 5, or 6; However, the compound of formula (I) satisfies the following conditions (i) to (iii): (i) X １ , X ２ , X ３ , X ４ , and X ｎ At least one of the groups is -SH or -S － (ii) Y １ , Y ２ , Y ３ , Y ４ , and Y ｎ and (iii) at least one of A, A １ , and A ２ and at least one of the following is -S-: (a) mixing a DNA template, a polymerase enzyme, an mRNA sequence initiator containing a phosphorothioate group, and nucleoside triphosphates (NTPs) at a specific molar ratio of the mRNA sequence initiator to the NTPs to form a mixture; (b) incubating the mixture at a specific temperature and for a specific period of time; (c) collecting and purifying the mRNA sequence having the 5' end region motif from the mixture; The method, wherein the NTP is GTP, ATP, CTP, UTP, or a modified NTP, or a combination thereof.

16. The method described in claim 15, which obtains a yield of at least 80% of an IVT reaction having a capping efficiency of at least 80%.

17. A method described in claim 15 or 16, which obtains a yield of at least 3 mg of mRNA per mL of IVT reaction with a capping efficiency of at least 80%.

18. The method described in claim 15 or 16, wherein the molar ratio of the mRNA sequence initiator to the NTP is about 1:5, about 1:2.5, about 1:1.67, about 1:1.25, or about 1:

1.

19. mRNA produced by the method of claim 15 or 16.

20. A pharmaceutical composition comprising the mRNA of claim 11 and one or more pharmaceutically acceptable excipients.

21. The pharmaceutical composition of claim 20 for use in reducing the risk of cardiovascular disease in a patient in need thereof.

22. The pharmaceutical composition of claim 20 for use in reducing the risk of coronary artery disease in a patient in need thereof.

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