Compositions and methods for capping RNA

Abstract

Provided herein are compositions and methods for the preparation of 5 '-terminal region modified mRNA. In particular, the present disclosure relates to novel mRNA 5'terminal region motifs and sequence initiators thereof, as well as assays that enable the measurement of functional aspects of these motifs and sequence initiators. Also provided herein are compositions and methods for treating conditions associated with coronary artery disease.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 315,323 filed on March 1, 2022 and No. 63 / 485,753 filed on February 17, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to RNA, and in particular to the 5' end region of mRNA molecules, including, for example, a novel mRNA 5' end region motif (or mRNA cap) and its initiator. Background Art

[0004] The 5' region of an mRNA can be an important structural and / or functional feature of a eukaryotic mRNA molecule, as it can provide stability to the mRNA (e.g., by providing protection against 5' exonucleases) and participate in RNA splicing, mRNA transport, and other activities that support protein translation. The structural elements of a conventional mRNA cap typically include an inverted 7-methylguanosine (m ) at the 5' end connected to a triphosphate (ppp) bridge. 7 G), which in turn is linked to the first nucleotide of the mRNA transcript (N 1 ). The 5' end region mRNA motifs and their sequence initiators described herein differ from conventional mRNA caps in several aspects, including, for example, that they include chemically modified reverse 7-methylguanosine (m7G) nucleoside structures and / or modified triphosphate (ppp) bonds. Conventional caps are typically one of the most expensive components in manufacturing mRNA molecules. Therefore, alternatives to conventional caps are important aspects of the art, each independently, while maintaining or improving capping efficiency and / or mRNA yield. Summary of the invention

[0005] This paper describes a new mRNA 5' region motif and its initiator.

[0006] In one aspect, described herein is an in vitro transcribed (IVT) mRNA sequence initiator comprising a compound of formula (I) or a salt or solvate thereof:

[0007]

[0008] in

[0009] B1 is

[0010] B2, B3 and Bn are each independently a natural, modified or unnatural nucleobase;

[0011] Z1 and Z2 are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3 or NHC(=O)CH3;

[0012] Z3, Z4 and Zn are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3;

[0013] Q1 and Q4 are each independently -CH2-, -CH=CH-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-;

[0014] Q2 and Q3 are each independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-;

[0015] X1, X2, X3, X4 and Xn are each independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3 or -OCH2CH3;

[0016] Y1, Y2, Y3, Y4 and Yn are each independently =O, =S, =NH or =NCH3;

[0017] A, A1 and A2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and

[0018] p=0, 1, 2, 3, 4, 5 or 6.

[0019] On the other hand, the compounds of formula (I) described herein satisfy one or more of the following restrictive 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-.

[0020] On the other hand, the IVT mRNA sequence initiator satisfies that at least one of X1, X2, X3, X4 and Xn is -SH or -S-; at least one of Y1, Y2, Y3, Y4 and Yn is =S; or at least one of A, A1 and A2 is -S-.

[0021] In one aspect, described herein is an in vitro transcribed (IVT) mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof:

[0022]

[0023] in

[0024] B1 is

[0025] B2, B3 and Bn are each independently a natural, modified or unnatural nucleobase;

[0026] Z1 and Z' are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3) or -SCH3;

[0027] Z2 and Z" are each independently fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3 or NHC(=O)CH3;

[0028] Z'' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3 or -OCH2CH3;

[0029] Z3, Z4 and Zn are each independently hydrogen, fluorine, -OH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3;

[0030] Q1 and Q4 are each independently -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-;

[0031] Q2 and Q3 are each independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-;

[0032] X1, X2, X3, X4 and Xn are each independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3 or -OCH2CH3;

[0033] Y1, Y2, Y3, Y4 and Yn are each independently =O, =S, =NH or =NCH3;

[0034] A, A1 and A2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and

[0035] p=0, 1, 2, 3, 4, 5 or 6.

[0036] In one aspect, described herein is an in vitro transcribed (IVT) mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof:

[0037]

[0038] in

[0039] B1 is

[0040] B2, B3 and Bn are each independently a natural, modified or unnatural nucleobase;

[0041] Z' and Z" are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3) or -SCH3;

[0042] Z'' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3 or -OCH2CH3;

[0043] Z1 and Z2 are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3 or NHC(=O)CH3;

[0044] Z3, Z4 and Zn are each independently hydrogen, fluorine, -OH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3;

[0045] Q1 and Q4 are each independently -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-;

[0046] Q2 and Q3 are each independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-;

[0047] X1, X2, X3, X4 and Xn are each independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3 or -OCH2CH3;

[0048] Y1, Y2, Y3, Y4 and Yn are each independently =O, =S, =NH or =NCH3;

[0049] A, A1 and A2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and

[0050] p=0, 1, 2, 3, 4, 5 or 6.

[0051] In one aspect, described herein is an in vitro transcribed (IVT) mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof:

[0052]

[0053] in

[0054] B 1 for

[0055] B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase;

[0056] Z' and Z" are each independently hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 ;

[0057] Z"' is hydrogen, fluorine, -CH 3 、-CH 2 CH 3 、-OCH 3 or -OCH 2 CH 3 ;

[0058] Z 1 and Z 2 are independently hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 ;

[0059] Z 3 , Z 4 and Z n are independently hydrogen, fluorine, -OH, -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 ;

[0060] Q 1 and Q 4 are independently -CH=CH-, -CH 2 -、-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 )-;

[0061] Q 2 and Q 3 are independently -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;

[0062] 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 ;

[0063] Y 1 , Y 2 , Y 3 , Y 4 and Y n are independently =O, =S, =NH or =NCH 3 ;

[0064] A. A 1 and A 2 are independently -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;and

[0065] p=0, 1, 2, 3, 4, 5 or 6.

[0066] In one aspect, described herein is an in vitro transcribed (IVT) mRNA sequence initiator comprising a compound of formula (III) or a salt or solvate thereof:

[0067]

[0068] in

[0069] B1 is

[0070] B2 is a modified or non-natural nucleobase;

[0071] B3 and Bn are each independently a natural, modified or unnatural nucleobase;

[0072] Z1 and Z2 are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3 or NHC(=O)CH3;

[0073] Z3, Z4 and Zn are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3;

[0074] Q1 and Q4 are each independently -CH2-, -CH=CH-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-;

[0075] Q2 and Q3 are each independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-;

[0076] X1, X2, X3, X4 and Xn are each independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3 or -OCH2CH3;

[0077] Y1, Y2, Y3, Y4 and Yn are each independently =O, =S, =NH or =NCH3;

[0078] A, A1 and A2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and

[0079] p=0, 1, 2, 3, 4, 5 or 6.

[0080] In another aspect, Z3 is hydrogen, fluorine, -OH, -OCH3, - or -OCH2CH3. In another aspect, wherein Z3 is -OCH3. In another aspect, Z4 and Zn are independently -OH or -OCH3. In another aspect, Z3, Z4 and Zn are each independently -OH or -OCH3. In another aspect, Y2, Y4 and Yn are independently =O or =S. In another aspect, X2 and X3 are independently -O- or -S-.

[0081] 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.

[0082] In another aspect, the IVT mRNA sequence initiator comprises a phosphorothioate. In another aspect, the phosphorothioate comprises a chiral phosphorus center. In another aspect, a composition is described herein that comprises an IVT mRNA sequence initiator, wherein the composition comprises a racemic mixture of an R diastereomer and an S diastereomer of the IVT mRNA sequence initiator. In another aspect, the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 60%. In another aspect, the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 80%. In another aspect, the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 90%. In another aspect, the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 95%. In another aspect, the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 98%.

[0083] In one aspect, described herein is an mRNA sequence having a 5' end region motif (I'):

[0084]

[0085] in

[0086] B1 is

[0087] B2, B3 and Bn are each independently a natural, modified or unnatural nucleobase;

[0088] Z1 and Z2 are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3 or NHC(=O)CH3;

[0089] Z3, Z4 and Zn are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3;

[0090] Q1 and Q4 are each independently -CH2-, -CH=CH-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-;

[0091] Q2 and Q3 are each independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-;

[0092] X1, X2, X3, X4 and Xn are each independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3 or -OCH2CH3;

[0093] Y1, Y2, Y3, Y4 and Yn are each independently =O, =S, =NH or =NCH3;

[0094] A, A1 and A2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and

[0095] p=0, 1, 2, 3, 4, 5 or 6.

[0096] In another aspect, formula (I) satisfies one or more of the following restrictive 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 aspect, at least one of X1, X2, X3, X4 and Xn is -SH or -S-; at least one of Y1, Y2, Y3, Y4 and Yn is =S; or at least one of A, A1 and A2 is -S-.

[0097] In one aspect, the present invention describes an mRNA sequence having a 5' terminal region motif (II'):

[0098]

[0099] in

[0100] B1 is

[0101] B2, B3 and Bn are each independently a natural, modified or unnatural nucleobase;

[0102] Z1 and Z' are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3) or -SCH3;

[0103] Z2 and Z" are each independently fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3 or NHC(=O)CH3;

[0104] Z'' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3 or -OCH2CH3;

[0105] Z3, Z4 and Zn are each independently hydrogen, fluorine, -OH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3;

[0106] Q1 and Q4 are each independently -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-;

[0107] Q2 and Q3 are each independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-;

[0108] X1, X2, X3, X4 and Xn are each independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3 or -OCH2CH3;

[0109] Y1, Y2, Y3, Y4 and Yn are each independently =O, =S, =NH or =NCH3;

[0110] A, A1 and A2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and

[0111] p=0, 1, 2, 3, 4, 5 or 6.

[0112] In one aspect, the present invention describes an mRNA sequence having a 5' terminal region motif (II'):

[0113]

[0114] in

[0115] B1 is

[0116] B2, B3 and Bn are each independently a natural, modified or unnatural nucleobase;

[0117] Z' and Z" are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3) or -SCH3;

[0118] Z'' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3 or -OCH2CH3;

[0119] Z1 and Z2 are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3 or NHC(=O)CH3;

[0120] Z3, Z4 and Zn are each independently hydrogen, fluorine, -OH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3;

[0121] Q1 and Q4 are each independently -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-;

[0122] Q2 and Q3 are each independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-;

[0123] X1, X2, X3, X4 and Xn are each independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3 or -OCH2CH3;

[0124] Y1, Y2, Y3, Y4 and Yn are each independently =O, =S, =NH or =NCH3;

[0125] A, A1 and A2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and

[0126] p=0, 1, 2, 3, 4, 5 or 6.

[0127] In one aspect, the present invention describes an mRNA sequence having a 5' terminal region motif (II'):

[0128]

[0129] in

[0130] B 1 for

[0131] B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase;

[0132] Z' and Z" are each independently hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 ;

[0133] Z"' is hydrogen, fluorine, -CH 3 、-CH 2 CH 3 、-OCH 3 or -OCH 2 CH 3 ;

[0134] Z 1 and Z 2 are independently hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 ;

[0135] Z 3 , Z 4 and Z n are independently hydrogen, fluorine, -OH, -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 ;

[0136] Q 1 and Q 4 are independently -CH=CH-, -CH 2 -、-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 )-;

[0137] Q2 and Q 3 are independently -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;

[0138] 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 ;

[0139] Y 1 , Y 2 , Y 3 , Y 4 and Y n are independently =O, =S, =NH or =NCH 3 ;

[0140] A. A 1 and A 2 are independently -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;and

[0141] p=0, 1, 2, 3, 4, 5 or 6.

[0142] In one aspect, the present invention describes an mRNA sequence having a 5' terminal region motif (III'):

[0143]

[0144] in

[0145] B1 is

[0146] B2 is a modified or non-natural nucleobase;

[0147] B3 and Bn are each independently a natural, modified or unnatural nucleobase;

[0148] Z1 and Z2 are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3 or NHC(=O)CH3;

[0149] Z3, Z4 and Zn are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3;

[0150] Q1 and Q4 are each independently -CH2-, -CH=CH-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-;

[0151] Q2 and Q3 are each independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-;

[0152] X1, X2, X3, X4 and Xn are each independently -OH, -SH, -O-, -S-, -NH2, -NHCH3, -NH(C(=O)CH3), -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -OCH3 or -OCH2CH3;

[0153] Y1, Y2, Y3, Y4 and Yn are each independently =O, =S, =NH or =NCH3;

[0154] A, A1 and A2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and

[0155] p=0, 1, 2, 3, 4, 5 or 6.

[0156] In one aspect, described herein is an mRNA sequence having a 5' region motif, wherein the 5' region motif is a compound from Table 1 or a salt or solvate thereof.

[0157] In another aspect, described herein is an mRNA sequence having a 5' terminal region motif, wherein the 5' terminal region motif is a compound from Table 2 or a salt or solvate thereof.

[0158] In another aspect, described herein is an mRNA sequence having a 5' terminal region motif, wherein the 5' terminal region motif is a compound from Table 3 or a salt or solvate thereof.

[0159] In another aspect, the mRNA sequence initiator is a salt. In some embodiments, the mRNA sequence initiator is a sodium salt.

[0160] On the other hand, in mRNA sequences having a 5' end region motif, Z 3 Hydrogen, fluorine, -OH, -OCH 3 , -or-OCH 2 CH 3 .

[0161] On the other hand, in mRNA sequences having a 5' end region motif, Z 3 OCH 3 .

[0162] On the other hand, in mRNA sequences having a 5' end region motif, Z 4 and Z n Each is independently -OH or -OCH 3 .

[0163] On the other hand, in mRNA sequences having a 5' end region motif, Z 3 , Z 4 and Z n Each is independently -OH or -OCH 3 .

[0164] On the other hand, in the mRNA sequence having the 5' end region motif, B 1 for

[0165] On the other hand, in the mRNA sequence having the 5' end region motif, B 1 for

[0166] On the other hand, in the mRNA sequence having the 5' end region motif, B 1 for

[0167] On the other hand, in mRNA sequences having a 5' end region motif, B 1 for

[0168] On the other hand, in mRNA sequences having a 5' end region motif, B 2 for

[0169] On the other hand, in mRNA sequences having a 5' end region motif, Z 1 Fluorine, -OH, -OCH 3 .

[0170] On the other hand, in mRNA sequences having a 5' end region motif, Z 1 For fluorine.

[0171] On the other hand, in mRNA sequences having a 5' end region motif, Z 1 It is -OH.

[0172] On the other hand, in mRNA sequences having a 5' end region motif, Z 1 OCH 3 .

[0173] On the other hand, in mRNA sequences having a 5' end region motif, Z 2 Fluorine, -OH, -OCH 3 .

[0174] On the other hand, in mRNA sequences having a 5' end region motif, Z 2 For fluorine.

[0175] On the other hand, in mRNA sequences having a 5' end region motif, Z 2 It is -OH.

[0176] On the other hand, in mRNA sequences having a 5' end region motif, Z 2 OCH 3 .

[0177] On the other hand, in mRNA sequences having a 5' end region motif, Q 1 and Q 4 -CH 2 O-.

[0178] On the other hand, in mRNA sequences having a 5' end region motif, Q 2 and Q 3 Each is -O-.

[0179] On the other hand, in the mRNA sequence having the 5' end region motif, Y 1 and Y 3 Each is =0.

[0180] On the other hand, in the mRNA sequence having the 5' end region motif, Y 2 , Y4 and Y n Each is independently =O or =S.

[0181] On the other hand, in the mRNA sequence having the 5' end region motif, Y 1 , Y 2 , Y 3 , Y 4 and Y n One or more of them is =S.

[0182] On the other hand, in the mRNA sequence having the 5' end region motif, Y 2 is =S.

[0183] On the other hand, in the mRNA sequence having the 5' end region motif, Y 4 is =S.

[0184] On the other hand, in the mRNA sequence having the 5' end region motif, Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is =0.

[0185] On the other hand, in mRNA sequences having a 5' end region motif, X 1 , X 4 and X n Each is -O - .

[0186] On the other hand, in mRNA sequences having a 5' end region motif, X 2 and X 3 Each independently is -O - or -S - .

[0187] On the other hand, in mRNA sequences having a 5' end region motif, X 3 -O - .

[0188] On the other hand, in mRNA sequences having a 5' end region motif, X 1 , X 2 , X 3 , X 4 and X n One or more of -S - .

[0189] On the other hand, in mRNA sequences having a 5' end region motif, X 2 For -S - .

[0190] On the other hand, in mRNA sequences having a 5' end region motif, X 4 For -S - .

[0191] On the other hand, in mRNA sequences having a 5' end region motif, X 1 , X 2 , X 3 , X 4 and X n Each is -O - .

[0192] On the other hand, in the mRNA sequence having the 5' end region motif, A, A 1 and A 2 Each is -O-.

[0193] On the other hand, in the mRNA sequence having the 5' end region motif, A, A 1 and A 2 One or more of them is -S-.

[0194] In another aspect of the mRNA sequence having the 5' end region motif, A is -S-, and A 1 and A 2 It is -O-.

[0195] On the other hand, in the mRNA sequence having the 5' end region motif, A 2 is -S-, and A and A 1 It is -O-.

[0196] On the other hand, in the mRNA sequence having the 5' end region motif, A, A 1 and A 2 It is -O-.

[0197] In another aspect of the mRNA sequence having the 5' end region motif, p is 0.

[0198] In another aspect of the mRNA sequence having the 5' end region motif, p is 1.

[0199] In another aspect of the mRNA sequence having the 5' end region motif, p is 2.

[0200] In another aspect of the mRNA sequence having a 5' region motif, wherein B 2 , B 3 and B n Each is independently adenine, cytosine, guanine, uracil, thymine, hypoxanthine or purine.

[0201] On the other hand, in mRNA sequences having a 5' end region motif, B 2is adenine, and B 3 For guanine.

[0202] On the other hand, in mRNA sequences having a 5' end region motif, B 2 is guanine, and B 3 Adenine.

[0203] On the other hand, in the case of mRNA sequences having the 5' end region motif, protein expression is increased.

[0204] On the other hand, in mRNA sequences having a 5' end region motif, Q 1 and Q 4 -CH 2 O-;Q 2 and Q 3 -O-;X n Each independently represents -OH, -SH, O - or S - ; Y n are each independently =O or =S; and B 1 for

[0205] On the other hand, in mRNA sequences having a 5' end region motif, Q 1 and Q 4 -CH 2 O-;Q 2 and Q 3 -O-;X n Each independently represents -OH, -SH, O - or S - ; Y n are each independently =O or =S; and B 1 for

[0206] On the other hand, in mRNA sequences having a 5' end region motif, Q 1 and Q 4 -CH 2 O-;Q 2 and Q 3 -O-;X n Each independently represents -OH, -SH, O - or S - ; Y n are each independently =O or =S; and B 1 for

[0207] In another aspect, described herein is a complex comprising an mRNA sequence having a 5' terminal region motif and a DNA template, wherein the mRNA sequence having a 5' terminal region motif comprises a compound described herein, wherein the DNA template comprises a promoter region, the promoter region comprising 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 wherein the mRNA sequence having a 5' terminal region motif hybridizes to the DNA template at least at nucleotide positions +1, +2, and +3.

[0208] In another aspect, described herein is a complex comprising an mRNA sequence having a 5' terminal region motif and a DNA template, wherein the mRNA sequence having a 5' terminal region motif comprises a compound described herein, wherein the DNA template comprises a promoter region, the promoter region comprising a transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and wherein the mRNA sequence having a 5' terminal region motif hybridizes to the DNA template at least at nucleotide positions +1 and +2.

[0209] 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.

[0210] On the other hand, a method of expressing mRNA is described herein, comprising introducing the mRNA described herein into a cell lysate to express the mRNA. On the other hand, the method also includes measuring the expression level of the mRNA. On the other hand, as described in the method herein, the expression level of the mRNA is at least 2 times, 3 times, 4 times, 6 times, 8 times or 20 times that of the corresponding mRNA without an mRNA sequence having a 5' end region motif. On the other hand, the method described herein relates to HeLa cells. On the other hand, described herein, the method involves producing up to 0.1wt% of dsRNA. On the other hand, described herein, the method involves producing up to 0.09wt% of dsRNA. On the other hand, described herein, the method involves producing up to 0.06wt% of dsRNA. On the other hand, described herein, the method involves producing up to 0.04wt% of dsRNA.

[0211] On the other hand, described herein is a method for detecting cellular immune stimulation produced by mRNA, the method comprising (a) contacting a preparation comprising capped mRNA according to a compound described herein with a cell reporter cell line, and (b) measuring RIG-I activation in the cell reporter cell line. On the other hand, the reporter cell line is a HEK-Lucia RIG-I model. On the other hand, compared to uncapped mRNA, the cellular immune stimulation described herein is reduced by at least 20%, 50%, 70%, 100% and 150%.

[0212] In another aspect, described herein is a method for producing an mRNA sequence having a 5' terminal region motif as described herein using an IVT reaction, the method comprising (a) mixing a DNA template, a polymerase, an mRNA sequence motif comprising a phosphorothioate group (PS), and a nucleoside triphosphate (NTP) at a specific molar ratio of the mRNA sequence motif to the NTP to produce a mixture, (b) incubating the mixture at a specific temperature and duration, and (c) collecting and purifying the mRNA sequence having a 5' terminal region motif from the mixture. In another aspect, the molar ratio is 1:5, and the method is capable of producing 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 is capable of producing 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 is capable of producing a yield of at least 80% with a capping efficiency of at least 90%. In another aspect, the molar ratio is 1:1.25, and the method is capable of producing a yield of at least 80% with a capping efficiency of at least 90%. In another aspect, the molar ratio is 1.0:1.0, and the method is capable of producing a yield of at least 80% with a capping efficiency of at least 80%. In another aspect, the molar ratio is 1:5, and the method is capable of producing 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 aspect, the molar ratio is 1:2.5, and the method is capable of producing 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 aspect, the molar ratio is 1:1.67, and the method is capable of producing 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 aspect, the molar ratio is 1:1.25, and the method is capable of producing 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 aspect, the molar ratio is 1.0:1.0, and the method can produce 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 aspect, the NTP is GTP, ATP, CTP, UTP, a modified NTP, or a combination thereof. In another aspect, the modified NTP is N1-methyl pseudouridine.

[0213] In another aspect, described herein is a cell containing an RNA molecule comprising an mRNA sequence described herein having a 5' end region motif.

[0214] In another aspect, described herein is a cell containing a polypeptide translated from an RNA molecule comprising an mRNA sequence having a 5' end region motif as described herein.

[0215] In another aspect, a pharmaceutical composition is described herein, comprising an RNA molecule comprising an mRNA sequence having a 5' terminal region motif as described herein and one or more pharmaceutically acceptable excipients. In another aspect, the drug comprises a lipid nanoparticle. In another aspect, the pharmaceutical composition is encapsulated in the lipid nanoparticle. In another aspect, the pharmaceutical composition further comprises one or more single guide RNAs, which are designed to target one or more specific locations of one or more genes of interest to induce a pharmacological effect after administration to a mammal.

[0216] On the other hand, a method for synthesizing RNA molecules is described herein, comprising: introducing an mRNA sequence having a 5' end region motif as described herein into a mixture comprising an RNA polymerase, and incubating the mixture for a time sufficient to allow transcription of the RNA molecule. On the other hand, the mixture also comprises a DNA template and nucleoside triphosphates.

[0217] In another aspect, described herein is a method of gene editing comprising introducing an RNA molecule or a pharmaceutical composition into a cell, wherein the RNA molecule comprises a guide RNA or a nuclease mRNA, wherein the RNA molecule is translated in the cell.

[0218] In another aspect, described herein is a method for reducing the risk of coronary artery disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition described herein.

[0219] Incorporated by Reference

[0220] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0221] The novel features of the present invention are specifically set forth in the appended claims and are embodied in the mRNA 5' terminal region structure described herein. A better understanding of the features and advantages of the present invention will be obtained by reference to the detailed description and accompanying drawings that set forth illustrative embodiments of 5' terminal region structures or compounds (sometimes referred to herein as motifs) in which the principles of the present invention are utilized, in which:

[0222] Figure 1a Shown are the phosphorothioate modified 5' initiator and the corresponding in vitro transcribed mRNA, which has a 5' region motif identified as 1007a in the specification.

[0223] Figure 1bShown are the phosphorothioate modified 5' initiator and the corresponding in vitro transcribed mRNA having a 5' region motif identified in the specification as 1107a.

[0224] Figure 2 Shown is a comparison of the production yields of mRNAs prepared using different 5' initiators.

[0225] Figure 3 Shown is a comparison of the full-length purity of mRNA between mRNAs prepared using different 5'-end initiators.

[0226] Figure 4 Shown is a comparison of mRNA capping efficiency between mRNAs prepared using different 5' end initiators.

[0227] Figure 5 Shown is the protein expression of mRNAs including different 5' region motifs in HeLa cell lysates.

[0228] Figure 6 Shown is protein expression in primary human hepatocytes of mRNAs including different 5' region motifs.

[0229] Figure 7 Shown is a comparison of ABE base editing in mice using proteins encoded by mRNAs including different 5' region motifs.

[0230] Figure 8 Shown is a comparison of ABE base editing in NHPs using proteins encoded by two ABE mRNAs that include different 5' region motifs.

[0231] Fig. 9 Shown is the immunostimulation of mRNAs including different 5' region motifs in HEK293 cells.

[0232] Fig.10 The chirality of the phosphorothioate in the 5' initiator 1007a is shown. 1007a_d1 represents the S diastereomer, while 1007a_d2 represents the R diastereomer.

[0233] Fig.11a Shown is the efficiency of analog incorporation of 5' initiator 5232 compared to analog 1002a at doses ranging from 0.125 mM to 2 mM as determined by ion-pairing reverse phase high performance liquid chromatography (IP-RP-HPLC).

[0234] Fig.11bShown is the efficiency of analog incorporation of 5' initiator 5232 compared to analog 1002a at doses of 0.5 mM, 2 mM and 4 mM as determined by IP-RP-HPLC mass chromatography.

[0235] Fig.12 Shown are the yield and quality of mRNA prepared using various 5' initiator analogs.

[0236] Fig.13 Shown are the percentages of dsRNA detected within mRNA samples generated using 5' initiator analogs.

[0237] Fig.14 Shown are in vitro RIG-I responses to mRNAs with various 5' initiator caps.

[0238] Fig.15 Shown is the in vitro stability of various 5' initiator caps as a measure of relative pyrophosphatase resistance.

[0239] Fig.16 Shown are the in vitro 5' initiator cap affinities for protein biogenesis factors obtained by measuring relative ribosome affinity.

[0240] Fig.17a Shown are the time- and dose-dependent expression of luciferase from mRNA synthesized with various 5' initiator caps, where cells were harvested 6 hours after exposure to the mRNA.

[0241] Fig.17b Shown is the time- and dose-dependent expression of luciferase from RNA synthesized with various 5' initiator caps, where cells were harvested 24 hours after exposure to mRNA.

[0242] Fig.18a Shown are the time- and dose-dependent expression of Cas9 from mRNA synthesized with various 5' initiator caps, where cells were harvested 6 hours after exposure to the mRNA.

[0243] Fig.18b Shown are the time- and dose-dependent expression of Cas9 from mRNA synthesized with various 5' initiator caps, where cells were harvested 24 hours after exposure to the mRNA.

[0244] Fig.19 Schematic diagrams of MA004 and MA079 are shown.

[0245] Fig. 20 Shown is the in vivo editing efficiency of ABEs synthesized using 5' initiator analogs in mice.

[0246] Fig.21aShown is the in vivo immunogenicity of ABEs synthesized using 5' initiator analogs in mice as a measure of TNF-α levels over time.

[0247] Figure 21b Shown is the in vivo immunogenicity of ABEs synthesized using 5' initiator analogs in mice as a measure of IFN-γ levels over time.

[0248] Fig.21c The in vivo immunogenicity of ABEs synthesized using 5' initiator analogs in mice as a measure of IFN-α levels over time is shown.

[0249] Fig.21d Shown is the in vivo immunogenicity of ABEs synthesized using 5' initiator analogs in mice as a measure of IFN-β levels over time.

[0250] Fig.21e Shown is the in vivo immunogenicity of ABEs synthesized using 5' initiator analogs in mice as a measure of MIP-1B levels over time.

[0251] Fig.21f Shown is the in vivo immunogenicity of ABEs synthesized using 5' initiator analogs in mice as a measure of IP-10 levels over time.

[0252] Figure 21g Shown is the in vivo immunogenicity of ABEs synthesized using 5' initiator analogs in mice as a measure of IL-10 levels over time.

[0253] Figure 21h Shown is the in vivo immunogenicity of ABEs synthesized using 5' initiator analogs in mice as a measure of IL-6 levels over time. DETAILED DESCRIPTION

[0254] Certain specific details of the present description are set forth in order to provide a thorough understanding of the various embodiments. However, those skilled in the art will appreciate that the present disclosure can be practiced without these details. In other cases, well-known structures and / or methods are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context otherwise requires, throughout the specification and the appended claims, the words "comprise" and variations thereof, such as "comprises" and "comprising" should be interpreted as open, inclusive meanings, i.e., "including, but not limited to". In addition, the headings provided herein are for convenience only and do not interpret the scope or meaning of the disclosure claimed. The section headings used herein are for organizational purposes only and should not be interpreted as limiting the subject matter described.

[0255] Messenger RNA (mRNA) encoding physiologically important proteins for therapeutic applications has shown significant advantages over DNA-based plasmid and viral vectors for delivering genetic material. Such important advantages include: (i) a potentially improved level of safety when compared to potential genomic damage that may result from viral or plasmid integration, (ii) more rapid protein expression upon mRNA delivery (unlike the delayed response that typically occurs with plasmids), (iii) robust dose-dependent control of protein expression, and (iv) simplification that can facilitate large-scale synthesis of mRNA compared to the manufacture of plasmid and viral vectors.

[0256] Messenger RNA can encode almost any known protein and can be delivered to specific cells, tissues and organs by a variety of methods known to those skilled in the art. Once delivered, such mRNA is able to direct ribosomal protein expression within the targeted cells or tissues, resulting in the production of potentially hundreds of copies of the encoded protein from a single mRNA molecule.

[0257] Several structural elements present in active mRNA molecules are used to effectively translate the encoded protein. One of these elements is the 5' end region of the mRNA. In naturally occurring mRNA, the 5' end region includes a cap structure, which is an important feature of eukaryotic mRNA molecules (and some viruses). Such cap structures are 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 a reverse 7-methylguanosine (m ) connected to the 5' end of the mRNA by a triphosphate (ppp) bridge. 7 G), and this phosphate bridge is linked to the first nucleotide (N 1 ). This is usually done by m 7 G(ppp)N 1The 5' cap portion represented is called cap-0. 1 Methylation of the 2'-hydroxyl group on the ribose ring (i.e., cap-1) can provide an identifier for self-RNA, which can thereby serve to protect mRNA from the innate immune system, which in turn can improve protein expression. 7 G(ppp)N 1 The m-cap-1 structure is a known conventional cap moiety used for in vitro transcription (IVT) of mRNA.

[0258] Here, we propose new mRNA 5' region motifs and initiators. These new designs include m 7 Modification of the purine base in the G portion; phosphorothioate (PS) substitution in the triphosphate bridge and chemical modification of the phosphodiester bond, substitution of the 5' end region nucleotide with an unconventional base, extension of the 5' end region nucleotide oligomer, and chemical modification of the ribose ring. Specifically, these mRNA 5' end region structures act as the terminal 5' end region 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 establishing a barrier that prevents or interferes with mRNA decapping by 5' exonuclease activity; affect capping efficiency and reduce the formation of immunostimulatory byproducts, which can improve the safety of mRNA; and promote the manufacturability of mRNA by regulating the binding affinity to the DNA template during the IVT reaction.

[0259] definition

[0260] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present 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., John Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide a general guide to many of the terms used in this application for those skilled in the art.

[0261] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and / or" unless the context clearly dictates otherwise.

[0262] When indicating the number of substituents, the term "one or more" refers to a range from one substituent to the highest possible number of substituents, for example, replacement of one hydrogen up to replacement of all hydrogens by substituents.

[0263] The term "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0264] As used herein, the term "nucleic acid" generally refers to one or more core bases, nucleosides or nucleotides, and the term includes polynucleobases, polynucleosides and polynucleotides. Nucleic acid can include polynucleotides, mononucleotides and oligonucleotides. Nucleic acid can include DNA, RNA or a mixture thereof, and can be single-stranded, double-stranded or partially single-stranded or double-stranded, and can form a secondary structure. In some embodiments, nucleic acid has multiple double-stranded segments and single-stranded segments. For example, nucleic acid can include polynucleotides, such as mRNA, wherein there are multiple double-stranded segments.

[0265] The terms "mRNA sequence initiator", "IVT mRNA sequence initiator" and "initiator" are used interchangeably herein and generally refer to a single-stranded ribo-oligonucleotide or deoxyribo-oligonucleotide or chimeric ribo / deoxyribo-oligonucleotide, which may be naturally occurring or synthetic and generally 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 contain one or more modifying groups. The mRNA sequence initiator may be a primer, such as an oligonucleotide primer. The mRNA sequence initiator, such as an oligonucleotide primer, may comprise RNA, DNA and / or other modified nucleosides. A skilled technician is able to design and prepare an mRNA sequence initiator suitable for transcription of a DNA template sequence.

[0266] The mRNA sequence initiator can be a capping primer or a capping oligonucleotide analog. For example, the capping mRNA sequence initiator can contain an initial capping oligonucleotide analog or an initial capping oligonucleotide, which has a cap 0, a cap 1, a cap 2 or a TMG cap structure on the 5' end. In some cases, the capping initiator, such as a capping primer or a capping oligonucleotide analog, has an unmodified or open 3'-OH group, and it can be extended by RNA polymerase by incorporating NTP on the 3' end. In some cases, the initiator as described herein can cause in vitro transcription under the control of the promoter in the transcription system, and the transcription system includes necessary components: DNA template (such as DNA plasmid), RNA polymerase, nucleoside 5'-triphosphate and appropriate buffer. The initiator can be an oligonucleotide carrying a terminal 3'-OH group, which is an effective substrate for RNA polymerase. In certain embodiments, the initiator is a substrate for RNA polymerase, and can be extended by incorporating NTP on the 3' end. In some embodiments, the initiator is complementary to the DNA template at the starting site.

[0267] The term "unsubstituted" or "unmodified" in the context of mRNA sequence initiators and nucleoside triphosphates (NTPs) as used herein generally refers to the starting capping initiator and unmodified NTPs.

[0268] As used herein, the term "modified start-capping initiator" generally refers to a start-capping mRNA sequence initiator that contains one or more additional modifying groups or moieties within the sequence initiator.

[0269] As used herein, the term "modifying group or moiety" generally refers to any chemical moiety that can be attached to or substituted to an initiator of an mRNA sequence, such as an initiator at a certain position, including but not limited to sugars, nucleoside bases, triphosphate bridges and / or internucleotide phosphates (e.g., U.S. Patent Application No. 20070281308). The modifying group of the capping initiator can be a group of any nature that is compatible with the transcription process.

[0270] The term "internucleotide linkage" as used herein generally refers to one or more bonds linking two nucleosides of an initiator (eg, an oligonucleotide primer or a nucleic acid), and may be a natural phosphodiester bond or a chemically modified nucleic acid backbone bond.

[0271] As used herein, the term "polynucleotide" generally refers to a molecule comprising two or more linked nucleic acid subunits (e.g., nucleotides), and can be used interchangeably with "oligonucleotide." For example, a polynucleotide can include one or more nucleotides selected from the group consisting of nucleotides carrying the corresponding nucleosides of the nucleobases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants and combinations thereof. Nucleotides generally include nucleosides and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphates (P 3) group. Nucleotide can comprise a nucleobase, a pentose (ribose or deoxyribose) and one or more phosphate groups. Ribonucleotides comprise nucleotides in which the sugar is ribose. Deoxyribonucleotides comprise nucleotides in which the sugar is deoxyribose. Nucleotides can be nucleoside monophosphates, nucleoside diphosphates, nucleoside triphosphates or nucleoside polyphosphates. For example, nucleotides can be deoxyribonucleoside polyphosphates, such as deoxyribonucleoside triphosphates (dNTPs), and exemplary dNTPs include deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP). dNTPs can also comprise detectable labels, such as luminescent labels or markers (e.g., fluorophores). For example, nucleotides can be purines (e.g., A or G, or variants thereof) or pyrimidines (e.g., C, T or U, or variants thereof). In some instances, the polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or its derivatives or variants. 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), and encompass nucleotide sequences and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, spiral, hairpin, stem loop, protuberance, etc. In some cases, the polynucleotide is circular. The polynucleotide can have various lengths. For example, the 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), 2kb, 3kb, 4kb, 5kb, 10kb, 50kb or more. The polynucleotide can be separated from a cell or tissue. For example, a polynucleotide sequence can include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules and / or synthetic DNA / RNA analogs.

[0272] The polynucleotide may comprise one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs and / or modified nucleotides, including acyl and carbocyclic nucleotides. Examples of modified nucleotides include, but are not limited to, 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-methyl Aminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 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, etc. In some cases, a nucleotide may include a modification in its phosphate portion, including a modification to the triphosphate portion. Non-limiting examples of such modifications include longer lengths of phosphate chains (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., α-thiotriphosphates and β-thiotriphosphates). Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that are typically available for hydrogen bonding with complementary nucleotides and / or at one or more atoms that are typically unable to hydrogen bond with complementary nucleotides), the sugar moiety, or the phosphate backbone. Nucleic acid molecules may also contain amine-modified groups, such as aminoallyl 1-dUTP (aa-dUTP) and aminohexyl acrylamide-dCTP (aha-dCTP), to allow covalent attachment of amine-reactive moieties, such as N-hydroxysuccinimide esters (NHS). Substitutes for standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure may provide a higher density of sites / cubic mm, higher safety (against accidental or intentional synthesis of natural toxins), easier differentiation of light-programmed polymerases, or lower secondary structures.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.

[0273] As used herein, the terms "polypeptide", "protein" and "peptide" are used interchangeably and refer to a polymer of amino acid residues connected via peptide bonds, and it may be composed of two or more polypeptide chains. The terms "polypeptide", "protein" and "peptide" refer to a polymer of at least two amino acid monomers bound together by amide bonds. Amino acids may be L-optical isomers or D-optical isomers. More specifically, the terms "polypeptide", "protein" and "peptide" refer to molecules composed of two or more amino acids in a specific order; for example, an order as determined by the base sequence of nucleotides in 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 its own unique function. Examples are hormones, enzymes, antibodies and any fragments thereof. In some cases, a protein may be a part of a protein, for example, a domain, subdomain or motif of a protein. In some cases, a protein may be a variant (or mutation) of a protein, in which one or more amino acid residues are inserted, deleted and / or substituted into the naturally occurring (or at least known) amino acid sequence of the protein. A protein or a variant thereof may be naturally occurring or recombinant.

[0274] As used herein, "hybridization" refers to the process by which the initiator of the capped mRNA sequence anneals to the DNA template under suitably stringent conditions according to the Watson-Crick base pairing rules during the transcription reaction. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainview, New York. One skilled in the art understands 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 those with lower complementarity will not stably hybridize. For examples of hybridization conditions and parameters, see, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, FM et al., 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J., all of which are incorporated herein by reference in their entirety. In certain embodiments, hybridization may occur between nucleic acid molecules of 20-100 nucleotides in length. 45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 99, 100, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides.

[0275] As used herein, "complement", "complementary" or "complementarity" in the context of, for example, a complex of a starting capped oligonucleotide primer and a DNA template refers to the standard Watson / Crick base pairing rules. 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; duplexes may contain mismatched base pairs, denatured or unmatched nucleotides. One skilled in the art can determine duplex stability empirically taking into account many variables including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, the incidence of mismatched base pairs, ionic strength, components of the hybridization buffer, and reaction conditions.

[0276] Complementarity can be "complete" or "total", in which all nucleotide bases of the two nucleic acid chains are matched according to the generally accepted base pairing rules; complementarity can be "partial", in which only some nucleotide bases of the capped mRNA sequence initiator and the DNA target are matched according to the generally accepted base pairing rules; or complementarity can be "non-existent", in which no nucleotide bases of the two nucleic acid chains are matched according to the generally accepted base pairing rules. The degree of complementarity between the capped mRNA sequence initiator (e.g., capping primer) and the DNA template may have a significant effect on the hybridization strength between the starting capped oligonucleotide and the DNA template and the corresponding reaction efficiency. The term complementarity can also be used to refer to individual nucleotides. For example, in contrast or comparison to the complementarity between the rest of the capped mRNA sequence initiator and the DNA chain, a specific nucleotide in the oligonucleotide can be noted for its complementarity or lack of complementarity with the nucleotides in the other chain.

[0277] As used herein, the terms "completely," "entirely," or "perfectly" complementary mean that every nucleotide base of the capped mRNA sequence initiator and the DNA target is exactly matched according to the generally accepted base pairing rules.

[0278] As used herein, the term "substantially complementary" refers to two sequences that hybridize under stringent hybridization conditions. It will be appreciated by those skilled in the art that substantially complementary sequences do not need to hybridize along their entire length. Specifically, substantially complementary sequences may include a continuous sequence of bases that do not hybridize to a target sequence, and may be located 3' or 5' to a continuous sequence of bases that hybridize to a target sequence under stringent hybridization conditions.

[0279] As used herein, the term "nucleoside" includes all naturally occurring nucleosides, including all forms of nucleoside bases and furanosides found in nature. The most commonly found base rings in naturally occurring nucleosides are purine rings and pyrimidine rings. Naturally occurring purine rings include, for example, adenine, guanine, and N- 6 -methyladenine. Naturally occurring pyrimidine rings include, for example, cytosine, thymine, 5-methylcytosine, pseudouracil. For example, naturally occurring nucleosides include, but are not limited to, ribose, 2'-O-methyl or 2'-deoxyribose derivatives of adenosine, guanosine, cytidine, thymidine, uridine, inosine, 7-methylguanosine or pseudouridine.

[0280] As used herein, the term "nucleoside analogs", "modified nucleosides" or "nucleoside derivatives" include synthetic nucleosides as described herein. Nucleoside derivatives also include nucleosides with modified bases and / or sugar moieties, with or without protecting groups, and include, for example, 2'-deoxy-2'-fluorouridine, 5-fluorouridine, and the like. The compounds and methods provided herein include such base rings and synthetic analogs thereof, as well as non-natural heterocyclic substituted base sugars and acyclic substituted base sugars. Other nucleoside derivatives that can be used in the present disclosure include, for example, LNA nucleosides, halogen substituted purines (e.g., 6-fluoropurine), halogen substituted pyrimidines, N 6 -ethyladenine, N 4 -(Alkyl)-cytosine, 5-ethylcytosine, etc. (U.S. Patent No. 6,762,298).

[0281] As used herein, the terms "universal base," "degenerate base," "universal base analog," and "degenerate base analog" include, for example, nucleoside analogs with artificial bases that, in certain embodiments, can be recognized by 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 disclosed in Loakes, D., Nucleic Acids Res., 29:2437-2447 (2001); Crey-Desbiolles, C. et al., Nucleic Acids Res., 33:1532-1543 (2005); Kincaid, K., et al., Nucleic Acids Res., 33:2620-2628 (2005); Preparata, FP, Oliver, JS, J. Comput. Biol. 753-765 (2004); and Hill, F. et al., Proc Natl Acad. Sci. USA, 95:4258-4263 (1998)).

[0282] As used herein, the term "modified NTP" refers to a nucleoside 5'-triphosphate having a chemical moiety group attached to any position, including a sugar, a base, a triphosphate chain, or any combination of these three positions. Examples of such NTPs can be found in, for example, "Nucleoside Triphosphates and Their Analogs: Chemistry, Biotechnology and Biological Applications," Vaghefi, M., ed., Taylor and Francis, Boca Raton (2005).

[0283] As used herein, the term "specificity" when used to refer to the 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 with a portion of a DNA template when the capped mRNA sequence initiator and the DNA chain are aligned. Possibly preferred higher levels of sequence identity 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.

[0284] 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 mimetics known to those skilled in the art are also suitable for use in the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide oligonucleotides with improved properties. For example, nuclease-resistant oligonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine or tuberculocidin) and any one of the oligomer modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used. When a natural base is substituted with a non-natural base and / or a universal base, the nucleotide is considered to include a modified nucleobase and / or nucleobase modification herein. Modified nucleobases and / or nucleobase modifications also include natural bases, non-natural bases and universal bases, including conjugated moieties, such as ligands described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups, which can be conjugated to nucleobases via suitable alkyl, alkenyl or linkers with amide bonds.

[0285] 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, but are not limited to, other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanosine, tuberculocidin, 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, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8 -(amino)adenine, 8-(halo)adenine, 8-(hydroxy)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-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxy guanine, 8-(thioalkyl)guanine, 8-(thio)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, 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-( 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-(guanidinoalkyl)uracil, 5-(l,uracil, 5-(3-oxadiazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halogenated)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-methylpseudouracil (N1-methylpseudouracil), 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylidene)-pseudouracil, 1-(aminocarbonylethylidene)-2(thio)-pseudouracil uracil, 1-(aminocarbonylethylidene)-4-(thio)pseudouracil, 1-(aminocarbonylethylidene)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylidene)-pseudouracil, 1-(aminoalkylamino-carbonylethylidene)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylidene)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylidene)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 1,3-(diaza)-2-(oxo)-phenothiazine-1-yl, 1-(aza)- substituted-1,3-(diaza)-2-(thioxo)-3-(aza)-phenoxazin-1-yl, 7-substituted-1-(aza)-2-(thioxo)-3-(aza)-phenoxazin-1-yl, 7-substituted-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted-1-(aza)-2-(thioxo)-3-(aza)-phenoxazin-1-yl, 7-substituted-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted-1-(aza)-2-(thioxo)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thioxo)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazine-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazine-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazine-1-yl, yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-l-yl, l,3,5-(triaza)-2,6-(dioxane)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tuberculocidin, isoguanine nucleoside, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolo[pi] pyrimidinyl, 3-(methyl)isoquinolone, 5-(methyl)isoquinolone, 3-(methyl)-7-(propynyl)isoquinolone, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyrazinyl, isoquinolone, 7-(propynyl)isoquinolone, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, stilbene, tetraphenylene, pentacene, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N2-substituted purine, N6-substituted purine, 06-substituted purine, substituted 1,2,4-triazole, pyrrolopyrimidin-2-one-3-yl, 6-phenyl-pyrrolopyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolopyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolopyrimidin-2-one-3-yl, di-ortho-substituted-6-phenyl-pyrrolopyrimidin-2-one-3-yl, p-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-one-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-one-3-yl, di-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O-alkylated 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 the oligonucleotide duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisoquinolone, 5-methylisoquinolone, 3-methyl-7-propynylisoquinolone, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9- methyl-imidazopyridinyl, pyrrolopyrazinyl, isoquinolone, 7-propynylisoquinolone, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, stilbene, tetracene, pentacene, and structural derivatives thereof (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447, which is incorporated herein by reference in its entirety). Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in International Application No. PCT US09 / 038425 filed March 26, 2009; those disclosed in Concise Encyclopedia Of Polymer Science And Engineering, pp. 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 by Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. All contents of the above documents are incorporated herein by reference.

[0286] 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 examined. Non-limiting examples include whole blood, plasma, saliva, cheek swabs, stool samples, urine samples, cell mass, or any other body fluid or tissue.

[0287] As used herein, the terms "administer", "administering", "administration", etc. refer to methods that can be used to deliver a compound or composition to a desired biological site of action. These methods include, but are not limited to, oral routes (po), intraduodenal routes (id), parenteral injections (including intravenous (iv), subcutaneous (sc), intraperitoneal (ip), intramuscular (im), intravascular or infusion (inf.)), topical (top.) and rectal (pr) administration. Those skilled in the art are familiar with administration techniques that can be used for the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally.

[0288] As used herein, the terms "co-administered," "co-administered," and the like are meant to encompass administration of selected therapeutic agents to a single patient, and are intended to encompass treatment regimens in which the agents are administered by the same or different routes of administration, or at the same or different times.

[0289] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of an agent or compound administered that will alleviate 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 a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use can be an amount of an agent that provides a clinically significant reduction in one or more disease symptoms. An appropriate "effective" amount can be determined in an individual case using techniques such as dose escalation studies.

[0290] The terms "enhance" or "enhancing" as used herein means to increase or prolong the amount, potency, or duration of a desired effect.

[0291] As used herein, "carbohydrate" refers to a compound that is itself a carbohydrate composed of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched or cyclic), wherein an oxygen, nitrogen or sulfur atom is bonded to each carbon atom; or a compound having as a part thereof a carbohydrate portion composed of one or more monosaccharide units, each monosaccharide unit having at least six carbon atoms (which may be linear, branched or cyclic), wherein an oxygen, nitrogen or sulfur atom is 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 C5 and above (preferably C5-C8) sugars; disaccharides and trisaccharides include sugars having two or three monosaccharide units (preferably C5-C8).

[0292] The term "monosaccharide" includes the radicals of allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fucose, galactosamine, D-galactitol, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucosaminol, glucose, glucose-6-phosphate, gulose, glyceraldehyde, L-glycero-D-mannos-heprose, glycerol, glycerone, gulose idose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinose, quinosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, throse, xylose and xylulose. Monosaccharides can be in D- or L- configuration. Monosaccharides can also be deoxy sugars (alcoholic hydroxyl groups are replaced by hydrogen), amino sugars (alcoholic hydroxyl groups are replaced by amino groups), thio sugars (alcoholic hydroxyl groups are replaced by thiol, or C=O is replaced by C=S, or the ring oxygen of the ring form is replaced by sulfur), seleno sugars, telluryl sugars, aza sugars (ring carbon is replaced by nitrogen), imino sugars (ring oxygen is replaced by nitrogen), phosphano sugars (ring oxygen is replaced by phosphorus), phospha sugars (ring carbon is replaced by phosphorus), C-substituted monosaccharides (hydrogen on non-terminal carbon atoms is replaced by carbon), unsaturated monosaccharides, sugar alcohols (carbonyl groups are replaced by CHOH groups), aldonic acids (aldehyde groups are replaced by carboxyl groups), ketoaldonic acids, uronic acids, aldaric acids, etc. Amino sugars include amino monosaccharides, preferably galactosamine, glutamine, mannosamine, fucosamine, quinavosamine, neuraminic acid, muramic acid, lactosediamine, acosamine, bacillosamine, daunosamine, erythromycin deoxysaccharide, forosamine, galactosamine, kanosamine, kanosamine, mycosamine, myosamine, persosamine, pneumosamine, purpurosamine, rhodosmine. It should be understood that monosaccharides and the like can also be substituted.

[0293] The terms "disaccharide", "trisaccharide" and "polysaccharide" include abeconose, acarbose, amicetose, amylopectin, amylose, apiose, arcanose, ascarose, ascorbic acid, boivinose, cellobiose, cellotriose, cellulose, potato triose, chalose, chitin, colisaccharide, cyclodextrin, macetosan, dextrin, 2-deoxyribose, 2-deoxyglucose, diglycan, digitalose, digitalisose, evalose, evemitrose, oligofructose, galto-oligosaccharide, gentiotriose, genitiobiose, dextran, gluicogen, glylcogen, hamamelisose, heparin, inulin, isolevoglucosenone, isomaltose, isomaltose, Maltotriose, isoglucosylmaltose, kojibiose, lactose, aminolactoside, lactosyldiamine, laminaribiose, levoglucosan, levoglucosone, β-maltose, maltotriose, mannan-oligosaccharide, amnninotriose, melezitose, melibiose, muramic acid, mycarose, allose, neuraminic acid, migerose, nojirimycon, noviose, oleanose, panose, palatose, carbenic acid The term "disaccharide" refers to a group of sucrose, ...

[0294] The term "subject" or "patient" encompasses mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (such as chimpanzees), and other ape and monkey species; farm animals, such as cattle, horses, sheep, goats, pigs; livestock, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs, etc. In one aspect, the mammal is a human. The term "animal" as used herein includes humans and non-human animals. In one embodiment, a "non-human animal" is a mammal, such as a rodent, such as a rat or a mouse. In one embodiment, the non-human animal is a mouse.

[0295] As used herein, the terms "treat," "treating," or "treatment" include alleviating, reducing, or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, e.g., preventing the development of a disease or condition, alleviating a disease or condition, causing regression of a disease or condition, alleviating a condition caused by a disease or condition, or prophylactically and / or therapeutically stopping a symptom of a disease or condition. The term "treatment" also encompasses the concepts of "prevent," "preventing," and "prevention," as described below. It should be understood that, although not excluded, treating a disorder or condition does not require complete elimination of the disorder, condition, or symptom associated therewith.

[0296] The terms "preventing" or "prevention" of a disease state means causing clinical symptoms of a disease state to not develop in a subject who is exposed to or susceptible to the disease state but does not yet experience or display symptoms of the disease state.

[0297] The terms "pharmaceutical composition" and "pharmaceutical formulation" (or "formulation") are used interchangeably and refer to a mixture or solution comprising a therapeutically effective amount of an active drug ingredient and one or more pharmaceutically acceptable excipients, for administration to a subject, such as a human in need thereof.

[0298] The term "drug combination" as used herein refers to a product produced by mixing or combining more than one pharmaceutically active ingredient, and includes fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients (e.g., compounds described herein and co-agents) are all administered to the patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients (e.g., compounds described herein and co-agents) are administered to the patient simultaneously, in parallel or sequentially as separate entities, without specific interval time restrictions, wherein such administration provides effective levels of two compounds in the patient's body. The latter is also applicable to mixture therapy, for example, the administration of three or more active ingredients.

[0299] The term "pharmaceutically acceptable" refers to the properties of materials useful for preparing pharmaceutical compositions that are generally safe, non-toxic, and neither biologically nor otherwise undesirable, and are acceptable for veterinary as well as human pharmaceutical use. "Pharmaceutically acceptable" may refer to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of a compound and is relatively non-toxic, for example, such a material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0300] The terms "pharmaceutically acceptable excipient," "pharmaceutically acceptable carrier," "pharmaceutically acceptable vehicle," and "therapeutically inert excipient" are used interchangeably and refer to any pharmaceutically acceptable ingredient in a pharmaceutical composition that has no therapeutic activity and is non-toxic to the subject to which it is administered, such as a disintegrant, binder, filler, solvent, buffer, tonicity agent, stabilizer, antioxidant, surfactant, carrier, diluent, excipient, preservative, or lubricant used to formulate a drug product.

[0301] The terms "base editing" and "base correction" are used interchangeably to indicate a base change or mutation that results in a base modification at a target sequence within a target gene. In certain embodiments, base editing occurs at a single base of the target sequence. In preferred embodiments, base editing does not involve double-strand breaks of the target sequence.

[0302] As used herein, the term "siRNA" refers to an agent that mediates the targeted cleavage of an RNA transcript. These agents are associated with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). The agent that effectively induces RNA interference is also referred to herein as siRNA, RNAi agent, or iRNA agent. As used herein, the term siRNA includes microRNA and precursor microRNA (pre-microRNA). As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0303] The term "2'-O-methoxyethyl" (also known as 2'-MOE, 2'-O(CH 2 ) 2 —OCH 3 and 2'-O-(2-methoxyethyl)) refers to an O-methoxy-ethyl modification of the 2' position of the furosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0304] The term "2'-O-methoxyethyl nucleotide" means a nucleotide comprising a 2'-O-methoxyethyl modified sugar moiety.

[0305] The term "5-methylcytosine" means cytosine modified with a methyl group attached to the 5' position. 5-Methylcytosine is a modified nucleobase.

[0306] The term "oxo" refers to a =0 substituent.

[0307] The term "alkyl" refers to a straight or branched hydrocarbon chain radical having from one to twenty carbon atoms and attached to the rest of the molecule by a single bond. Alkyl groups comprising up to 10 carbon atoms are referred to as C 1 -C 10Alkyl groups, for example, up to 6 carbon atoms are C 1 -C 6 Alkyl. Alkyl (and other moieties defined herein) including other numbers of carbon atoms are similarly represented. Alkyl includes but is not limited to C 1 -C 10 Alkyl, C 1 -C 9 Alkyl, C 1 -C 8 Alkyl, C 1 -C 7 Alkyl, C 1 -C 6 Alkyl, C 1 -C 5 Alkyl, C 1 -C 4 Alkyl, C 1 -C 3 Alkyl, C 1 -C 2 Alkyl, C 2 -C 8 Alkyl, C 3 -C 8 Alkyl and C 4 -C 8 Alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, isobutyl, sec-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethylpropyl, and the like. In some embodiments, the alkyl group is methyl or ethyl. In some embodiments, the alkyl group is -CH(CH 3 ) 2 or -C(CH 3 ) 3 Unless otherwise specifically stated in the specification, an alkyl group 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 group. In some embodiments, an alkylene group is -CH 2 -、-CH 2 CH 2 -or-CH 2 CH 2 CH 2 In some embodiments, the alkylene group is -CH 2 In some embodiments, the alkylene group is -CH 2 CH 2 In some embodiments, the alkylene group is -CH 2 CH 2 CH 2 -.

[0308] The term "alkoxy" refers to a group of formula -OR, wherein R is an alkyl group as defined. Unless otherwise specifically stated in this specification, the alkoxy group may be optionally substituted as described below. Representative alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy. In some embodiments, the alkoxy group is a methoxy group. In some embodiments, the alkoxy group is an ethoxy group.

[0309] The term "alkylamino" refers to a group of the formula -NHR or -NRR, wherein each R is independently an alkyl group as defined above. Unless stated otherwise specifically in the specification, an alkylamino group may be optionally substituted as described below.

[0310] The term "alkenyl" refers to a type of alkyl group in which at least one carbon-carbon double bond is present. In one embodiment, the alkenyl group has the formula -C(R)=CR 2 , wherein R refers to the remainder of the alkenyl group, which may be the same or different. In some embodiments, R is H or an alkyl group. In some embodiments, the alkenyl group is selected from ethylene (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, and the like. Non-limiting examples of alkenyl groups include -CH=CH 2 、-C(CH 3 )=CH 2 、-CH=CHCH 3 、-C(CH 3 )=CHCH 3 and -CH 2 CH=CH 2 Depending on the structure, an alkenyl group can be monovalent or divalent (ie, alkenylene).

[0311] The term "alkynyl" refers to a class of alkyl groups in which at least one carbon-carbon triple bond is present. Thus, "alkynylene" may refer to a divalent alkynyl group. In one embodiment, the alkenyl group has the formula -C≡CR, wherein R refers to the remainder of the alkynyl group. In some embodiments, R is H or an alkyl group. In some embodiments, the alkynyl group is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Non-limiting examples of alkynyl groups include -C≡CH, -C≡CCH 3 -C≡CCH 2 CH 3 、-CH 2 C≡CH.

[0312] The term "aryl" refers to an aromatic ring in which each of the atoms forming the ring is a carbon atom. The aryl group may be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl and naphthyl. In some embodiments, the aryl group is phenyl. Depending on the structure, the aryl group may be monovalent or divalent (i.e., "arylene"). Unless otherwise specifically stated in this specification, the term "aryl" or the prefix "ar-" (e.g., in "aralkyl") means an aryl group that includes optional substitution. In some embodiments, the aryl group is partially reduced to form a cycloalkyl group as defined herein. In some embodiments, the aryl group is fully reduced to form a cycloalkyl group as defined herein. In some embodiments, the aryl group is C 6 -C 14 In some embodiments, aryl is C 6 -C 10 Aryl.

[0313] The term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic group, wherein each of the atoms forming the ring (i.e., skeleton 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 this case, the cycloalkyl is bonded by a non-aromatic ring carbon atom). The cycloalkyl includes a group with 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls with three to ten carbon atoms, three to eight carbon atoms, three to six carbon atoms, or three to five carbon atoms. Monocyclic cycloalkyls 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-dihydronaphthyl, 1,4-dihydronaphthyl, tetrainyl, decalinyl, 3,4-dihydronaphthyl-1(2H)-one, spiro[2.2]pentyl, norbornyl, and bicyclo[1.1.1]pentyl. Unless specifically stated otherwise in this specification, cycloalkyl groups may be optionally substituted. Depending on the structure, cycloalkyl groups may be monovalent or divalent (i.e., cycloalkylene groups).

[0314] The term "haloalkyl" means an alkyl group in which at least one of the hydrogen atoms of the alkyl group has been replaced by identical or different halogen atoms, in particular fluorine atoms. Examples of haloalkyl groups 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" means an alkyl group in which all the hydrogen atoms of the alkyl group have been replaced by identical or different halogen atoms.

[0315] The term "heteroalkylene" refers to an alkyl group as described above in which one or more carbon atoms of the alkyl group are replaced by an O, N or S atom. "Heteroalkylene" or "heteroalkylene chain" refers to a straight or branched divalent heteroalkyl chain that connects the rest of the molecule to a radical. Unless otherwise specifically stated in this specification, a heteroalkyl or heteroalkylene group may be optionally substituted as described below. Representative heteroalkylene groups include, but are not limited to, -OCH 2 CH 2 O-、-OCH 2 CH 2 OCH 2 CH 2 O- or -OCH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 O-.

[0316] The term "heterocycloalkyl" refers to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen and sulfur. Unless specifically stated otherwise in this specification, the heterocycloalkyl group may be a monocyclic or bicyclic ring system, which may contain a fused (when fused to an aryl or heteroaryl ring, the heterocycloalkyl group is bonded through a non-aromatic ring atom) or a bridged ring system. The nitrogen, carbon or sulfur atoms in the heterocyclyl group may optionally be oxidized. The nitrogen atom may optionally be quaternized. The heterocycloalkyl group is partially or fully saturated. Examples of heterocycloalkyl include, but are not limited to, dioxolanyl, thienyl [1,3] dithianyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, 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 indicated, the ring of heterocycloalkyl has 2 to 12 carbons. In some embodiments, the ring of heterocycloalkyl has 2 to 10 carbons. In some embodiments, the ring of heterocycloalkyl has 2 to 10 carbons and 1 or 2 N atoms. In some embodiments, the ring of heterocycloalkyl has 2 to 10 carbons and 3 or 4 N atoms. In some embodiments, the ring of heterocycloalkyl has 2 to 12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms and 0-1 S atoms. In some embodiments, the ring of heterocycloalkyl has 2 to 12 carbons, 1-3 N atoms, 0-1 O atoms and 0-1 S atoms. It should be understood that when referring to the number of carbon atoms in heterocycloalkyl, the number of carbon atoms in heterocycloalkyl is different from the total number of atoms (including heteroatoms) constituting heterocycloalkyl (i.e., the skeleton atoms of heterocycloalkyl ring). Unless otherwise specifically stated in this specification, heterocycloalkyl may be optionally substituted. As used herein, the term "heterocycloalkylene" may refer to a divalent heterocycloalkyl group.

[0317] The term "heteroaryl" refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen and sulfur. Heteroaryl is monocyclic or bicyclic. Illustrative examples of monocyclic heteroaryl groups include pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazolyl, 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 pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl and furazolyl. 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 pyridyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl or furanyl. In some embodiments, the ring of heteroaryl contains 0-6 N atoms. In some embodiments, the ring of heteroaryl contains 1-4 N atoms. In some embodiments, the heteroaryl ring contains 4-6 N atoms. In some embodiments, the heteroaryl ring contains 0-4 N atoms, 0-1 O atoms, 0-1 P atoms, and 0-1 S atoms. In some embodiments, the heteroaryl ring contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms. In some embodiments, the heteroaryl is C 1 -C 9 In some embodiments, the monocyclic heteroaryl is C 1 -C 5 In some embodiments, the monocyclic heteroaryl is a 5-membered or 6-membered heteroaryl. In some embodiments, the bicyclic heteroaryl is C 6 -C 9 Heteroaryl. In some embodiments, the heteroaryl is partially reduced to form a heterocycloalkyl as defined herein. In some embodiments, the heteroaryl is fully reduced to form a heterocycloalkyl as defined herein. Depending on the structure, the heteroaryl can be monovalent or divalent (i.e., a "heteroarylene" group).

[0318] Unless otherwise indicated, the terms "substituted," "substituent," and the like may refer to the individual and independent replacement of one or more hydrogen radicals in a given structure with a radical of a specified substituent, including but not limited to: D, halogen, -CN, -NH 2 、-NH(alkyl), -N(alkyl) 2 、-OH、-CO 2H, -CO 2 Alkyl, -C(=O)NH 2 、-C(=O)NH(alkyl)、-C(=O)N(alkyl) 2 、-S(=O) 2 NH 2 、-S(=O) 2 NH(alkyl), -S(=O) 2 N(alkyl) 2 In some other embodiments, the optional substituents are independently selected from D, halogen, -CN, -NH 2 、-NH(CH 3 )、-N(CH 3 ) 2 、-OH、-CO 2 H, -CO 2 (C 1 -C 4 alkyl), -C(=O)NH 2 、-C(=O)NH(C 1 -C 4 alkyl), -C(=O)N(C 1 -C 4 alkyl) 2 、-S(=O) 2 NH 2 、-S(=O) 2 NH(C 1 -C 4 Alkyl), -S(=O) 2 N(C 1 -C 4 alkyl) 2 , C 1 -C 4 Alkyl, C 3 -C 6 Cycloalkyl, C 1 -C 4 Fluoroalkyl, C 1 -C 4 Heteroalkyl, C 1 -C 4 Alkoxy, C 1 -C 4 Fluoroalkoxy, -SC 1 -C 4 Alkyl, -S(=O)C 1 -C 4 Alkyl and -S(=O) 2 (C 1-C 4 In some embodiments, the optional substituents are independently selected from D, halogen, -CN, -NH 2 、-OH、-NH(CH 3 )、-N(CH 3 ) 2 、-NH(cyclopropyl), -CH 3 、-CH 2 CH 3 , -CF 3 、-OCH 3 and-OCF 3 In some embodiments, the substituted groups are substituted with one or two of the aforementioned groups. In some embodiments, the optional substituents on aliphatic carbon atoms (acyclic or cyclic) comprise oxo (=0).

[0319] The term "unsubstituted" means that the specified group does not carry a substituent. The term "optionally substituted" means that the specified group is unsubstituted or substituted with one or more substituents, which are independently selected from possible substituents. When indicating the number of substituents, the term "one or more" means from one substituent to the highest possible number of substitutions, i.e., one hydrogen is replaced by a substituent until all hydrogens are replaced by substituents.

[0320] "About" means within ±10% of a value. For example, if it is stated that "a marker can be increased by about 50%", it implies that the marker can be increased by 45%-55%.

[0321] "Active pharmaceutical agent" refers to the substance in a pharmaceutical composition that provides a therapeutic benefit when administered to a subject.

[0322] "Dosage unit" refers to the form in which a pharmaceutical formulation is provided, such as a pill, tablet, or other dosage unit known in the art. In certain embodiments, a dosage unit is a vial containing a lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial containing a reconstituted antisense oligonucleotide.

[0323] "Dose" refers to a specific amount of a pharmaceutical preparation provided in a single administration or within a specific time period. In certain embodiments, the dose can be administered in one, two or more boluses, tablets or injections. For example, in certain embodiments requiring subcutaneous administration, the desired dose requires a volume that is not easily accommodated by a single injection, and therefore, two or more injections can be used to achieve the desired dose. In certain embodiments, the pharmaceutical preparation is administered by continuous infusion over an extended time period. The dose can be stated as the amount of the pharmaceutical preparation per hour, day, week or month. The dose can also be expressed as the mass (e.g. mg / kg or g / kg) of the drug product or drug substance per unit mass of the subject's tissue.

[0324] "Modified internucleoside linkage" refers to a substitution or any alteration of a naturally occurring internucleoside linkage. For example, a phosphorothioate linkage is a modified internucleoside linkage.

[0325] "Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymine, 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).

[0326] "Modified nucleoside" refers to a nucleoside having at least one modified sugar moiety and / or a modified nucleobase.

[0327] "Modified nucleotide" refers to a nucleotide having at least one modified sugar moiety, a modified internucleoside linkage, and / or a modified nucleobase.

[0328] A "modified oligonucleotide" refers to an oligonucleotide comprising at least one modified nucleotide.

[0329] "Modified sugar" refers to a substitution or alteration of a natural sugar. For example, a 2'-O-methoxyethyl modified sugar is a modified sugar.

[0330] "Motif" refers to the pattern of chemically distinct regions in an antisense compound.

[0331] "Statin" refers to an agent that inhibits the activity of HMG-CoA reductase.

[0332] "Symptoms of a cardiovascular disease or condition" refers to phenomena caused by and accompanying a cardiovascular disease or condition and serving as an indicator of a cardiovascular disease or condition. For example, angina; chest pain; shortness of breath; palpitations; weakness; dizziness; nausea; sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling of the lower limbs; cyanosis; fatigue; fainting; facial paralysis; limb paralysis; claudication or muscle cramps; abdominal distension; or fever are symptoms of a cardiovascular disease or condition.

[0333] "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 an ANGPTL3 gene can be targeted by a guide nucleotide associated with a Cas9 nuclease.

[0334] Methods for detecting and / or measuring polypeptides in biological materials are well known in the art and include, but are 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 an 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.

[0335] Methods for detecting and / or measuring RNA in biological material are well known in the art and include, but are not limited to, Western blot, RNA protection assay, RT PCR. Suitable methods are described in Michael R. Green, Joseph Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Peter MacCallum 2012, page 2,028, ISBN 978-1-936113-42-2.

[0336] Ribonucleoprotein (RNP) refers to a nuclear protein containing RNA. RNP can be a complex of ribonucleic acid and RNA binding protein. This combination can also be called protein-RNA complex. These complexes can play many biological functions, including but not limited to DNA replication, DNA modification, gene expression, metabolism and RNA modification, and pre-mRNA splicing.

[0337] As used herein, the terms "biomarker" or "marker" are used interchangeably and refer to any biochemical marker, serological marker, genetic marker, or other clinical or echographic characteristic 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).

[0338] As used herein, the term "antibody" includes but is not limited to a population of immunoglobulin molecules or a fragment of an immunoglobulin molecule, which may be polyclonal or monoclonal and have any class and isotype. Immunoglobulins have five major classes: IgA, IgD, IgE, IgG and IgM, and several of these may also be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 (human), IgA2 (human), IgAa (dog), IgAb (dog), IgAc (dog) and IgAd (dog). Such fragments typically include a portion of an antibody molecule that specifically binds to an antigen. For example, a fragment of an immunoglobulin molecule referred to in the art as Fab, Fab' or F(ab')2 is included within the meaning of the term antibody.

[0339] As used herein, the term "label" refers to a detectable compound, composition or solid support that can be directly or indirectly conjugated to a monoclonal antibody or protein (e.g., via covalent or non-covalent means, alone or encapsulated). The label can be detectable by itself (e.g., radioisotope labels, chemiluminescent dyes, electrochemical labels, metal chelates, latex particles or fluorescent markers), or in the case of enzyme labels, can catalyze chemical changes in detectable substrate compounds or compositions (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.). The label used in the present disclosure can be, but is not limited to, alkaline phosphatase; glucose-6-phosphate dehydrogenase ("G6PDH"); horseradish peroxidase (HRP); chemiluminescent agents (e.g., isoluminol), fluorescent agents (e.g., fluorescein and rhodamine compounds); ribozymes; and dyes. The label can also be a specific binding molecule that is detectable itself (e.g., biotin, avidin, streptavidin, digoxigenin, maltose, oligohistidine (e.g., hexahistidine), 2,4-dinitrobenzene, phenylarsenate, ssDNA, dsDNA, etc.). The use of a label generates a signal that can be detected by means such as detection of electromagnetic radiation or direct visualization, and which can optionally be measured.

[0340] "Substantial binding" or "substantially binding" refers to the amount of specific binding or recognition between molecules in a mixture determined under specific assay conditions. In its broadest aspect, substantial binding involves the difference between the inability of a first molecule to bind to or recognize a second molecule and the ability of the first molecule to bind to or recognize a third molecule, such that the difference is sufficient to allow a meaningful assay to distinguish specific binding under a specific set of assay conditions, including the relative concentrations of the molecules and the time and temperature of incubation. On the other hand, in the sense of cross-reactivity, a molecule is substantially unable to bind to or recognize another molecule, wherein the first molecule exhibits less than 25% of the reactivity exhibited to the third molecule under a specific set of assay conditions including the relative concentrations of the molecules and incubation, such as less than 10%, e.g., less than 5%. Specific binding can be tested using a number of widely known methods, e.g., immunohistochemical assays, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), or Western blot assays.

[0341] As used herein, the term "substantially identical amino acid sequences" encompass amino acid sequences that are similar but not identical to naturally occurring amino acid sequences. For example, an amino acid sequence (e.g., a polypeptide) having an amino acid sequence substantially identical to a flagellin protein may have one or more modifications, such as amino acid additions, deletions, or substitutions, relative to the amino acid sequence of a naturally occurring flagellin protein, provided that the modified polypeptide substantially retains at least one biological activity of the flagellin protein, such as immunoreactivity. The "percent similarity" between two sequences is a function of the number of positions containing matching residues or conserved residues common to the two sequences divided by the number of comparison positions multiplied by 100. In this regard, a conserved residue in a sequence is a residue that is physically or functionally similar to a corresponding reference residue, for example, it has a similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.

[0342] 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.

[0343] The term "heterologous" refers to any two or more nucleic acid or polypeptide sequences that are not generally found in the same relationship to each other in nature. For example, a heterologous nucleic acid is generally recombinantly produced and has two or more sequences, e.g., sequences from unrelated genes, that are arranged to produce a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous polypeptide generally refers to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0344] As used herein, the term "fragment" refers to a peptide, polypeptide or protein segment comprising the 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.

[0345] An "epitope" is an antigenic determinant on a polypeptide that is recognized and bound by a paratope on a polypeptide-specific antibody (eg, an IBD-related antibody).

[0346] The term "clinical factors" includes symptoms of a patient associated with a disease. Examples of clinical factors of cardiovascular disease include, but are not limited to, angina; chest pain; shortness of breath; palpitations; weakness; dizziness; nausea; sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling of the lower limbs; cyanosis; fatigue; syncope; facial paralysis; quadriplegia; claudication or muscle cramps; abdominal distension; or fever. In some embodiments, the diagnosis of cardiovascular disease is based on the use of a statistical algorithm to analyze the presence or level of one or more markers in a patient and determine whether the patient has a combination of one or more clinical factors.

[0347] The term "prognosis" includes a prediction of the likely course and outcome of a pathological condition (e.g., cardiovascular disease), or the likelihood of recovery from a disease. In some embodiments, the use of a statistical algorithm provides a prognosis for a patient's cardiovascular disease. For example, the prognosis may be surgery, the development of one or more clinical factors, the development of intestinal cancer, or recovery from a disease.

[0348] The term "CRISPR-Cas system" referred to herein includes CRISPR-associated proteins translated by mRNA encoding the protein and a single guide RNA. In some embodiments, the CRISPR-associated protein may have inherent endonuclease activity. In some embodiments, the CRISPR-Cas system promotes guide RNA-mediated gene changes. The protein or the protein produced by the mRNA encoding the protein can promote bases or nucleobases and / or gene editing within the targeted segment of the gene of interest.

[0349] Provided herein are methods and compositions for targeted delivery of therapeutic agents (such as nucleic acid agents). As used herein, therapeutic agents can be connected or associated with a targeting moiety to help targeted delivery. For example, a therapeutic agent and a targeting moiety can form a conjugate. The therapeutic agent can include a programmable nuclease system guided by a nucleic acid 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 drug for treating any disease, disorder or condition associated with a gene, wherein the gene can be changed, manipulated, edited and modified by the insertion or deletion of DNA. According to another aspect of the present disclosure, the modified guide RNA can be used to change a gene by missing, replacing, repairing or inserting a segment of one or more nucleotides or DNA. This can be performed in a microorganism or animal, particularly a mammal, and more particularly in a human. The guide RNA of the present disclosure and the CRISPR / Cas system known in the art can be used to genetically alter or correct human cells or tissues in vitro, and then inserted back into patients in need. In another aspect of the present disclosure, a pharmaceutical composition is provided, which includes a modified guide RNA and a CRISPR-Cas system according to the present disclosure and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may include a vector or cell having a modified guide RNA of the present disclosure.

[0350] Compound

[0351] In one aspect, described herein is an IVT mRNA sequence initiator of formula (I) or a salt or solvate thereof:

[0352]

[0353] in

[0354] B 1 for

[0355] B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase;

[0356] 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 NHCH3 or NHC(=O)CH 3 ;

[0357] 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 ;

[0358] Q 1 and Q 4 Each is 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 )-;

[0359] Q 2 and Q 3 are independently -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;

[0360] X 1 , X 2 , X 3 , X4 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 ;

[0361] Y 1 , Y 2 , Y 3 , Y 4 and Y n are independently =O, =S, =NH or =NCH 3 ;

[0362] A. A 1 and A 2 are independently -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;and

[0363] p = 0, 1, 2, 3, 4, 5 or 6,

[0364] The condition is (i) Z 1 , Z 2 and Z 3 is hydrogen; (ii) Q 1 and Q 4 -CH 2 O-; (iii) Q 2 and Q 3 is oxygen; (iv) X 1 , X 2 , X 3 , X 4 and X n At least one of -SH or -S - ; (v) Y 1 , Y 2 , Y 3 , Y 4 and Y nAt least one of =S; or (vi) A, A 1 and A 2 At least one of them is -S-.

[0365] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (Ia):

[0366]

[0367] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (Ib):

[0368]

[0369] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (Ic):

[0370] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (Id):

[0371] In some embodiments, the IVT mRNA sequence initiator of formula (Id) has structure.

[0372] In some embodiments, the IVT mRNA sequence initiator of formula (Id) has structure.

[0373] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (Ie):

[0374] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (If):

[0375]

[0376] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (Ig):

[0377]

[0378] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (Ih):

[0379]

[0380] In some embodiments of the 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.

[0381] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Z 1 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 In some embodiments, Z 1 In some embodiments, Z 1 is F. In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 -CH 3 In some embodiments, Z 1 -CH 2 CH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 1 OCH 2 CH 3 In some embodiments, Z 1 For-SCH 3 In some embodiments, Z 1 For -NH 2 In some embodiments, Z 1 For NHCH 3 In some embodiments, Z 1 NHC(=O)CH 3 .

[0382] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ia), (Id), (Ie), (If), (Ig), or (Ih), Z 2Hydrogen, 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 In some embodiments, Z 2 In some embodiments, Z 2 is F. In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 -CH 3 In some embodiments, Z 2 -CH 2 CH 3 In some embodiments, Z 2 OCH 3 In some embodiments, Z 2 OCH 2 CH 3 In some embodiments, Z 2 For-SCH 3 In some embodiments, Z 2 For -NH 2 In some embodiments, Z 2 For NHCH 3 In some embodiments, Z 2 NHC(=O)CH 3 .

[0383] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Z 3 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 CH2 CH 3 、-OCH(CH 3 ) 2 、-SCH 3 or -OCH 2 CH 2 OCH 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 -CH 3 In some embodiments, Z 3 -CH 2 CH 3 In some embodiments, Z 3 OCH 2 OCH 3 In some embodiments, Z 3 OCH 2 CH 2 CH 3 In some embodiments, Z 3 -OCH(CH 3 ) 2 In some embodiments, Z 3 For-SCH 3 In some embodiments, Z 3 OCH 2 CH 2 OCH 3 .

[0384] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Z 4 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 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 -CH 3 In some embodiments, Z 4 -CH 2 CH 3 In some embodiments, Z 4 OCH 2 OCH 3 In some embodiments, Z 4 OCH 2 CH 2 CH 3 In some embodiments, Z 4 -OCH(CH 3 ) 2 In some embodiments, Z 4 For-SCH 3 In some embodiments, Z 4 OCH 2 CH 2 OCH 3 .

[0385] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Z n 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 In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n -CH 3 In some embodiments, Z n -CH 2 CH 3 In some embodiments, Z n OCH 2 OCH 3 In some embodiments, Z n OCH 2 CH 2 CH 3 In some embodiments, Z n -OCH(CH 3 ) 2 In some embodiments, Z n For-SCH 3 In some embodiments, Z n OCH 2 CH 2 OCH 3 .

[0386] 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 In some embodiments, B 2 In some embodiments, B 2 In some embodiments, B 2 is uracil, in some embodiments, B 2 is thymine, in some embodiments, B 2 In some embodiments, B 2 For purine.

[0387] 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 In some embodiments, B 3 In some embodiments, B 3 In some embodiments, B 3 is uracil, in some embodiments, B 3 is thymine, in some embodiments, B 3 In some embodiments, B 3 For purine.

[0388] 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 In some embodiments, B n In some embodiments, B n In some embodiments, B n is uracil, in some embodiments, B n is thymine, in some embodiments, B n In some embodiments, B n For purine.

[0389] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), B 2 , B 3 and B n In some embodiments, at least one of B is adenine. 2 , B 3 and B n In some embodiments, at least one of B is guanine. 2 In some embodiments, B 3 Adenine.

[0390] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Q 1 -CH2 -、-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 )-. In some embodiments, Q 1 -CH 2 -. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 CH 2 -. In some embodiments, Q 1 -CH 2 CF 2 -. In some embodiments, Q 1 -CH 2 NH 2 -. In some embodiments, Q 1 -CH 2 NH(CH 3 )-. In some embodiments, Q 1 -CH 2 N(C(=O)CH 3 )-.

[0391] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Q 4 -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 )-. In some embodiments, Q 4 -CH 2 -. In some embodiments, Q 4 is -CH=CH-. In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 CH 2 -. In some embodiments, Q 4 -CH 2 CF 2 -. In some embodiments, Q 4 -CH 2 NH 2 -. In some embodiments, Q 4 -CH 2 NH(CH 3 )-. In some embodiments, Q 4 -CH 2 N(C(=O)CH 3 )-.

[0392] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Q 2 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 2 In some embodiments, Q 2 In some embodiments, Q 2 -CH 2 -. In some embodiments, Q 2 For-CF 2 -. In some embodiments, Q 2 In some embodiments, Q 2 -N(CH 3 )-. In some embodiments, Q 2 -N(C(=O)CH 3 ).

[0393] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Q 3 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 3 In some embodiments, Q 3 In some embodiments, Q 3 -CH 2 -. In some embodiments, Q 3 For-CF 2 -. In some embodiments, Q 3 In some embodiments, Q 3 -N(CH 3 )-. In some embodiments, Q 3 -N(C(=O)CH 3 ).

[0394] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), X 1 -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 In some embodiments, X 1 In some embodiments, X 1 In some embodiments, X 1 -O - In some embodiments, X 1 For -S - In some embodiments, X 1 For -NH2 In some embodiments, X 1 -NHCH 3 In some embodiments, X 1 -NH(C(=O)CH 3 ). In some embodiments, X 1 -CH 3 In some embodiments, X 1 -CH 2 CH 3 In some embodiments, X 1 -CH 2 CH 2 CH 3 In some embodiments, X 1 -CH(CH 3 ) 2 In some embodiments, X 1 OCH 3 In some embodiments, X 1 OCH 2 CH 3 .

[0395] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), X 2 -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 In some embodiments, X 2 In some embodiments, X 2 In some embodiments, X 2 -O - In some embodiments, X 2 For -S - In some embodiments, X 2 For -NH 2In some embodiments, X 2 -NHCH 3 In some embodiments, X 2 -NH(C(=O)CH 3 ). In some embodiments, X 2 -CH 3 In some embodiments, X 2 -CH 2 CH 3 In some embodiments, X 2 -CH 2 CH 2 CH 3 In some embodiments, X 2 -CH(CH 3 ) 2 In some embodiments, X 2 OCH 3 In some embodiments, X 2 OCH 2 CH 3 .

[0396] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), X 3 -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 In some embodiments, X 3 In some embodiments, X 3 In some embodiments, X 3 -O - In some embodiments, X 3 For -S - In some embodiments, X 3 For -NH 2 In some embodiments, X3 -NHCH 3 In some embodiments, X 3 -NH(C(=O)CH 3 ). In some embodiments, X 3 -CH 3 In some embodiments, X 3 -CH 2 CH 3 In some embodiments, X 3 -CH 2 CH 2 CH 3 In some embodiments, X 3 -CH(CH 3 ) 2 In some embodiments, X 3 OCH 3 In some embodiments, X 3 OCH 2 CH 3 .

[0397] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), X 4 -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 In some embodiments, X 4 In some embodiments, X 4 In some embodiments, X 4 -O - In some embodiments, X 4 For -S - In some embodiments, X 4 For -NH 2 In some embodiments, X 4-NHCH 3 In some embodiments, X 4 -NH(C(=O)CH 3 ). In some embodiments, X 4 -CH 3 In some embodiments, X 4 -CH 2 CH 3 In some embodiments, X 4 -CH 2 CH 2 CH 3 In some embodiments, X 4 -CH(CH 3 ) 2 In some embodiments, X 4 OCH 3 In some embodiments, X 4 OCH 2 CH 3 .

[0398] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), X n -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 In some embodiments, X n In some embodiments, X n In some embodiments, X n -O - In some embodiments, X n For -S - In some embodiments, X n For -NH 2 In some embodiments, X n -NHCH3 In some embodiments, X n -NH(C(=O)CH 3 ). In some embodiments, X n -CH 3 In some embodiments, X n -CH 2 CH 3 In some embodiments, X n -CH 2 CH 2 CH 3 In some embodiments, X n -CH(CH 3 ) 2 In some embodiments, X n OCH 3 In some embodiments, X n OCH 2 CH 3 .

[0399] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Y 1 =O, =S, =NH or =NCH 3 In some embodiments, Y 1 =0. In some embodiments, Y 1 =S. In some embodiments, Y 1 =NH. In some embodiments, Y 1 =NHCH 3 .

[0400] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Y 2 =O, =S, =NH or =NCH 3 In some embodiments, Y 2 =0. In some embodiments, Y 2 =S. In some embodiments, Y 2 =NH. In some embodiments, Y 2 =NHCH 3 .

[0401] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Y3 =O, =S, =NH or =NCH 3 In some embodiments, Y 3 =0. In some embodiments, Y 3 =S. In some embodiments, Y 3 =NH. In some embodiments, Y 3 =NHCH 3 .

[0402] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Y 4 =O, =S, =NH or =NCH 3 In some embodiments, Y 4 =0. In some embodiments, Y 4 =S. In some embodiments, Y 4 =NH. In some embodiments, Y 4 =NHCH 3 .

[0403] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), Y n =O, =S, =NH or =NCH 3 In some embodiments, Y n =0. In some embodiments, Y n =S. In some embodiments, Y n =NH. In some embodiments, Y n =NHCH 3 .

[0404] In some embodiments of the compounds of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), A is -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH 2 -. In some embodiments, A is -NH-. In some embodiments, A is -N(CH 3 In some embodiments, A is -N(C(=O)CH 3 )-.

[0405] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), A 1 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 1 In some embodiments, A 1 In some embodiments, A 1 -CH 2 -. In some embodiments, A 1 In some embodiments, A 1 -N(CH 3 )-. In some embodiments, A 1 -N(C(=O)CH 3 )-.

[0406] In some embodiments of the compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), or (Ih), A 2 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 2 In some embodiments, A 2 In some embodiments, A 2 -CH 2 -. In some embodiments, A 2 In some embodiments, A 2 -N(CH 3 )-. In some embodiments, A 2 -N(C(=O)CH 3 )-.

[0407] In some embodiments of compounds 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.

[0408] In some embodiments of the compound of Formula (I), X 1 , X 2 , X 3 , X 4 and X n At least one of them is -SH.

[0409] In some embodiments of the compound of Formula (I), X 1 , X 2 , X 3 , X 4 and X n At least one of them is -S-.

[0410] In some embodiments of the compound of Formula (I), Y 1 , Y 2 , Y 3 , Y 4 and Y n At least one of them is =S.

[0411] In some embodiments of the compound of Formula (I), A, A 1 and A 2 At least one of them is -S-.

[0412] In some embodiments of the compound of Formula (I), Z 1 , Z 2 , Z 3 , Z 4 and Z n At least one of them is -OCH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 3 and Z 1 OCH 3 In some embodiments of the compound of formula (I), Z 1 , Z 2 , Z 3 , Z 4 and Z n In some embodiments, at least one of Z is -OH.1 , Z 2 and Z 4 In some embodiments, Z 2 and Z 4 It is -OH.

[0413] In some embodiments of the compound of Formula (I), Q 1 , Q 2 , Q 3 and Q 4 At least one of them is -OCH 3 In some embodiments of the compound of Formula (I), Q 1 , Q 2 , Q 3 and Q 4 In some embodiments, at least one of Q 1 and Q 4 OCH 3 In some embodiments, Q 2 and Q 3 It is -O-.

[0414] In some embodiments of the compound of Formula (I), Y 1 , Y 2 , Y 3 , Y 4 and Y n In some embodiments, at least one of Y is =O. 1 , Y 2 , Y 3 and Y 4 =0. In some embodiments, Y 1 , Y 2 , Y 3 , Y 4 and Y n =S. In some embodiments, Y 2 =S. In some embodiments, Y 4 is =S.

[0415] In some embodiments of the compound of Formula (I), X 1 , X 2 , X 3 , X 4 and X n At least one of -O - In some embodiments, X 1 , X 2 , X 3 and X 4 -O - In some embodiments, X 1 For -S- In some embodiments, X 2 For -S - In some embodiments, X 3 For -S - In some embodiments, X 4 For -S - .

[0416] In some embodiments of the compound of formula (I), in some embodiments of the compound of formula (I), A, A 1 and A 2 In some embodiments, at least one of A, A 1 and A 2 It is -O-.

[0417] In one aspect, the present invention describes an mRNA sequence having a 5' region motif of the motif (I'):

[0418]

[0419] in

[0420] B 1 for

[0421] B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase;

[0422] 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 ;

[0423] 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 ;

[0424] Q 1 and Q 4 Each is 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 )-;

[0425] Q 2 and Q 3 are independently -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;

[0426] 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 CH3 、-CH(CH 3 ) 2 、-OCH 3 or -OCH 2 CH 3 ;

[0427] Y 1 , Y 2 , Y 3 , Y 4 and Y n are independently =O, =S, =NH or =NCH 3 ;

[0428] A. A 1 and A 2 are independently -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;and

[0429] p = 0, 1, 2, 3, 4, 5 or 6,

[0430] The condition is (i) Z 1 , Z 2 and Z 3 is hydrogen; (ii) Q 1 and Q 4 -CH 2 O-; (iii) Q 2 and Q 3 is oxygen; (iv) X 1 , X 2 , X 3 , X 4 and X n At least one of -SH or -S - ; (v) Y 1 , Y 2 , Y 3 , Y 4 and Y n At least one of =S; or (vi) A, A 1 and A 2 At least one of them is -S-.

[0431] In some embodiments, the mRNA sequence having a 5' region motif has the structure of the motif (I'-a):

[0432]

[0433] In some embodiments, the mRNA sequence having the 5' end region motif has the structure of the motif (I'-b):

[0434]

[0435] In some embodiments, the mRNA sequence having the 5' region motif has the structure of the motif (I'-c):

[0436] In some embodiments, the mRNA sequence having the 5' end region motif has the structure of the motif (I'-d):

[0437] In some embodiments, the mRNA sequence having the 5' end region motif has the structure of the motif (I'-e):

[0438] In some embodiments, the mRNA sequence having a 5' region motif has the structure of the motif (I'-f):

[0439] In some embodiments, the mRNA sequence having the 5' end region motif has the structure of the motif (I'-g):

[0440]

[0441] In some embodiments, the mRNA sequence having a 5' end region motif has the structure of the motif (I'-h):

[0442]

[0443] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Z 1 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 In some embodiments, Z 1 In some embodiments, Z 1 is F. In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 -CH3 In some embodiments, Z 1 -CH 2 CH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 1 OCH 2 CH 3 In some embodiments, Z 1 For-SCH 3 In some embodiments, Z 1 For -NH 2 In some embodiments, Z 1 For NHCH 3 In some embodiments, Z 1 NHC(=O)CH 3 .

[0444] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-a), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Z 2 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 In some embodiments, Z 2 In some embodiments, Z 2 is F. In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 -CH 3 In some embodiments, Z 2 -CH 2 CH 3 In some embodiments, Z 2 OCH 3 In some embodiments, Z 2 OCH 2 CH 3 In some embodiments, Z 2 For-SCH 3 In some embodiments, Z2 For -NH 2 In some embodiments, Z 2 For NHCH 3 In some embodiments, Z 2 NHC(=O)CH 3 .

[0445] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Z 3 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 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 -CH 3 In some embodiments, Z 3 -CH 2 CH 3 In some embodiments, Z 3 OCH 2 OCH 3 In some embodiments, Z 3 OCH 2 CH 2 CH 3 In some embodiments, Z 3 -OCH(CH 3 ) 2 In some embodiments, Z3 For-SCH 3 In some embodiments, Z 3 OCH 2 CH 2 OCH 3 .

[0446] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Z 4 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 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 -CH 3 In some embodiments, Z 4 -CH 2 CH 3 In some embodiments, Z 4 OCH 2 OCH 3 In some embodiments, Z 4 OCH 2 CH 2 CH 3 In some embodiments, Z 4 -OCH(CH 3 ) 2 In some embodiments, Z 4 For-SCH3 In some embodiments, Z 4 OCH 2 CH 2 OCH 3 .

[0447] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Z n 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 In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n -CH 3 In some embodiments, Z n -CH 2 CH 3 In some embodiments, Z n OCH 2 OCH 3 In some embodiments, Z n OCH 2 CH 2 CH 3 In some embodiments, Z n -OCH(CH 3 ) 2 In some embodiments, Z n For-SCH 3In some embodiments, Z n OCH 2 CH 2 OCH 3 .

[0448] 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 In some embodiments, B 2 In some embodiments, B 2 In some embodiments, B 2 is uracil, in some embodiments, B 2 is thymine, in some embodiments, B 2 In some embodiments, B 2 For purine.

[0449] 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 In some embodiments, B 3 In some embodiments, B 3 In some embodiments, B 3 is uracil, in some embodiments, B 3 is thymine, in some embodiments, B 3 In some embodiments, B 3 For purine.

[0450] 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 In some embodiments, B n In some embodiments, B n In some embodiments, B n is uracil, in some embodiments, Bn is thymine, in some embodiments, B n In some embodiments, B n For purine.

[0451] 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 In some embodiments, at least one of B is adenine. 2 , B 3 and B n In some embodiments, at least one of B is guanine. 2 In some embodiments, B 3 Adenine.

[0452] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Q 1 -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 )-. In some embodiments, Q 1 -CH 2 -. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 CH 2 -. In some embodiments, Q 1 -CH 2 CF 2 -. In some embodiments, Q1 -CH 2 NH 2 -. In some embodiments, Q 1 -CH 2 NH(CH 3 )-. In some embodiments, Q 1 -CH 2 N(C(=O)CH 3 )-.

[0453] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Q 4 -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 )-. In some embodiments, Q 4 -CH 2 -. In some embodiments, Q 4 is -CH=CH-. In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 CH 2 -. In some embodiments, Q 4 -CH 2 CF 2 -. In some embodiments, Q 4 -CH 2 NH 2 -. In some embodiments, Q 4 -CH 2 NH(CH 3 )-. In some embodiments, Q 4 -CH 2 N(C(=O)CH 3 )-.

[0454] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Q 2 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 2 In some embodiments, Q 2 In some embodiments, Q 2 -CH 2 -. In some embodiments, Q 2 For-CF 2 -. In some embodiments, Q 2 In some embodiments, Q 2 -N(CH 3 )-. In some embodiments, Q 2 -N(C(=O)CH 3 ).

[0455] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Q 3 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 3 In some embodiments, Q 3 In some embodiments, Q 3 -CH 2 -. In some embodiments, Q 3 For-CF 2 -. In some embodiments, Q 3 In some embodiments, Q 3 -N(CH 3 )-. In some embodiments, Q 3 -N(C(=O)CH 3 ).

[0456] In some embodiments of the 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 - 、-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 In some embodiments, X 1 In some embodiments, X 1 In some embodiments, X 1 -O - In some embodiments, X 1 For -S - In some embodiments, X 1 For -NH 2 In some embodiments, X 1 -NHCH 3 In some embodiments, X 1 -NH(C(=O)CH 3 ). In some embodiments, X 1 -CH 3 In some embodiments, X 1 -CH 2 CH 3 In some embodiments, X 1 -CH 2 CH 2 CH 3 In some embodiments, X 1 -CH(CH 3 ) 2 In some embodiments, X 1 OCH 3 In some embodiments, X 1 OCH 2 CH 3 .

[0457] In some embodiments of the 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 - 、-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 In some embodiments, X 2 In some embodiments, X 2 In some embodiments, X 2 -O - In some embodiments, X 2 For -S - In some embodiments, X 2 For -NH 2 In some embodiments, X 2 -NHCH 3 In some embodiments, X 2 -NH(C(=O)CH 3 ). In some embodiments, X 2 -CH 3 In some embodiments, X 2 -CH 2 CH 3 In some embodiments, X 2 -CH 2 CH 2 CH 3 In some embodiments, X 2 -CH(CH 3 ) 2 In some embodiments, X 2 OCH 3 In some embodiments, X 2 OCH 2 CH 3 .

[0458] In some embodiments of the 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 - 、-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 In some embodiments, X 3 In some embodiments, X 3 In some embodiments, X 3 -O - In some embodiments, X 3 For -S - In some embodiments, X 3 For -NH 2 In some embodiments, X 3 -NHCH 3 In some embodiments, X 3 -NH(C(=O)CH 3 ). In some embodiments, X 3 -CH 3 In some embodiments, X 3 -CH 2 CH 3 In some embodiments, X 3 -CH 2 CH 2 CH 3 In some embodiments, X 3 -CH(CH 3 ) 2 In some embodiments, X 3 OCH 3 In some embodiments, X 3 OCH 2 CH 3 .

[0459] In some embodiments of the 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 - 、-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 In some embodiments, X 4 In some embodiments, X 4 In some embodiments, X 4 -O - In some embodiments, X 4 For -S - In some embodiments, X 4 For -NH 2 In some embodiments, X 4 -NHCH 3 In some embodiments, X 4 -NH(C(=O)CH 3 ). In some embodiments, X 4 -CH 3 In some embodiments, X 4 -CH 2 CH 3 In some embodiments, X 4 -CH 2 CH 2 CH 3 In some embodiments, X 4 -CH(CH 3 ) 2 In some embodiments, X 4 OCH 3 In some embodiments, X 4 OCH 2 CH 3 .

[0460] In some embodiments of the 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 - 、-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 In some embodiments, X n In some embodiments, X n In some embodiments, X n -O - In some embodiments, X n For -S - In some embodiments, X n For -NH 2 In some embodiments, X n -NHCH 3 In some embodiments, X n -NH(C(=O)CH 3 ). In some embodiments, X n -CH 3 In some embodiments, X n -CH 2 CH 3 In some embodiments, X n -CH 2 CH 2 CH 3 In some embodiments, X n -CH(CH 3 ) 2 In some embodiments, X n OCH 3 In some embodiments, X n OCH 2 CH 3 .

[0461] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Y 1 =O, =S, =NH or =NCH 3 In some embodiments, Y 1 =0. In some embodiments, Y 1 =S. In some embodiments, Y 1 =NH. In some embodiments, Y 1 =NHCH 3 .

[0462] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Y 2 =O, =S, =NH or =NCH 3 In some embodiments, Y 2 =0. In some embodiments, Y 2 =S. In some embodiments, Y 2 =NH. In some embodiments, Y 2 =NHCH 3 .

[0463] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Y 3 =O, =S, =NH or =NCH 3 In some embodiments, Y 3 =0. In some embodiments, Y 3 =S. In some embodiments, Y 3 =NH. In some embodiments, Y 3 =NHCH 3 .

[0464] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Y 4 =O, =S, =NH or =NCH 3 In some embodiments, Y 4 =0. In some embodiments, Y 4 =S. In some embodiments, Y4 =NH. In some embodiments, Y 4 =NHCH 3 .

[0465] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g) or (I'-h), Y n =O, =S, =NH or =NCH 3 In some embodiments, Y n =0. In some embodiments, Y n =S. In some embodiments, Y n =NH. In some embodiments, Y n =NHCH 3 .

[0466] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), A is -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH 2 -. In some embodiments, A is -NH-. In some embodiments, A is -N(CH 3 In some embodiments, A is -N(C(=O)CH 3 )-.

[0467] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), A 1 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 1 In some embodiments, A 1 In some embodiments, A 1 -CH 2 -. In some embodiments, A 1 In some embodiments, A 1-N(CH 3 )-. In some embodiments, A 1 -N(C(=O)CH 3 )-.

[0468] In some embodiments of the compounds of motif (I'), (I'-a), (I'-b), (I'-c), (I'-d), (I'-e), (I'-f), (I'-g), or (I'-h), A 2 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 2 In some embodiments, A 2 In some embodiments, A 2 -CH 2 -. In some embodiments, A 2 In some embodiments, A 2 -N(CH 3 )-. In some embodiments, A 2 -N(C(=O)CH 3 )-.

[0469] 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.

[0470] In some embodiments of the compounds of motif (I'), X 1 , X 2 , X 3 , X 4 and X n At least one of them is -SH.

[0471] In some embodiments of the compounds of motif (I'), X 1 , X 2 , X 3 , X 4 and X n At least one of them is -S-.

[0472] 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.

[0473] In some embodiments of the compounds of motif (I'), A, A 1 and A 2 At least one of them is -S-.

[0474] In some embodiments of the compounds of motif (I'), Z 1 , Z 2 , Z 3 , Z 4 and Z n At least one of them is -OCH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 3 and Z 1 OCH 3 In some embodiments of the compounds of motif (I'), Z 1 , Z 2 , Z 3 , Z 4 and Z n In some embodiments, at least one of Z is -OH. 1 , Z 2 and Z 4 In some embodiments, Z 2 and Z 4 It is -OH.

[0475] In some embodiments of the compounds of motif (I'), Q 1 , Q 2 , Q 3 and Q 4 At least one of them is -OCH 3 In some embodiments of the compound of Formula (I), Q 1 , Q 2 , Q 3 and Q 4 In some embodiments, at least one of Q 1 and Q 4 OCH 3 In some embodiments, Q 2 and Q 3 It is -O-.

[0476] 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 Y is =O. 1 , Y 2 , Y 3 and Y 4 =0. In some embodiments, Y 1 , Y 2 , Y 3 , Y 4 and Y n =S. In some embodiments, Y 2 =S. In some embodiments, Y 4 is =S.

[0477] In some embodiments of the compounds of motif (I'), X 1 , X 2 , X 3 , X 4 and X n At least one of -O - In some embodiments, X 1 , X 2 , X 3 and X 4 -O - In some embodiments, X 1 For -S - In some embodiments, X 2 For -S - In some embodiments, X 3 For -S - In some embodiments, X 4 For -S - .

[0478] In some embodiments of the compounds of motif (I'), 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 It is -O-.

[0479] In one aspect, described herein is an mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof:

[0480]

[0481] in

[0482] B 1 for

[0483] B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase;

[0484] Z 1 and Z' are each independently hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 ;

[0485] Z 2 and Z" are each independently fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 ;

[0486] Z"' is hydrogen, fluorine, -CH 3 、-CH 2 CH 3 、-OCH 3 or -OCH 2 CH 3 ;

[0487] Z 3 , Z 4 and Z n are independently hydrogen, fluorine, -OH, -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 ;

[0488] Q 1 and Q 4 are independently -CH=CH-, -CH 2 -、-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 )-;

[0489] Q 2 and Q 3 are independently -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;

[0490] 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 ;

[0491] Y 1 , Y 2 , Y 3 , Y 4 and Y n are independently =O, =S, =NH or =NCH 3 ;

[0492] A. A 1 and A 2 are independently -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;and

[0493] p=0, 1, 2, 3, 4, 5 or 6.

[0494] In some embodiments of the 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.

[0495] In some embodiments of the 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 In some embodiments, B 2 In some embodiments, B 2 In some embodiments, B 2 is uracil, in some embodiments, B 2 is thymine, in some embodiments, B 2 In some embodiments, B 2 For purine.

[0496] In some embodiments of the 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 In some embodiments, B 3 In some embodiments, B 3 In some embodiments, B 3 is uracil, in some embodiments, B 3 is thymine, in some embodiments, B 3 In some embodiments, B 3 For purine.

[0497] In some embodiments of the 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 In some embodiments, B n In some embodiments, B n In some embodiments, B n is uracil, in some embodiments, B n is thymine, in some embodiments, B n In some embodiments, B n For purine.

[0498] In some embodiments of the 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 Hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 -CH 3 In some embodiments, Z 1 -CH 2 CH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 1 -NH(CH 3 ), in some embodiments, Z 1 For -NH 2 -. In some embodiments, Z 1 -NH(C(=O)CH 3 ). In some embodiments, Z 1 For-SCH 3 .

[0499] In some embodiments of compounds 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 3 、-CH2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 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 3 In some embodiments, Z' is -CH 2 CH 3 In some embodiments, Z' is -OCH 3 In some embodiments, Z' is -NH(CH 3 ), in some embodiments, Z' is -NH 2 -. In some embodiments, Z' is -NH(C(=O)CH 3 In some embodiments, Z' is -SCH 3 .

[0500] In some embodiments of the 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 Fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 -CH 3 In some embodiments, Z 2 -CH 2 CH 3In some embodiments, Z 2 OCH 3 In some embodiments, Z 2 For-SCH 3 In some embodiments, Z 2 OCH 2 CH 3 In some embodiments, Z 2 For -NH 2 In some embodiments, Z 2 For NHCH 3 In some embodiments, Z 2 NHC(=O)CH 3 .

[0501] In some embodiments of the compounds 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 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 In some embodiments, Z" is fluorine. In some embodiments, Z" is -OH. In some embodiments, Z" is -SH. In some embodiments, Z" is -CH 3 In some embodiments, Z" is -CH 2 CH 3 In some embodiments, Z" is -OCH 3 In some embodiments, Z" is -SCH 3 In some embodiments, Z" is -OCH 2 CH 3 In some embodiments, Z" is -NH 2 In some embodiments, Z" is NHCH 3 In some embodiments, Z" is NHC(=O)CH 3 .

[0502] In some embodiments of the compounds 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 3 、-CH 2 CH 3 、-OCH 3 or -OCH 2 CH 3 In some embodiments, Z'' is hydrogen. In some embodiments, Z'' is fluorine. In some embodiments, Z'' is -CH 3 In some embodiments, Z'' is -CH 2 CH 3 In some embodiments, Z'' is -OCH 3 In some embodiments, Z'' is -OCH 2 CH 3 .

[0503] In some embodiments of the 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 For hydrogen, fluorine, -OH, -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 2OCH 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 -CH 3 In some embodiments, Z 3 -CH 2 CH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 3 For -NH 2 In some embodiments, Z 3 -NHCH 3 In some embodiments, Z 3 -NH(C(=O)CH 3 ). In some embodiments, Z 3 OCH 2 CH 3 In some embodiments, Z 3 OCH 2 OCH 3 In some embodiments, Z 3 OCH 2 CH 2 CH 3 In some embodiments, Z 3 -OCH(CH 3 ) 2 In some embodiments, Z 3 For-SCH 3 In some embodiments, Z 3 OCH 2 CH 2 OCH 3 .

[0504] In some embodiments of the 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 For hydrogen, fluorine, -OH, -CH 3 、-CH 2 CH 3 、-OCH3 、-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 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 -CH 3 In some embodiments, Z 4 -CH 2 CH 3 In some embodiments, Z 4 OCH 3 In some embodiments, Z 4 For -NH 2 In some embodiments, Z 4 -NHCH 3 In some embodiments, Z 4 -NH(C(=O)CH 3 ). In some embodiments, Z 4 OCH 2 CH 3 In some embodiments, Z 4 OCH 2 OCH 3 In some embodiments, Z 4 OCH 2 CH 2 CH 3 In some embodiments, Z 4 -OCH(CH 3 ) 2 In some embodiments, Z 4 For-SCH 3 In some embodiments, Z 4 OCH 2 CH 2 OCH3 .

[0505] In some embodiments of the 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 For hydrogen, fluorine, -OH, -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 In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n -CH 3 In some embodiments, Z n -CH 2 CH 3 In some embodiments, Z n OCH 3 In some embodiments, Z n For -NH 2 In some embodiments, Z n -NHCH 3 In some embodiments, Z n -NH(C(=O)CH 3 ). In some embodiments, Z n OCH 2 CH 3 In some embodiments, Zn OCH 2 OCH 3 In some embodiments, Z n OCH 2 CH 2 CH 3 In some embodiments, Z n -OCH(CH 3 ) 2 In some embodiments, Z n For-SCH 3 In some embodiments, Z n OCH 2 CH 2 OCH 3 .

[0506] In some embodiments of the 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 -CH=CH-, -CH 2 -、-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 )-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 -CH 2 -. In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 CH 2 -. In some embodiments, Q 1 -CH 2 CF2 -. In some embodiments, Q 1 -CH 2 NH 2 -. In some embodiments, Q 1 -CH 2 NH(CH 3 )-. In some embodiments, Q 1 -CH 2 N(C(=O)CH 3 )-.

[0507] In some embodiments of the 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 -CH=CH-, -CH 2 -、-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 )-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 4 -CH 2 -. In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 CH 2 -. In some embodiments, Q 4 -CH 2 CF 2 -. In some embodiments, Q 4 -CH 2 NH 2 -. In some embodiments, Q 4-CH 2 NH(CH 3 )-. In some embodiments, Q 4 -CH 2 N(C(=O)CH 3 )-.

[0508] In some embodiments of the 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 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 2 In some embodiments, Q 2 In some embodiments, Q 2 -CH 2 -. In some embodiments, Q 2 For-CF 2 -. In some embodiments, Q 2 In some embodiments, Q 2 -N(CH 3 )-. In some embodiments, Q 2 -N(C(=O)CH 3 )-.

[0509] In some embodiments of the 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 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 3In some embodiments, Q 3 In some embodiments, Q 3 -CH 2 -. In some embodiments, Q 3 For-CF 2 -. In some embodiments, Q 3 In some embodiments, Q 3 -N(CH 3 )-. In some embodiments, Q 3 -N(C(=O)CH 3 )-.

[0510] In some embodiments of the 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 - 、-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 In some embodiments, X 1 In some embodiments, X 1 In some embodiments, X 1 -O - In some embodiments, X 1 For -S - In some embodiments, X 1 For -NH 2 In some embodiments, X 1 -NHCH 3 In some embodiments, X 1 -NH(C(=O)CH 3). In some embodiments, X 1 -CH 3 In some embodiments, X 1 -CH 2 CH 3 In some embodiments, X 1 -CH 2 CH 2 CH 3 In some embodiments, X 1 -CH(CH 3 ) 2 In some embodiments, X 1 OCH 3 In some embodiments, X 1 OCH 2 CH 3 .

[0511] In some embodiments of the 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 - 、-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 In some embodiments, X 2 In some embodiments, X 2 In some embodiments, X 2 -O - In some embodiments, X 2 For -S - In some embodiments, X 2 For -NH 2In some embodiments, X 2 -NHCH 3 In some embodiments, X 2 -NH(C(=O)CH 3 ). In some embodiments, X 2 -CH 3 In some embodiments, X 2 -CH 2 CH 3 In some embodiments, X 2 -CH 2 CH 2 CH 3 In some embodiments, X 2 -CH(CH 3 ) 2 In some embodiments, X 2 OCH 3 In some embodiments, X 2 OCH 2 CH 3 .

[0512] In some embodiments of the 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 - 、-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 In some embodiments, X 3 In some embodiments, X 3 In some embodiments, X 3 -O- In some embodiments, X 3 For -S - In some embodiments, X 3 For -NH 2 In some embodiments, X 3 -NHCH 3 In some embodiments, X 3 -NH(C(=O)CH 3 ). In some embodiments, X 3 -CH 3 In some embodiments, X 3 -CH 2 CH 3 In some embodiments, X 3 -CH 2 CH 2 CH 3 In some embodiments, X 3 -CH(CH 3 ) 2 In some embodiments, X 3 OCH 3 In some embodiments, X 3 OCH 2 CH 3 .

[0513] In some embodiments of the 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 - 、-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 3In some embodiments, X 4 In some embodiments, X 4 In some embodiments, X 4 -O - In some embodiments, X 4 For -S - In some embodiments, X 4 For -NH 2 In some embodiments, X 4 -NHCH 3 In some embodiments, X 4 -NH(C(=O)CH 3 ). In some embodiments, X 4 -CH 3 In some embodiments, X 4 -CH 2 CH 3 In some embodiments, X 4 -CH 2 CH 2 CH 3 In some embodiments, X 4 -CH(CH 3 ) 2 In some embodiments, X 4 OCH 3 In some embodiments, X 4 OCH 2 CH 3 .

[0514] In some embodiments of the 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 - 、-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 In some embodiments, X n In some embodiments, X n In some embodiments, X n -O - In some embodiments, X n For -S - In some embodiments, X n For -NH 2 In some embodiments, X n -NHCH 3 In some embodiments, X n -NH(C(=O)CH 3 ). In some embodiments, X n -CH 3 In some embodiments, X n -CH 2 CH 3 In some embodiments, X n -CH 2 CH 2 CH 3 In some embodiments, X n -CH(CH 3 ) 2 In some embodiments, X n OCH 3 In some embodiments, X n OCH 2 CH 3 .

[0515] In some embodiments of the compounds 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 1 =0. In some embodiments, Y 1 =S. In some embodiments, Y 1=NH. In some embodiments, Y 1 =NCH 3 .

[0516] In some embodiments of the compounds 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 2 =0. In some embodiments, Y 2 =S. In some embodiments, Y 2 =NH. In some embodiments, Y 2 =NCH 3 .

[0517] In some embodiments of the compounds 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 3 =0. In some embodiments, Y 3 =S. In some embodiments, Y 3 =NH. In some embodiments, Y 3 =NCH 3 .

[0518] In some embodiments of the compounds 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 =O, =S, =NH or =NCH 3In some embodiments, Y 4 =0. In some embodiments, Y 4 =S. In some embodiments, Y 4 =NH. In some embodiments, Y 4 =NCH 3 .

[0519] In some embodiments of the compounds 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 =O, =S, =NH or =NCH 3 In some embodiments, Y n =0. In some embodiments, Y n =S. In some embodiments, Y n =NH. In some embodiments, Y n =NCH 3 .

[0520] In some embodiments of the compounds 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 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH 2 -. In some embodiments, A is -NH-. In some embodiments, A is -N(CH 3 In some embodiments, A is -N(C(=O)CH 3 )-.

[0521] In some embodiments of the 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 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 1 In some embodiments, A 1 In some embodiments, A 1 -CH 2 -. In some embodiments, A 1 In some embodiments, A 1 -N(CH 3 )-. In some embodiments, A 1 -N(C(=O)CH 3 )-.

[0522] In some embodiments of the 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 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 2 In some embodiments, A 2 In some embodiments, A 2 -CH 2 -. In some embodiments, A 2 In some embodiments, A 2 -N(CH 3 )-. In some embodiments, A 2 -N(C(=O)CH 3 )-.

[0523] In some embodiments of compounds 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.

[0524] In some embodiments, B 1 for In some embodiments, B 1 for In some embodiments, B 2 and B 3 Each is independently adenine or guanine. In some embodiments, B 2 In some embodiments, B 3 For guanine.

[0525] In some embodiments, Z 1 , Z 2 , Z 3 and Z 4 Each is independently -OH or -OCH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 2 In some embodiments, Z 1 , Z 2 and Z 4 is -OH, and Z 3 OCH 3 In some embodiments, Z 1 and Z 3 OCH 3 , and Z 2 and Z 4 In some embodiments, Z', Z", and Z'" are hydrogen.

[0526] In some embodiments, Q 1 and Q 4OCH 2 -. In some embodiments, Q 2 and Q 3 It is -O-.

[0527] In some embodiments, X 1 , X 2 , X 3 and X 4 In some embodiments, X 1 , X 2 , X 3 and X 4 In some embodiments, X 1 , X 3 , X 4 is -O-, and X 2 For -S-.

[0528] In some embodiments, Y 1 , Y 2 , Y 3 and Y 4 =0. In some embodiments, Y 1 , Y 2 , Y 3 and Y 4 =S. In some embodiments, Y 1 , Y 3 and Y 4 =O, and Y 2 =S. In some embodiments, Y 1 , Y 2 , Y 3 =O, and Y 4 is =S.

[0529] In some embodiments, A, A 1 and A 2 It is -O-.

[0530] In some embodiments, p is 0.

[0531] In one aspect, the present invention describes an mRNA sequence having a 5' terminal region motif (II'):

[0532]

[0533] in

[0534] B 1 for

[0535] B 2 , B 3 and B neach independently a natural, modified or unnatural nucleobase;

[0536] Z 1 and Z' are each independently hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 ;

[0537] Z 2 and Z" are each independently fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 ;

[0538] Z"' is hydrogen, fluorine, -CH 3 、-CH 2 CH 3 、-OCH 3 or -OCH 2 CH 3 ;

[0539] Z 3 , Z 4 and Z n are independently hydrogen, fluorine, -OH, -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 2CH 2 OCH 3 ;

[0540] Q 1 and Q 4 are independently -CH=CH-, -CH 2 -、-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 )-;

[0541] Q 2 and Q 3 are independently -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;

[0542] 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 ;

[0543] Y 1 , Y 2 , Y 3 , Y 4 and Y n are independently =O, =S, =NH or =NCH3 ;

[0544] A. A 1 and A 2 are independently -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;and

[0545] p=0, 1, 2, 3, 4, 5 or 6.

[0546] 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), B 2 are independently natural, modified or non-natural nucleobases. In some embodiments, B 2 In some embodiments, B 2 In some embodiments, B 2 In some embodiments, B 2 is uracil, in some embodiments, B 2 is thymine, in some embodiments, B 2 In some embodiments, B 2 For purine.

[0547] 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), B 3 are independently natural, modified or non-natural nucleobases. In some embodiments, B 3 In some embodiments, B 3 In some embodiments, B 3 In some embodiments, B 3is uracil, in some embodiments, B 3 is thymine, in some embodiments, B 3 In some embodiments, B 3 For purine.

[0548] 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), B n are independently natural, modified or non-natural nucleobases. In some embodiments, B n In some embodiments, B n In some embodiments, B n In some embodiments, B n is uracil, in some embodiments, B n is thymine, in some embodiments, B n In some embodiments, B n For purine.

[0549] 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 Hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1In some embodiments, Z 1 In some embodiments, Z 1 -CH 3 In some embodiments, Z 1 -CH 2 CH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 1 -NH(CH 3 ), in some embodiments, Z 1 For -NH 2 -. In some embodiments, Z 1 -NH(C(=O)CH 3 ). In some embodiments, Z 1 For-SCH 3 .

[0550] 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 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 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 3 In some embodiments, Z' is -CH 2 CH 3 In some embodiments, Z' is -OCH 3 In some embodiments, Z' is -NH(CH 3 ), in some embodiments, Z' is -NH 2 -. In some embodiments, Z' is -NH(C(=O)CH 3In some embodiments, Z' is -SCH 3 .

[0551] 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 Fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 -CH 3 In some embodiments, Z 2 -CH 2 CH 3 In some embodiments, Z 2 OCH 3 In some embodiments, Z 2 For-SCH 3 In some embodiments, Z 2 OCH 2 CH 3 In some embodiments, Z 2 For -NH 2 In some embodiments, Z 2 For NHCH 3 In some embodiments, Z 2 NHC(=O)CH 3 .

[0552] 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 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 In some embodiments, Z" is fluorine. In some embodiments, Z" is -OH. In some embodiments, Z" is -SH. In some embodiments, Z" is -CH 3 In some embodiments, Z" is -CH 2 CH 3 In some embodiments, Z" is -OCH 3 In some embodiments, Z" is -SCH 3 In some embodiments, Z" is -OCH 2 CH 3 In some embodiments, Z" is -NH 2 In some embodiments, Z" is NHCH 3 In some embodiments, Z" is NHC(=O)CH 3 .

[0553] 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 3 、-CH 2 CH 3 、-OCH 3 or -OCH 2CH 3 In some embodiments, Z'' is hydrogen. In some embodiments, Z'' is fluorine. In some embodiments, Z'' is -CH 3 In some embodiments, Z'' is -CH 2 CH 3 In some embodiments, Z'' is -OCH 3 In some embodiments, Z'' is -OCH 2 CH 3 .

[0554] 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 For hydrogen, fluorine, -OH, -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 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 -CH 3 In some embodiments, Z 3 -CH 2 CH 3 In some embodiments, Z 3 OCH 3In some embodiments, Z 3 For -NH 2 In some embodiments, Z 3 -NHCH 3 In some embodiments, Z 3 -NH(C(=O)CH 3 ). In some embodiments, Z 3 OCH 2 CH 3 In some embodiments, Z 3 OCH 2 OCH 3 In some embodiments, Z 3 OCH 2 CH 2 CH 3 In some embodiments, Z 3 -OCH(CH 3 ) 2 In some embodiments, Z 3 For-SCH 3 In some embodiments, Z 3 OCH 2 CH 2 OCH 3 .

[0555] 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 For hydrogen, fluorine, -OH, -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 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 -CH 3 In some embodiments, Z 4 -CH 2 CH 3 In some embodiments, Z 4 OCH 3 In some embodiments, Z 4 For -NH 2 In some embodiments, Z 4 -NHCH 3 In some embodiments, Z 4 -NH(C(=O)CH 3 ). In some embodiments, Z 4 OCH 2 CH 3 In some embodiments, Z 4 OCH 2 OCH 3 In some embodiments, Z 4 OCH 2 CH 2 CH 3 In some embodiments, Z 4 -OCH(CH 3 ) 2 In some embodiments, Z 4 For-SCH 3 In some embodiments, Z 4 OCH 2 CH 2 OCH 3 .

[0556] 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 For hydrogen, fluorine, -OH, -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 In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n -CH 3 In some embodiments, Z n -CH 2 CH 3 In some embodiments, Z n OCH 3 In some embodiments, Z n For -NH 2 In some embodiments, Z n -NHCH 3 In some embodiments, Z n -NH(C(=O)CH 3 ). In some embodiments, Z n OCH 2 CH 3 In some embodiments, Z n OCH2 OCH 3 In some embodiments, Z n OCH 2 CH 2 CH 3 In some embodiments, Z n -OCH(CH 3 ) 2 In some embodiments, Z n For-SCH 3 In some embodiments, Z n OCH 2 CH 2 OCH 3 .

[0557] 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), Q 1 -CH=CH-, -CH 2 -、-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 )-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 -CH 2 -. In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 CH 2 -. In some embodiments, Q 1 -CH 2CF 2 -. In some embodiments, Q 1 -CH 2 NH 2 -. In some embodiments, Q 1 -CH 2 NH(CH 3 )-. In some embodiments, Q 1 -CH 2 N(C(=O)CH 3 )-.

[0558] 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), Q 4 -CH=CH-, -CH 2 -、-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 )-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 4 -CH 2 -. In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 CH 2 -. In some embodiments, Q 4 -CH 2 CF 2 -. In some embodiments, Q 4 -CH 2 NH 2-. In some embodiments, Q 4 -CH 2 NH(CH 3 )-. In some embodiments, Q 4 -CH 2 N(C(=O)CH 3 )-.

[0559] 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), Q 2 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 2 In some embodiments, Q 2 In some embodiments, Q 2 -CH 2 -. In some embodiments, Q 2 For-CF 2 -. In some embodiments, Q 2 In some embodiments, Q 2 -N(CH 3 )-. In some embodiments, Q 2 -N(C(=O)CH 3 )-.

[0560] 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), Q 3 -O-, -S-, -CH 2 -、-CF 2-、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 3 In some embodiments, Q 3 In some embodiments, Q 3 -CH 2 -. In some embodiments, Q 3 For-CF 2 -. In some embodiments, Q 3 In some embodiments, Q 3 -N(CH 3 )-. In some embodiments, Q 3 -N(C(=O)CH 3 )-.

[0561] 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 - 、-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 In some embodiments, X 1 In some embodiments, X 1 In some embodiments, X 1 -O - In some embodiments, X 1 For -S - In some embodiments, X 1 For -NH2 In some embodiments, X 1 -NHCH 3 In some embodiments, X 1 -NH(C(=O)CH 3 ). In some embodiments, X 1 -CH 3 In some embodiments, X 1 -CH 2 CH 3 In some embodiments, X 1 -CH 2 CH 2 CH 3 In some embodiments, X 1 -CH(CH 3 ) 2 In some embodiments, X 1 OCH 3 In some embodiments, X 1 OCH 2 CH 3 .

[0562] 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 - 、-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 In some embodiments, X 2 In some embodiments, X 2In some embodiments, X 2 -O - In some embodiments, X 2 For -S - In some embodiments, X 2 For -NH 2 In some embodiments, X 2 -NHCH 3 In some embodiments, X 2 -NH(C(=O)CH 3 ). In some embodiments, X 2 -CH 3 In some embodiments, X 2 -CH 2 CH 3 In some embodiments, X 2 -CH 2 CH 2 CH 3 In some embodiments, X 2 -CH(CH 3 ) 2 In some embodiments, X 2 OCH 3 In some embodiments, X 2 OCH 2 CH 3 .

[0563] 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 - 、-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 In some embodiments, X 3 In some embodiments, X 3 In some embodiments, X 3 -O - In some embodiments, X 3 For -S - In some embodiments, X 3 For -NH 2 In some embodiments, X 3 -NHCH 3 In some embodiments, X 3 -NH(C(=O)CH 3 ). In some embodiments, X 3 -CH 3 In some embodiments, X 3 -CH 2 CH 3 In some embodiments, X 3 -CH 2 CH 2 CH 3 In some embodiments, X 3 -CH(CH 3 ) 2 In some embodiments, X 3 OCH 3 In some embodiments, X 3 OCH 2 CH 3 .

[0564] 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 - 、-NH 2 、-NHCH 3 、-NH(C(=O)CH 3 ),-CH3 、-CH 2 CH 3 、-CH 2 CH 2 CH 3 、-CH(CH 3 ) 2 、-OCH 3 or -OCH 2 CH 3 In some embodiments, X 4 In some embodiments, X 4 In some embodiments, X 4 -O - In some embodiments, X 4 For -S - In some embodiments, X 4 For -NH 2 In some embodiments, X 4 -NHCH 3 In some embodiments, X 4 -NH(C(=O)CH 3 ). In some embodiments, X 4 -CH 3 In some embodiments, X 4 -CH 2 CH 3 In some embodiments, X 4 -CH 2 CH 2 CH 3 In some embodiments, X 4 -CH(CH 3 ) 2 In some embodiments, X 4 OCH 3 In some embodiments, X 4 OCH 2 CH 3 .

[0565] 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 - 、-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 In some embodiments, X n In some embodiments, X n In some embodiments, X n -O - In some embodiments, X n For -S - In some embodiments, X n For -NH 2 In some embodiments, X n -NHCH 3 In some embodiments, X n -NH(C(=O)CH 3 ). In some embodiments, X n -CH 3 In some embodiments, X n -CH 2 CH 3 In some embodiments, X n -CH 2 CH 2 CH 3 In some embodiments, X n -CH(CH 3 ) 2 In some embodiments, X n OCH 3 In some embodiments, X n OCH 2 CH 3 .

[0566] 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 1 =0. In some embodiments, Y 1 =S. In some embodiments, Y 1 =NH. In some embodiments, Y 1 =NCH 3 .

[0567] 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 2 =0. In some embodiments, Y 2 =S. In some embodiments, Y 2 =NH. In some embodiments, Y 2 =NCH 3 .

[0568] 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 =O, =S, =NH or =NCH 3In some embodiments, Y 3 =0. In some embodiments, Y 3 =S. In some embodiments, Y 3 =NH. In some embodiments, Y 3 =NCH 3 .

[0569] 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 4 =0. In some embodiments, Y 4 =S. In some embodiments, Y 4 =NH. In some embodiments, Y 4 =NCH 3 .

[0570] 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 =O, =S, =NH or =NCH 3 In some embodiments, Y n =0. In some embodiments, Y n =S. In some embodiments, Y n =NH. In some embodiments, Y n =NCH 3 .

[0571] 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), A is -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH 2 -. In some embodiments, A is -NH-. In some embodiments, A is -N(CH 3 In some embodiments, A is -N(C(=O)CH 3 )-.

[0572] 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), A 1 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 1 In some embodiments, A 1 In some embodiments, A 1 -CH 2 -. In some embodiments, A 1 In some embodiments, A 1 -N(CH 3 )-. In some embodiments, A 1 -N(C(=O)CH 3 )-.

[0573] 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), A 2 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 2 In some embodiments, A 2 In some embodiments, A 2 -CH 2 -. In some embodiments, A 2 In some embodiments, A 2 -N(CH 3 )-. In some embodiments, A 2 -N(C(=O)CH 3 )-.

[0574] 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), 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.

[0575] In some embodiments, B 1 for In some embodiments, B 2 and B 3 Each is independently adenine or guanine. In some embodiments, B 2 In some embodiments, B 3 For guanine.

[0576] In some embodiments, Z 1 , Z 2 , Z 3 and Z 4 Each is independently -OH or -OCH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 2 In some embodiments, Z 1 , Z 2 and Z 4 is -OH, and Z 3 OCH 3 In some embodiments, Z 1 and Z 3 OCH 3 , and Z 2 and Z 4 In some embodiments, Z', Z", and Z'" are hydrogen.

[0577] In some embodiments, Q 1 and Q 4 OCH 2 -. In some embodiments, Q 2 and Q 3 It is -O-.

[0578] In some embodiments, X 1 , X 2 , X 3 and X 4 In some embodiments, X 1 , X 2 , X 3 and X 4 In some embodiments, X 1 , X 3 , X 4 is -O-, and X 2 For -S-.

[0579] In some embodiments, Y 1 , Y 2 , Y 3 and Y 4 =0. In some embodiments, Y 1 , Y 2 , Y 3 and Y 4 =S. In some embodiments, Y1 , Y 3 and Y 4 =O, and Y 2 =S. In some embodiments, Y 1 , Y 2 , Y 3 =O, and Y 4 is =S.

[0580] In some embodiments, A, A 1 and A 2 It is -O-.

[0581] In some embodiments, p is 0.

[0582] In one aspect, described herein is an mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof:

[0583]

[0584] in

[0585] B 1 for

[0586] B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase;

[0587] Z' and Z" are each independently hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 ;

[0588] Z"' is hydrogen, fluorine, -CH 3 、-CH 2 CH 3 、-OCH 3 or -OCH 2 CH 3 ;

[0589] Z 1 and Z 2 are independently hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 ;

[0590] Z 3 , Z 4 and Z n are independently hydrogen, fluorine, -OH, -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 ;

[0591] Q 1 and Q 4 are independently -CH=CH-, -CH 2 -、-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 )-;

[0592] Q 2 and Q 3 are independently -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH3 )-;

[0593] 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 ;

[0594] Y 1 , Y 2 , Y 3 , Y 4 and Y n are independently =O, =S, =NH or =NCH 3 ;

[0595] A. A 1 and A 2 are independently -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;and

[0596] p=0, 1, 2, 3, 4, 5 or 6.

[0597] In some embodiments of the 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 for In some embodiments, B 1 for In some embodiments, B 1 for In some embodiments, B 1 for

[0598] In some embodiments of the 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 In some embodiments, B 2 In some embodiments, B 2 In some embodiments, B 2 is uracil, in some embodiments, B 2 is thymine, in some embodiments, B 2 In some embodiments, B 2 For purine.

[0599] In some embodiments of the 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 In some embodiments, B 3 In some embodiments, B 3 In some embodiments, B 3 is uracil, in some embodiments, B 3 is thymine, in some embodiments, B 3 In some embodiments, B 3 For purine.

[0600] In some embodiments of the 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 In some embodiments, B n In some embodiments, B n In some embodiments, B n is uracil, in some embodiments, B n is thymine, in some embodiments, B n In some embodiments, B n For purine.

[0601] In some embodiments of the compounds 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 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 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 3 In some embodiments, Z' is -CH 2 CH 3 In some embodiments, Z' is -OCH 3 In some embodiments, Z' is -NH(CH 3), in some embodiments, Z' is -NH 2 -. In some embodiments, Z' is -NH(C(=O)CH 3 In some embodiments, Z' is -SCH 3 .

[0602] In some embodiments of the compounds 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 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 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 3 In some embodiments, Z" is -CH 2 CH 3 In some embodiments, Z" is -OCH 3 In some embodiments, Z" is -NH(CH 3 ), in some embodiments, Z" is -NH 2 -. In some embodiments, Z" is -NH(C(=O)CH 3 In some embodiments, Z" is -SCH 3 .

[0603] In some embodiments of the compounds 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, -CH3 、-CH 2 CH 3 、-OCH 3 or -OCH 2 CH 3 In some embodiments, Z'' is hydrogen. In some embodiments, Z'' is fluorine. In some embodiments, Z'' is -CH 3 In some embodiments, Z'' is -CH 2 CH 3 In some embodiments, Z'' is -OCH 3 In some embodiments, Z'' is -OCH 2 CH 3 .

[0604] In some embodiments of the 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 Hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 -CH 3 In some embodiments, Z 1 -CH 2 CH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 1 For-SCH 3 In some embodiments, Z1 OCH 2 CH 3 In some embodiments, Z 1 For -NH 2 In some embodiments, Z 1 For NHCH 3 In some embodiments, Z 1 NHC(=O)CH 3 .

[0605] In some embodiments of the 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 Hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 -CH 3 In some embodiments, Z 2 -CH 2 CH 3 In some embodiments, Z 2 OCH 3 In some embodiments, Z 2 For-SCH 3 In some embodiments, Z 2 OCH 2 CH 3 In some embodiments, Z 2 For -NH 2 In some embodiments, Z 2For NHCH 3 In some embodiments, Z 2 NHC(=O)CH 3 .

[0606] In some embodiments of the 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 For hydrogen, fluorine, -OH, -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 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 -CH 3 In some embodiments, Z 3 -CH 2 CH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 3 For -NH 2 In some embodiments, Z 3 -NHCH 3 In some embodiments, Z 3 -NH(C(=O)CH 3). In some embodiments, Z 3 OCH 2 CH 3 In some embodiments, Z 3 OCH 2 OCH 3 In some embodiments, Z 3 OCH 2 CH 2 CH 3 In some embodiments, Z 3 -OCH(CH 3 ) 2 In some embodiments, Z 3 For-SCH 3 In some embodiments, Z 3 OCH 2 CH 2 OCH 3 .

[0607] In some embodiments of the 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 For hydrogen, fluorine, -OH, -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 In some embodiments, Z 4 In some embodiments, Z 4In some embodiments, Z 4 In some embodiments, Z 4 -CH 3 In some embodiments, Z 4 -CH 2 CH 3 In some embodiments, Z 4 OCH 3 In some embodiments, Z 4 For -NH 2 In some embodiments, Z 4 -NHCH 3 In some embodiments, Z 4 -NH(C(=O)CH 3 ). In some embodiments, Z 4 OCH 2 CH 3 In some embodiments, Z 4 OCH 2 OCH 3 In some embodiments, Z 4 OCH 2 CH 2 CH 3 In some embodiments, Z 4 -OCH(CH 3 ) 2 In some embodiments, Z 4 For-SCH 3 In some embodiments, Z 4 OCH 2 CH 2 OCH 3 .

[0608] In some embodiments of the 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 For hydrogen, fluorine, -OH, -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 In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n -CH 3 In some embodiments, Z n -CH 2 CH 3 In some embodiments, Z n OCH 3 In some embodiments, Z n For -NH 2 In some embodiments, Z n -NHCH 3 In some embodiments, Z n -NH(C(=O)CH 3 ). In some embodiments, Z n OCH 2 CH 3 In some embodiments, Z n OCH 2 OCH 3 In some embodiments, Z n OCH 2 CH 2 CH 3 In some embodiments, Z n -OCH(CH 3 ) 2 In some embodiments, Z n For-SCH 3 In some embodiments, Z n OCH 2 CH 2 OCH 3 .

[0609] In some embodiments of the 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 -CH=CH-, -CH 2 -、-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 )-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 -CH 2 -. In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 CH 2 -. In some embodiments, Q 1 -CH 2 CF 2 -. In some embodiments, Q 1 -CH 2 NH 2 -. In some embodiments, Q 1 -CH 2 NH(CH 3 )-. In some embodiments, Q 1 -CH 2 N(C(=O)CH 3 )-.

[0610] In some embodiments of the 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 -CH=CH-, -CH 2 -、-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 )-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 4 -CH 2 -. In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 CH 2 -. In some embodiments, Q 4 -CH 2 CF 2 -. In some embodiments, Q 4 -CH 2 NH 2 -. In some embodiments, Q 4 -CH 2 NH(CH 3 )-. In some embodiments, Q 4 -CH 2 N(C(=O)CH 3 )-.

[0611] In some embodiments of the 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 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 2 In some embodiments, Q 2 In some embodiments, Q 2 -CH 2 -. In some embodiments, Q 2 For-CF 2 -. In some embodiments, Q 2 In some embodiments, Q 2 -N(CH 3 )-. In some embodiments, Q 2 -N(C(=O)CH 3 )-.

[0612] In some embodiments of the 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 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 3 In some embodiments, Q 3 In some embodiments, Q 3 -CH 2 -. In some embodiments, Q3 For-CF 2 -. In some embodiments, Q 3 In some embodiments, Q 3 -N(CH 3 )-. In some embodiments, Q 3 -N(C(=O)CH 3 )-.

[0613] In some embodiments of the 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 - 、-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 In some embodiments, X 1 In some embodiments, X 1 In some embodiments, X 1 -O - In some embodiments, X 1 For -S - In some embodiments, X 1 For -NH 2 In some embodiments, X 1 -NHCH 3 In some embodiments, X 1 -NH(C(=O)CH 3 ). In some embodiments, X 1 -CH 3 In some embodiments, X 1-CH 2 CH 3 In some embodiments, X 1 -CH 2 CH 2 CH 3 In some embodiments, X 1 -CH(CH 3 ) 2 In some embodiments, X 1 OCH 3 In some embodiments, X 1 OCH 2 CH 3 .

[0614] In some embodiments of the 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 - 、-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 In some embodiments, X 2 In some embodiments, X 2 In some embodiments, X 2 -O - In some embodiments, X 2 For -S - In some embodiments, X 2 For -NH 2 In some embodiments, X 2 -NHCH 3In some embodiments, X 2 -NH(C(=O)CH 3 ). In some embodiments, X 2 -CH 3 In some embodiments, X 2 -CH 2 CH 3 In some embodiments, X 2 -CH 2 CH 2 CH 3 In some embodiments, X 2 -CH(CH 3 ) 2 In some embodiments, X 2 OCH 3 In some embodiments, X 2 OCH 2 CH 3 .

[0615] In some embodiments of the 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 - 、-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 In some embodiments, X 3 In some embodiments, X 3 In some embodiments, X 3 -O - In some embodiments, X3 For -S - In some embodiments, X 3 For -NH 2 In some embodiments, X 3 -NHCH 3 In some embodiments, X 3 -NH(C(=O)CH 3 ). In some embodiments, X 3 -CH 3 In some embodiments, X 3 -CH 2 CH 3 In some embodiments, X 3 -CH 2 CH 2 CH 3 In some embodiments, X 3 -CH(CH 3 ) 2 In some embodiments, X 3 OCH 3 In some embodiments, X 3 OCH 2 CH 3 .

[0616] In some embodiments of the 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 - 、-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 3In some embodiments, X 4 In some embodiments, X 4 In some embodiments, X 4 -O - In some embodiments, X 4 For -S - In some embodiments, X 4 For -NH 2 In some embodiments, X 4 -NHCH 3 In some embodiments, X 4 -NH(C(=O)CH 3 ). In some embodiments, X 4 -CH 3 In some embodiments, X 4 -CH 2 CH 3 In some embodiments, X 4 -CH 2 CH 2 CH 3 In some embodiments, X 4 -CH(CH 3 ) 2 In some embodiments, X 4 OCH 3 In some embodiments, X 4 OCH 2 CH 3 .

[0617] In some embodiments of the 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 - 、-NH 2 、-NHCH 3 、-NH(C(=O)CH 3 ),-CH 3 、-CH 2 CH 3 、-CH 2 CH2 CH 3 、-CH(CH 3 ) 2 、-OCH 3 or -OCH 2 CH 3 In some embodiments, X n In some embodiments, X n In some embodiments, X n -O - In some embodiments, X n For -S - In some embodiments, X n For -NH 2 In some embodiments, X n -NHCH 3 In some embodiments, X n -NH(C(=O)CH 3 ). In some embodiments, X n -CH 3 In some embodiments, X n -CH 2 CH 3 In some embodiments, X n -CH 2 CH 2 CH 3 In some embodiments, X n -CH(CH 3 ) 2 In some embodiments, X n OCH 3 In some embodiments, X n OCH 2 CH 3 .

[0618] In some embodiments of the 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 1 =0. In some embodiments, Y1 =S. In some embodiments, Y 1 =NH. In some embodiments, Y 1 =NCH 3 .

[0619] In some embodiments of the 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 2 =0. In some embodiments, Y 2 =S. In some embodiments, Y 2 =NH. In some embodiments, Y 2 =NCH 3 .

[0620] In some embodiments of the 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 3 =0. In some embodiments, Y 3 =S. In some embodiments, Y 3 =NH. In some embodiments, Y 3 =NCH 3 .

[0621] In some embodiments of the 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 4 =0. In some embodiments, Y 4 =S. In some embodiments, Y 4 =NH. In some embodiments, Y 4 =NCH 3 .

[0622] In some embodiments of the 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 =O, =S, =NH or =NCH 3 In some embodiments, Y n =0. In some embodiments, Y n =S. In some embodiments, Y n =NH. In some embodiments, Y n =NCH 3 .

[0623] In some embodiments of the 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 2 -、-NH-、-N(CH 3)-or-N(C(=O)CH 3 )-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH 2 -. In some embodiments, A is -NH-. In some embodiments, A is -N(CH 3 In some embodiments, A is -N(C(=O)CH 3 )-.

[0624] In some embodiments of the 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 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 1 In some embodiments, A 1 In some embodiments, A 1 -CH 2 -. In some embodiments, A 1 In some embodiments, A 1 -N(CH 3 )-. In some embodiments, A 1 -N(C(=O)CH 3 )-.

[0625] In some embodiments of the 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 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH3 )-. In some embodiments, A 2 In some embodiments, A 2 In some embodiments, A 2 -CH 2 -. In some embodiments, A 2 In some embodiments, A 2 -N(CH 3 )-. In some embodiments, A 2 -N(C(=O)CH 3 )-.

[0626] In some embodiments of compounds 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.

[0627] In some embodiments, B 1 for In some embodiments, B 2 and B 3 Each is independently adenine or guanine. In some embodiments, B 2 In some embodiments, B 3 For guanine.

[0628] In some embodiments, Z 1 , Z 2 , Z 3 and Z 4 Each is independently -OH or -OCH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 2 In some embodiments, Z 1 , Z 2 and Z4 is -OH, and Z 3 OCH 3 In some embodiments, Z 1 and Z 3 OCH 3 , and Z 2 and Z 4 In some embodiments, Z', Z", and Z'" are hydrogen.

[0629] In some embodiments, Q 1 and Q 4 OCH 2 -. In some embodiments, Q 2 and Q 3 It is -O-.

[0630] In some embodiments, X 1 , X 2 , X 3 and X 4 In some embodiments, X 1 , X 2 , X 3 and X 4 In some embodiments, X 1 , X 3 , X 4 is -O-, and X 2 For -S-.

[0631] In some embodiments, Y 1 , Y 2 , Y 3 and Y 4 =0. In some embodiments, Y 1 , Y 2 , Y 3 and Y 4 =S. In some embodiments, Y 1 , Y 3 and Y 4 =O, and Y 2 =S. In some embodiments, Y 1 , Y 2 , Y 3 =O, and Y 4 is =S.

[0632] In some embodiments, A, A 1 and A 2 It is -O-.

[0633] In some embodiments, p is 0.

[0634] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-a):

[0635]

[0636] In some embodiments, the IVT mRNA sequence initiator has a structure of formula (II-a'):

[0637]

[0638] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-b):

[0639]

[0640] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-b'):

[0641]

[0642] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-c):

[0643]

[0644] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-c'):

[0645]

[0646] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-d):

[0647]

[0648] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-d'):

[0649]

[0650] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-e):

[0651]

[0652] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-e'):

[0653]

[0654] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-f):

[0655]

[0656] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-g):

[0657]

[0658] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-g'):

[0659]

[0660] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-h):

[0661]

[0662] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-h'):

[0663]

[0664] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-i):

[0665]

[0666] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-I'):

[0667]

[0668] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-j):

[0669]

[0670] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-j'):

[0671]

[0672] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-k):

[0673]

[0674] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-k'):

[0675]

[0676] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-1):

[0677]

[0678] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-1'):

[0679]

[0680] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-m):

[0681]

[0682] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-m'):

[0683]

[0684] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-n):

[0685]

[0686] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-n'):

[0687]

[0688] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-o):

[0689]

[0690] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-o'):

[0691]

[0692] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-p):

[0693]

[0694] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-p'):

[0695]

[0696] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-q):

[0697]

[0698] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-q'):

[0699]

[0700] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-r):

[0701]

[0702] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-r'):

[0703]

[0704] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-s):

[0705]

[0706] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-s'):

[0707]

[0708] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-t):

[0709]

[0710] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-t'):

[0711]

[0712] In some embodiments, the IVT mRNA sequence initiator has the structure of Formula (II-u):

[0713]

[0714] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (II-u'):

[0715]

[0716] In some embodiments of formula (II) or formula (II-a) to (II-u'), each B is independently a natural nucleobase. In some embodiments of formula (II) or formula (II-a) to (II-u'), each B is independently a modified nucleobase. In some embodiments of formula (II) or formula (II-a) to (II-u'), each B is independently a non-natural nucleobase.

[0717] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), B 1 for

[0718] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), B 1 for.

[0719] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), B 1 for

[0720] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Z 1 For fluorine.

[0721] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Z 1 It is -OH.

[0722] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Z 1 OCH 3 .

[0723] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Z 2 For fluorine.

[0724] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Z 2 It is -OH.

[0725] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Z 2 OCH 3 .

[0726] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 1 -CH 2 O-.

[0727] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 1 It is -O-.

[0728] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 4 -CH 2 O-.

[0729] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 4 It is -O-.

[0730] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 2 It is -O-.

[0731] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 3 It is -O-.

[0732] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y 1 is =O.

[0733] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y 3 is =O.

[0734] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y 2 is =O.

[0735] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y 4 is =O.

[0736] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y n is =O.

[0737] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y 1 , Y 2 , Y 3 , Y 4 and Y n One or more of them is =S.

[0738] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y 2 is =S.

[0739] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y 4 is =S.

[0740] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y n is =S.

[0741] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is =0.

[0742] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), X 1 , X 4 and X n Each is -O - .

[0743] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), X 2 It is -O-.

[0744] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), X 3 -O - .

[0745] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), X 1 , X 2 , X 3 , X 4 and X n One or more of -S - .

[0746] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), X 2 For -S - .

[0747] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), X 4 For -S - .

[0748] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), X 3 For -S-.

[0749] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), X 1 , X 2 , X 3 , X 4 and X n Each is -O - .

[0750] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), A, A 1 , A 2 Each is -O-.

[0751] In some embodiments of Formula (II) or Formula (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 In some embodiments, A 2 In some embodiments, A 2 In some embodiments, A is -S-. In some embodiments, A is -O-. 1 It is -O-.

[0752] In some embodiments of Formula (II) or Formulas (II-a) to (II-u'), p is 0. In some embodiments, p is 1. In some embodiments, p is 2.

[0753] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), B 2 It is adenine, cytosine, guanine, uracil, thymine, hypoxanthine or purine.

[0754] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), B 3 It is adenine, cytosine, guanine, uracil, thymine, hypoxanthine or purine.

[0755] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), B n It is adenine, cytosine, guanine, uracil, thymine, hypoxanthine or purine.

[0756] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 1-CH 2 In some embodiments, Q 4 -CH 2 O-.

[0757] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 2 In some embodiments, Q 3 It is -O-.

[0758] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), X n Each independently represents -OH, -SH, O - or S - .

[0759] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Y n Each is independently =O or =S.

[0760] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), B 1 for

[0761] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 1 -CH 2 O-;Q 4 -CH 2 O-;Q 2 Q 3 -O-;X n Each independently represents -OH, -SH, O - or S - ; Y n Each independently represents =O or =S; B 1 for

[0762] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 1 -CH 2 O-;Q 4 -CH 2 O-;Q 2 Q 3 For-O-'X n Each independently represents -OH, -SH, O - or S - ; Y n Each independently represents =O or =S; B 1 for

[0763] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), Q 1 -CH 2 O-;Q 4 -CH 2 O-;Q 2 Q 3 For-O-'X n Each independently represents -OH, -SH, O - or S - ; Y n Each independently represents =O or =S; B 1 for

[0764] In some embodiments of Formula (II) or Formula (II-a) to (II-u'), B 2 In some embodiments of Formula (II) or Formula (II-a) to (II-u'), B 3 For guanine.

[0765] In one aspect, the present invention describes an mRNA sequence having a 5' terminal region motif (motif (II'):

[0766]

[0767] in

[0768] B 1 for

[0769] B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase;

[0770] Z' and Z" are each independently hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 ;

[0771] Z"' is hydrogen, fluorine, -CH 3 、-CH 2 CH 3 、-OCH 3 or -OCH 2 CH3 ;

[0772] Z 1 and Z 2 are independently hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 ;

[0773] Z 3 , Z 4 and Z n are independently hydrogen, fluorine, -OH, -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 ;

[0774] Q 1 and Q 4 are independently -CH=CH-, -CH 2 -、-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 )-;

[0775] Q 2 and Q 3 are independently -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;

[0776] 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 ;

[0777] Y 1 , Y 2 , Y 3 , Y 4 and Y n are independently =O, =S, =NH or =NCH 3 ;

[0778] A. A 1 and A 2 are independently -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;and

[0779] p=0, 1, 2, 3, 4, 5 or 6.

[0780] In some embodiments of compounds having the 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 for In some embodiments, B 1 for In some embodiments, B 1 for In some embodiments, B 1 for

[0781] In some embodiments of compounds having the 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 In some embodiments, B 2 In some embodiments, B 2 In some embodiments, B 2 is uracil, in some embodiments, B 2 is thymine, in some embodiments, B 2 In some embodiments, B 2 For purine.

[0782] In some embodiments of compounds having the 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 In some embodiments, B 3 In some embodiments, B 3 In some embodiments, B 3 is uracil, in some embodiments, B 3 is thymine, in some embodiments, B 3 In some embodiments, B 3 For purine.

[0783] In some embodiments of compounds having the 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 In some embodiments, B n In some embodiments, B n In some embodiments, B n is uracil, in some embodiments, B n is thymine, in some embodiments, B n In some embodiments, B n For purine.

[0784] In some embodiments of compounds of the 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 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH 3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 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 3 In some embodiments, Z' is -CH 2 CH 3 In some embodiments, Z' is -OCH 3 In some embodiments, Z' is -NH(CH 3 ), in some embodiments, Z' is -NH 2 -. In some embodiments, Z' is -NH(C(=O)CH 3 In some embodiments, Z' is -SCH 3 .

[0785] In some embodiments of compounds of the 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 3 、-CH 2 CH 3 、-OCH 3 、-NH(CH3 ), -NH 2 、-NH(C(=O)CH 3 ) or -SCH 3 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 3 In some embodiments, Z" is -CH 2 CH 3 In some embodiments, Z" is -OCH 3 In some embodiments, Z" is -NH(CH 3 ), in some embodiments, Z" is -NH 2 -. In some embodiments, Z" is -NH(C(=O)CH 3 In some embodiments, Z" is -SCH 3 .

[0786] In some embodiments of compounds of the 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 3 、-CH 2 CH 3 、-OCH 3 or -OCH 2 CH 3 In some embodiments, Z'' is hydrogen. In some embodiments, Z'' is fluorine. In some embodiments, Z'' is -CH 3 In some embodiments, Z'' is -CH 2 CH 3 In some embodiments, Z'' is -OCH 3 In some embodiments, Z'' is -OCH 2 CH 3 .

[0787] In some embodiments of compounds having the 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 Hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 -CH 3 In some embodiments, Z 1 -CH 2 CH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 1 For-SCH 3 In some embodiments, Z 1 OCH 2 CH 3 In some embodiments, Z 1 For -NH 2 In some embodiments, Z 1 For NHCH 3 In some embodiments, Z 1 NHC(=O)CH 3 .

[0788] In some embodiments of compounds having the 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 Hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-SCH 3 、-OCH 2 CH 3 、-NH 2 NHCH 3 or NHC(=O)CH 3 In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 -CH 3 In some embodiments, Z 2 -CH 2 CH 3 In some embodiments, Z 2 OCH 3 In some embodiments, Z 2 For-SCH 3 In some embodiments, Z 2 OCH 2 CH 3 In some embodiments, Z 2 For -NH 2 In some embodiments, Z 2 For NHCH 3 In some embodiments, Z 2 NHC(=O)CH 3 .

[0789] In some embodiments of compounds having the 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 For hydrogen, fluorine, -OH, -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 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 -CH 3 In some embodiments, Z 3 -CH 2 CH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 3 For -NH 2 In some embodiments, Z 3 -NHCH 3 In some embodiments, Z 3 -NH(C(=O)CH 3 ). In some embodiments, Z 3 OCH 2 CH 3 In some embodiments, Z3 OCH 2 OCH 3 In some embodiments, Z 3 OCH 2 CH 2 CH 3 In some embodiments, Z 3 -OCH(CH 3 ) 2 In some embodiments, Z 3 For-SCH 3 In some embodiments, Z 3 OCH 2 CH 2 OCH 3 .

[0790] In some embodiments of compounds having the 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 For hydrogen, fluorine, -OH, -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 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4-CH 3 In some embodiments, Z 4 -CH 2 CH 3 In some embodiments, Z 4 OCH 3 In some embodiments, Z 4 For -NH 2 In some embodiments, Z 4 -NHCH 3 In some embodiments, Z 4 -NH(C(=O)CH 3 ). In some embodiments, Z 4 OCH 2 CH 3 In some embodiments, Z 4 OCH 2 OCH 3 In some embodiments, Z 4 OCH 2 CH 2 CH 3 In some embodiments, Z 4 -OCH(CH 3 ) 2 In some embodiments, Z 4 For-SCH 3 In some embodiments, Z 4 OCH 2 CH 2 OCH 3 .

[0791] In some embodiments of compounds having the 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 For hydrogen, fluorine, -OH, -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 In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n -CH 3 In some embodiments, Z n -CH 2 CH 3 In some embodiments, Z n OCH 3 In some embodiments, Z n For -NH 2 In some embodiments, Z n -NHCH 3 In some embodiments, Z n -NH(C(=O)CH 3 ). In some embodiments, Z n OCH 2 CH 3 In some embodiments, Z n OCH 2 OCH 3 In some embodiments, Z n OCH 2 CH 2 CH 3 In some embodiments, Z n -OCH(CH 3 ) 2 In some embodiments, Z n For-SCH 3 In some embodiments, Z n OCH 2 CH 2 OCH 3 .

[0792] In some embodiments of compounds having the 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 -CH=CH-, -CH 2 -、-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 )-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 -CH 2 -. In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 CH 2 -. In some embodiments, Q 1 -CH 2 CF 2 -. In some embodiments, Q 1 -CH 2 NH 2 -. In some embodiments, Q 1 -CH 2 NH(CH 3 )-. In some embodiments, Q 1 -CH 2 N(C(=O)CH 3 )-.

[0793] In some embodiments of compounds having the 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 -CH=CH-, -CH 2 -、-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 )-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 4 -CH 2 -. In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 CH 2 -. In some embodiments, Q 4 -CH 2 CF 2 -. In some embodiments, Q 4 -CH 2 NH 2 -. In some embodiments, Q 4 -CH 2 NH(CH 3 )-. In some embodiments, Q 4 -CH 2 N(C(=O)CH 3 )-.

[0794] In some embodiments of compounds having the 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 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 2 In some embodiments, Q 2 In some embodiments, Q 2 -CH 2 -. In some embodiments, Q 2 For-CF 2 -. In some embodiments, Q 2 In some embodiments, Q 2 -N(CH 3 )-. In some embodiments, Q 2 -N(C(=O)CH 3 )-.

[0795] In some embodiments of compounds having the 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 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 3 In some embodiments, Q 3 In some embodiments, Q3 -CH 2 -. In some embodiments, Q 3 For-CF 2 -. In some embodiments, Q 3 In some embodiments, Q 3 -N(CH 3 )-. In some embodiments, Q 3 -N(C(=O)CH 3 )-.

[0796] In some embodiments of compounds having the 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 - 、-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 In some embodiments, X 1 In some embodiments, X 1 In some embodiments, X 1 -O - In some embodiments, X 1 For -S - In some embodiments, X 1 For -NH 2 In some embodiments, X 1 -NHCH 3 In some embodiments, X 1 -NH(C(=O)CH 3). In some embodiments, X 1 -CH 3 In some embodiments, X 1 -CH 2 CH 3 In some embodiments, X 1 -CH 2 CH 2 CH 3 In some embodiments, X 1 -CH(CH 3 ) 2 In some embodiments, X 1 OCH 3 In some embodiments, X 1 OCH 2 CH 3 .

[0797] In some embodiments of compounds having the 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 - 、-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 In some embodiments, X 2 In some embodiments, X 2 In some embodiments, X 2 -O - In some embodiments, X 2 For -S -In some embodiments, X 2 For -NH 2 In some embodiments, X 2 -NHCH 3 In some embodiments, X 2 -NH(C(=O)CH 3 ). In some embodiments, X 2 -CH 3 In some embodiments, X 2 -CH 2 CH 3 In some embodiments, X 2 -CH 2 CH 2 CH 3 In some embodiments, X 2 -CH(CH 3 ) 2 In some embodiments, X 2 OCH 3 In some embodiments, X 2 OCH 2 CH 3 .

[0798] In some embodiments of compounds having the 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 - 、-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 3In some embodiments, X 3 In some embodiments, X 3 In some embodiments, X 3 -O - In some embodiments, X 3 For -S - In some embodiments, X 3 For -NH 2 In some embodiments, X 3 -NHCH 3 In some embodiments, X 3 -NH(C(=O)CH 3 ). In some embodiments, X 3 -CH 3 In some embodiments, X 3 -CH 2 CH 3 In some embodiments, X 3 -CH 2 CH 2 CH 3 In some embodiments, X 3 -CH(CH 3 ) 2 In some embodiments, X 3 OCH 3 In some embodiments, X 3 OCH 2 CH 3 .

[0799] In some embodiments of compounds having the 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 - 、-NH 2 、-NHCH 3 、-NH(C(=O)CH 3 ),-CH 3 、-CH 2 CH 3 、-CH2 CH 2 CH 3 、-CH(CH 3 ) 2 、-OCH 3 or -OCH 2 CH 3 In some embodiments, X 4 In some embodiments, X 4 In some embodiments, X 4 -O - In some embodiments, X 4 For -S - In some embodiments, X 4 For -NH 2 In some embodiments, X 4 -NHCH 3 In some embodiments, X 4 -NH(C(=O)CH 3 ). In some embodiments, X 4 -CH 3 In some embodiments, X 4 -CH 2 CH 3 In some embodiments, X 4 -CH 2 CH 2 CH 3 In some embodiments, X 4 -CH(CH 3 ) 2 In some embodiments, X 4 OCH 3 In some embodiments, X 4 OCH 2 CH 3 .

[0800] In some embodiments of compounds having the 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- 、-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 In some embodiments, X n In some embodiments, X n In some embodiments, X n -O - In some embodiments, X n For -S - In some embodiments, X n For -NH 2 In some embodiments, X n -NHCH 3 In some embodiments, X n -NH(C(=O)CH 3 ). In some embodiments, X n -CH 3 In some embodiments, X n -CH 2 CH 3 In some embodiments, X n -CH 2 CH 2 CH 3 In some embodiments, X n -CH(CH 3 ) 2 In some embodiments, X n OCH 3 In some embodiments, X n OCH 2 CH 3 .

[0801] In some embodiments of compounds having the 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 1 =0. In some embodiments, Y 1 =S. In some embodiments, Y 1 =NH. In some embodiments, Y 1 =NCH 3 .

[0802] In some embodiments of compounds having the 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 2 =0. In some embodiments, Y 2 =S. In some embodiments, Y 2 =NH. In some embodiments, Y 2 =NCH 3 .

[0803] In some embodiments of compounds having the 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 3 =0. In some embodiments, Y 3 =S. In some embodiments, Y 3 =NH. In some embodiments, Y 3 =NCH 3 .

[0804] In some embodiments of compounds having the 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 =O, =S, =NH or =NCH 3 In some embodiments, Y 4 =0. In some embodiments, Y 4 =S. In some embodiments, Y 4 =NH. In some embodiments, Y 4 =NCH 3 .

[0805] In some embodiments of compounds having the 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 =O, =S, =NH or =NCH 3 In some embodiments, Y n =0. In some embodiments, Y n =S. In some embodiments, Y n =NH. In some embodiments, Y n =NCH 3 .

[0806] In some embodiments of compounds of the 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- 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH 2 -. In some embodiments, A is -NH-. In some embodiments, A is -N(CH 3 In some embodiments, A is -N(C(=O)CH 3 )-.

[0807] In some embodiments of compounds having the 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 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 1 In some embodiments, A 1 In some embodiments, A 1 -CH 2 -. In some embodiments, A 1 In some embodiments, A 1 -N(CH 3 )-. In some embodiments, A 1 -N(C(=O)CH 3 )-.

[0808] In some embodiments of compounds having the 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 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 2 In some embodiments, A 2 In some embodiments, A 2 -CH 2 -. In some embodiments, A 2 In some embodiments, A2 -N(CH 3 )-. In some embodiments, A 2 -N(C(=O)CH 3 )-.

[0809] 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'), 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.

[0810] In some embodiments, B 1 for In some embodiments, B 2 and B 3 Each is independently adenine or guanine. In some embodiments, B 2 In some embodiments, B 3 For guanine.

[0811] In some embodiments, B 1 for In some embodiments, B 2 and B 3 Each is independently adenine or guanine. In some embodiments, B 2 In some embodiments, B 3 For guanine.

[0812] In some embodiments, Z 1 , Z 2 , Z 3 and Z 4 Each is independently -OH or -OCH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 2In some embodiments, Z 1 , Z 2 and Z 4 is -OH, and Z 3 OCH 3 In some embodiments, Z 1 and Z 3 OCH 3 , and Z 2 and Z 4 In some embodiments, Z', Z", and Z'" are hydrogen.

[0813] In some embodiments, Q 1 and Q 4 OCH 2 -. In some embodiments, Q 2 and Q 3 It is -O-.

[0814] In some embodiments, X 1 , X 2 , X 3 and X 4 In some embodiments, X 1 , X 2 , X 3 and X 4 In some embodiments, X 1 , X 3 , X 4 is -O-, and X 2 For -S-.

[0815] In some embodiments, Y 1 , Y 2 , Y 3 and Y 4 =0. In some embodiments, Y 1 , Y 2 , Y 3 and Y 4 =S. In some embodiments, Y 1 , Y 3 and Y 4 =O, and Y 2 =S. In some embodiments, Y 1 , Y 2 , Y 3 =O, and Y 4 is =S.

[0816] In some embodiments, A, A 1 and A 2 It is -O-.

[0817] In some embodiments, p is 0.

[0818] One aspect described herein is an IVT mRNA sequence initiator of formula (I) or a salt or solvate thereof:

[0819]

[0820] in

[0821] B 1 for

[0822] B 2 is a modified or non-natural nucleobase;

[0823] B 3 and B n each independently a natural, modified or unnatural nucleobase;

[0824] 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 ;

[0825] 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 CH2 OCH 3 ;

[0826] Q 1 and Q 4 Each is 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 )-;

[0827] Q 2 and Q 3 are independently -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;

[0828] 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 ;

[0829] Y 1 , Y 2 , Y 3 , Y 4 and Y n are independently =O, =S, =NH or =NCH3 ;

[0830] A. A 1 and A 2 are independently -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;and

[0831] p=0, 1, 2, 3, 4, 5 or 6.

[0832] In some embodiments, the IVT mRNA sequence initiator has the structure of formula (III-a):

[0833]

[0834] In some embodiments of the compound of Formula (III) or (III-a), Z 1 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 In some embodiments, Z 1 In some embodiments, Z 1 is F. In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 -CH 3 In some embodiments, Z 1 -CH 2 CH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 1 OCH 2 CH 3 In some embodiments, Z 1 For-SCH 3 In some embodiments, Z 1 For -NH 2 In some embodiments, Z 1 For NHCH 3 In some embodiments, Z1 NHC(=O)CH 3 .

[0835] In some embodiments of the compound of Formula (III) or (III-a), Z 2 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 In some embodiments, Z 2 In some embodiments, Z 2 is F. In some embodiments, Z 2 In some embodiments, Z 2 In some embodiments, Z 2 -CH 3 In some embodiments, Z 2 -CH 2 CH 3 In some embodiments, Z 2 OCH 3 In some embodiments, Z 2 OCH 2 CH 3 In some embodiments, Z 2 For-SCH 3 In some embodiments, Z 2 For -NH 2 In some embodiments, Z 2 For NHCH 3 In some embodiments, Z 2 NHC(=O)CH 3 .

[0836] In some embodiments of the compound of Formula (III) or (III-a), Z 3 Hydrogen, fluorine, -OH, -SH, -CH 3 、-CH 2 CH 3 、-OCH 3 、-NH 2 、-NHCH 3 、-NH(C(=O)CH 3 ),-OCH 2 CH3 、-OCH 2 OCH 3 、-OCH 2 CH 2 CH 3 、-OCH(CH 3 ) 2 、-SCH 3 or -OCH 2 CH 2 OCH 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 -CH 3 In some embodiments, Z 3 -CH 2 CH 3 In some embodiments, Z 3 OCH 2 OCH 3 In some embodiments, Z 3 OCH 2 CH 2 CH 3 In some embodiments, Z 3 -OCH(CH 3 ) 2 In some embodiments, Z 3 For-SCH 3 In some embodiments, Z 3 OCH 2 CH 2 OCH 3 .

[0837] In some embodiments of the compound of (III) or (III-a), Z 4 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 CH2 CH 3 、-OCH(CH 3 ) 2 、-SCH 3 or -OCH 2 CH 2 OCH 3 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 -CH 3 In some embodiments, Z 4 -CH 2 CH 3 In some embodiments, Z 4 OCH 2 OCH 3 In some embodiments, Z 4 OCH 2 CH 2 CH 3 In some embodiments, Z 4 -OCH(CH 3 ) 2 In some embodiments, Z 4 For-SCH 3 In some embodiments, Z 4 OCH 2 CH 2 OCH 3 .

[0838] In some embodiments of the compound of Formula (III) or (III-a), Z n 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 In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n -CH 3 In some embodiments, Z n -CH 2 CH 3 In some embodiments, Z n OCH 2 OCH 3 In some embodiments, Z n OCH 2 CH 2 CH 3 In some embodiments, Z n -OCH(CH 3 ) 2 In some embodiments, Z n For-SCH 3 In some embodiments, Z n OCH 2 CH 2 OCH 3 .

[0839] In some embodiments of the compound of Formula (III) or (III-a), B 2 is a modified or non-natural nucleobase. 2 is a modified guanine. In some embodiments, B 2 is a modified adenine. In some embodiments, B 2 is a modified cytosine. 2 is a modified uracil. In some embodiments, B 2 is a modified thymine, in some embodiments, B 2 is a modified hypoxanthine. 2 is a modified purine. 2 is 2-aminoadenine. In some embodiments, B 2 for

[0840] 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 In some embodiments, B 3 In some embodiments, B 3 In some embodiments, B 3 is uracil, in some embodiments, B 3 is thymine, in some embodiments, B 3 In some embodiments, B 3 For purine.

[0841] 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 In some embodiments, B n In some embodiments, B n In some embodiments, B n is uracil, in some embodiments, B n is thymine, in some embodiments, B n In some embodiments, B n For purine.

[0842] In some embodiments of the compound of Formula (III) or (III-a), B 2 is a modified adenine. In some embodiments, B 3 For guanine.

[0843] In some embodiments of the compound of Formula (III) or (III-a), Q 1 -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 )-. In some embodiments, Q 1 -CH 2-. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 CH 2 -. In some embodiments, Q 1 -CH 2 CF 2 -. In some embodiments, Q 1 -CH 2 NH 2 -. In some embodiments, Q 1 -CH 2 NH(CH 3 )-. In some embodiments, Q 1 -CH 2 N(C(=O)CH 3 )-.

[0844] In some embodiments of the compound of Formula (III) or (III-a), Q 4 -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 )-. In some embodiments, Q 4 -CH 2 -. In some embodiments, Q 4 is -CH=CH-. In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 CH 2 -. In some embodiments, Q 4 -CH 2 CF 2-. In some embodiments, Q 4 -CH 2 NH 2 -. In some embodiments, Q 4 -CH 2 NH(CH 3 )-. In some embodiments, Q 4 -CH 2 N(C(=O)CH 3 )-.

[0845] In some embodiments of the compound of Formula (III) or (III-a), Q 2 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 2 In some embodiments, Q 2 In some embodiments, Q 2 -CH 2 -. In some embodiments, Q 2 For-CF 2 -. In some embodiments, Q 2 In some embodiments, Q 2 -N(CH 3 )-. In some embodiments, Q 2 -N(C(=O)CH 3 ).

[0846] In some embodiments of the compound of Formula (III) or (III-a), Q 3 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 3 In some embodiments, Q 3 In some embodiments, Q 3 -CH 2 -. In some embodiments, Q 3 For-CF 2 -. In some embodiments, Q 3 In some embodiments, Q 3 -N(CH 3)-. In some embodiments, Q 3 -N(C(=O)CH 3 ).

[0847] In some embodiments of the compound of Formula (III) or (III-a), X 1 -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 In some embodiments, X 1 In some embodiments, X 1 In some embodiments, X 1 -O - In some embodiments, X 1 For -S - In some embodiments, X 1 For -NH 2 In some embodiments, X 1 -NHCH 3 In some embodiments, X 1 -NH(C(=O)CH 3 ). In some embodiments, X 1 -CH 3 In some embodiments, X 1 -CH 2 CH 3 In some embodiments, X 1 -CH 2 CH 2 CH 3 In some embodiments, X 1 -CH(CH 3 ) 2 In some embodiments, X 1 OCH 3 In some embodiments, X 1 OCH 2 CH 3 .

[0848] In some embodiments of the compound of Formula (III) or (III-a), X 2 -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 In some embodiments, X 2 In some embodiments, X 2 In some embodiments, X 2 -O - In some embodiments, X 2 For -S - In some embodiments, X 2 For -NH 2 In some embodiments, X 2 -NHCH 3 In some embodiments, X 2 -NH(C(=O)CH 3 ). In some embodiments, X 2 -CH 3 In some embodiments, X 2 -CH 2 CH 3 In some embodiments, X 2 -CH 2 CH 2 CH 3 In some embodiments, X 2 -CH(CH 3 ) 2 In some embodiments, X 2 OCH 3 In some embodiments, X 2 OCH 2 CH 3 .

[0849] In some embodiments of the compound of Formula (III) or (III-a), X3 -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 In some embodiments, X 3 In some embodiments, X 3 In some embodiments, X 3 -O - In some embodiments, X 3 For -S - In some embodiments, X 3 For -NH 2 In some embodiments, X 3 -NHCH 3 In some embodiments, X 3 -NH(C(=O)CH 3 ). In some embodiments, X 3 -CH 3 In some embodiments, X 3 -CH 2 CH 3 In some embodiments, X 3 -CH 2 CH 2 CH 3 In some embodiments, X 3 -CH(CH 3 ) 2 In some embodiments, X 3 OCH 3 In some embodiments, X 3 OCH 2 CH 3 .

[0850] In some embodiments of the compound of Formula (III) or (III-a), X 4 -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 In some embodiments, X 4 In some embodiments, X 4 In some embodiments, X 4 -O - In some embodiments, X 4 For -S - In some embodiments, X 4 For -NH 2 In some embodiments, X 4 -NHCH 3 In some embodiments, X 4 -NH(C(=O)CH 3 ). In some embodiments, X 4 -CH 3 In some embodiments, X 4 -CH 2 CH 3 In some embodiments, X 4 -CH 2 CH 2 CH 3 In some embodiments, X 4 -CH(CH 3 ) 2 In some embodiments, X 4 OCH 3 In some embodiments, X 4 OCH 2 CH 3 .

[0851] In some embodiments of the compound of Formula (III) or (III-a), X n -OH, -SH, -O - , -S - 、-NH 2 、-NHCH 3 、-NH(C(=O)CH3 ),-CH 3 、-CH 2 CH 3 、-CH 2 CH 2 CH 3 、-CH(CH 3 ) 2 、-OCH 3 or -OCH 2 CH 3 In some embodiments, X n In some embodiments, X n In some embodiments, X n -O - In some embodiments, X n For -S - In some embodiments, X n For -NH 2 In some embodiments, X n -NHCH 3 In some embodiments, X n -NH(C(=O)CH 3 ). In some embodiments, X n -CH 3 In some embodiments, X n -CH 2 CH 3 In some embodiments, X n -CH 2 CH 2 CH 3 In some embodiments, X n -CH(CH 3 ) 2 In some embodiments, X n OCH 3 In some embodiments, X n OCH 2 CH 3 .

[0852] In some embodiments of the compound of (III) or (III-a), Y 1 =O, =S, =NH or =NCH 3 In some embodiments, Y 1 =0. In some embodiments, Y 1 =S. In some embodiments, Y 1 =NH. In some embodiments, Y 1 =NHCH3 .

[0853] In some embodiments of the compound of Formula (III) or (III-a), Y 2 =O, =S, =NH or =NCH 3 In some embodiments, Y 2 =0. In some embodiments, Y 2 =S. In some embodiments, Y 2 =NH. In some embodiments, Y 2 =NHCH 3 .

[0854] In some embodiments of the compound of (III) or (III-a), Y 3 =O, =S, =NH or =NCH 3 In some embodiments, Y 3 =0. In some embodiments, Y 3 =S. In some embodiments, Y 3 =NH. In some embodiments, Y 3 =NHCH 3 .

[0855] In some embodiments of the compound of Formula (III) or (III-a), Y 4 =O, =S, =NH or =NCH 3 In some embodiments, Y 4 =0. In some embodiments, Y 4 =S. In some embodiments, Y 4 =NH. In some embodiments, Y 4 =NHCH 3 .

[0856] In some embodiments of the compound of Formula (III) or (III-a), Y n =O, =S, =NH or =NCH 3 In some embodiments, Y n =0. In some embodiments, Y n =S. In some embodiments, Y n =NH. In some embodiments, Y n =NHCH 3 .

[0857] In some embodiments of the compound of formula (III) or (III-a), A is -O-, -S-, -CH 2 -、-NH-、-N(CH3 )-or-N(C(=O)CH 3 )-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH 2 -. In some embodiments, A is -NH-. In some embodiments, A is -N(CH 3 In some embodiments, A is -N(C(=O)CH 3 )-.

[0858] In some embodiments of the compound of (III) or (III-a), A 1 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 1 In some embodiments, A 1 In some embodiments, A 1 -CH 2 -. In some embodiments, A 1 In some embodiments, A 1 -N(CH 3 )-. In some embodiments, A 1 -N(C(=O)CH 3 )-.

[0859] In some embodiments of the compound of (III) or (III-a), A 2 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 2 In some embodiments, A 2 In some embodiments, A 2 -CH 2 -. In some embodiments, A 2 In some embodiments, A 2 -N(CH 3 )-. In some embodiments, A 2 -N(C(=O)CH 3 )-.

[0860] In some embodiments of compounds 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.

[0861] In some embodiments of the compound of Formula (III) or (III-a), Z 1 , Z 2 , Z 3 , Z 4 and Z n At least one of them is -OCH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 3 and Z 1 OCH 3 In some embodiments of the compound of formula (I), Z 1 , Z 2 , Z 3 , Z 4 and Z n In some embodiments, at least one of Z is -OH. 1 , Z 2 and Z 4 In some embodiments, Z 2 and Z 4 It is -OH.

[0862] In some embodiments of the compound of Formula (III) or (III-a), Q 1 , Q 2 , Q 3 and Q 4 At least one of them is -OCH 3 In some embodiments of the compound of Formula (I), Q 1 , Q 2 , Q 3 and Q 4 In some embodiments, at least one of Q 1 and Q 4 OCH 3 In some embodiments, Q 2 and Q 3 It is -O-.

[0863] In some embodiments of the compound 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 is =O. 1 , Y 2 , Y 3 and Y 4 =0. In some embodiments, Y 1 , Y 2 , Y 3 , Y 4 and Y n is =S.

[0864] In some embodiments of the compound of Formula (III) or (III-a), X 1 , X 2 , X 3 , X 4 and X n At least one of -O - In some embodiments, X 1 , X 2 , X 3 and X 4 -O - .

[0865] In some embodiments of the compound of formula (III) or (III-a), in some embodiments of the compound of formula (I), A, A 1 and A 2 In some embodiments, at least one of A, A 1 and A 2 It is -O-.

[0866] In one aspect, described herein is an mRNA sequence having a 5' region motif of the motif (III'):

[0867]

[0868] in

[0869] B 1 for

[0870] B 2 is a modified or non-natural nucleobase;

[0871] B 3 and B n each independently a natural, modified or unnatural nucleobase;

[0872] Z 1 and Z 2are 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 ;

[0873] 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 ;

[0874] Q 1 and Q 4 Each is 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 )-;

[0875] Q 2 and Q 3are independently -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;

[0876] 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 ;

[0877] Y 1 , Y 2 , Y 3 , Y 4 and Y n are independently =O, =S, =NH or =NCH 3 ;

[0878] A. A 1 and A 2 are independently -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-;and

[0879] p=0, 1, 2, 3, 4, 5 or 6.

[0880] In some embodiments, the mRNA sequence having the 5' end region motif has the structure of the motif (III'-a):

[0881]

[0882] In some embodiments of the compounds of motif (III') or (III'-a), Z 1Hydrogen, 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 In some embodiments, Z 1 In some embodiments, Z 1 is F. In some embodiments, Z 1 In some embodiments, Z 1 In some embodiments, Z 1 -CH 3 In some embodiments, Z 1 -CH 2 CH 3 In some embodiments, Z 1 OCH 3 In some embodiments, Z 1 OCH 2 CH 3 In some embodiments, Z 1 For-SCH 3 In some embodiments, Z 1 For -NH 2 In some embodiments, Z 1 For NHCH 3 In some embodiments, Z 1 NHC(=O)CH 3 .

[0883] In some embodiments of the compounds of motif (III') or (III'-a), Z 2 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 In some embodiments, Z 2 In some embodiments, Z 2 is F. In some embodiments, Z 2In some embodiments, Z 2 In some embodiments, Z 2 -CH 3 In some embodiments, Z 2 -CH 2 CH 3 In some embodiments, Z 2 OCH 3 In some embodiments, Z 2 OCH 2 CH 3 In some embodiments, Z 2 For-SCH 3 In some embodiments, Z 2 For -NH 2 In some embodiments, Z 2 For NHCH 3 In some embodiments, Z 2 NHC(=O)CH 3 .

[0884] In some embodiments of the compounds of motif (III') or (III'-a), Z 3 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 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 In some embodiments, Z 3 -CH 3 In some embodiments, Z 3-CH 2 CH 3 In some embodiments, Z 3 OCH 2 OCH 3 In some embodiments, Z 3 OCH 2 CH 2 CH 3 In some embodiments, Z 3 -OCH(CH 3 ) 2 In some embodiments, Z 3 For-SCH 3 In some embodiments, Z 3 OCH 2 CH 2 OCH 3 .

[0885] In some embodiments of the compounds of motif (III') or (III'-a), Z 4 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 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 In some embodiments, Z 4 -CH 3 In some embodiments, Z 4 -CH 2 CH 3 In some embodiments, Z 4 OCH2 OCH 3 In some embodiments, Z 4 OCH 2 CH 2 CH 3 In some embodiments, Z 4 -OCH(CH 3 ) 2 In some embodiments, Z 4 For-SCH 3 In some embodiments, Z 4 OCH 2 CH 2 OCH 3 .

[0886] In some embodiments of the compounds of motif (III') or (III'-a), Z n 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 In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n In some embodiments, Z n -CH 3 In some embodiments, Z n -CH 2 CH 3 In some embodiments, Z n OCH 2 OCH 3 In some embodiments, Z n OCH2 CH 2 CH 3 In some embodiments, Z n -OCH(CH 3 ) 2 In some embodiments, Z n For-SCH 3 In some embodiments, Z n OCH 2 CH 2 OCH 3 .

[0887] In some embodiments of the compounds of motif (III') or (III'-a), B 2 is a modified or non-natural nucleobase. 2 is a modified guanine. In some embodiments, B 2 is a modified adenine. In some embodiments, B 2 is a modified cytosine. 2 is a modified uracil. In some embodiments, B 2 is a modified thymine, in some embodiments, B 2 is a modified hypoxanthine. 2 is a modified purine. 2 is 2-aminoadenine. In some embodiments, B 2 for

[0888] In some embodiments of the compounds of motif (III') or (III'-a), B 3 are independently natural, modified or non-natural nucleobases. In some embodiments, B 3 In some embodiments, B 3 In some embodiments, B 3 In some embodiments, B 3 is uracil, in some embodiments, B 3 is thymine, in some embodiments, B 3 In some embodiments, B 3 For purine.

[0889] In some embodiments of the compounds of motif (III') or (III'-a), B n are independently natural, modified or non-natural nucleobases. In some embodiments, B nIn some embodiments, B n In some embodiments, B n In some embodiments, B n is uracil, in some embodiments, B n is thymine, in some embodiments, B n In some embodiments, B n For purine.

[0890] In some embodiments of the compounds of motif (III') or (III'-a), B 2 , B 3 and B n In some embodiments, at least one of B is adenine. 2 , B 3 and B n In some embodiments, at least one of B is guanine. 2 is a modified adenine. In some embodiments, B 3 Adenine.

[0891] In some embodiments of the compounds of motif (III') or (III'-a), Q 1 -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 )-. In some embodiments, Q 1 -CH 2 -. In some embodiments, Q 1 is -CH=CH-. In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 In some embodiments, Q 1 -CH 2 CH 2 -. In some embodiments, Q 1 -CH 2 CF 2-. In some embodiments, Q 1 -CH 2 NH 2 -. In some embodiments, Q 1 -CH 2 NH(CH 3 )-. In some embodiments, Q 1 -CH 2 N(C(=O)CH 3 )-.

[0892] In some embodiments of the compounds of motif (III') or (III'-a), Q 4 -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 )-. In some embodiments, Q 4 -CH 2 -. In some embodiments, Q 4 is -CH=CH-. In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 In some embodiments, Q 4 -CH 2 CH 2 -. In some embodiments, Q 4 -CH 2 CF 2 -. In some embodiments, Q 4 -CH 2 NH 2 -. In some embodiments, Q 4 -CH 2 NH(CH 3 )-. In some embodiments, Q 4 -CH 2 N(C(=O)CH 3 )-.

[0893] In some embodiments of the compounds of motif (III') or (III'-a), Q 2 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 2 In some embodiments, Q 2 In some embodiments, Q 2 -CH 2 -. In some embodiments, Q 2 For-CF 2 -. In some embodiments, Q 2 In some embodiments, Q 2 -N(CH 3 )-. In some embodiments, Q 2 -N(C(=O)CH 3 ).

[0894] In some embodiments of the compounds of motif (III') or (III'-a), Q 3 -O-, -S-, -CH 2 -、-CF 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, Q 3 In some embodiments, Q 3 In some embodiments, Q 3 -CH 2 -. In some embodiments, Q 3 For-CF 2 -. In some embodiments, Q 3 In some embodiments, Q 3 -N(CH 3 )-. In some embodiments, Q 3 -N(C(=O)CH 3 ).

[0895] In some embodiments of the compounds of motif (III') or (III'-a), X 1 -OH, -SH, -O - , -S - 、-NH 2 、-NHCH 3 、-NH(C(=O)CH3 ),-CH 3 、-CH 2 CH 3 、-CH 2 CH 2 CH 3 、-CH(CH 3 ) 2 、-OCH 3 or -OCH 2 CH 3 In some embodiments, X 1 In some embodiments, X 1 In some embodiments, X 1 -O - In some embodiments, X 1 For -S - In some embodiments, X 1 For -NH 2 In some embodiments, X 1 -NHCH 3 In some embodiments, X 1 -NH(C(=O)CH 3 ). In some embodiments, X 1 -CH 3 In some embodiments, X 1 -CH 2 CH 3 In some embodiments, X 1 -CH 2 CH 2 CH 3 In some embodiments, X 1 -CH(CH 3 ) 2 In some embodiments, X 1 OCH 3 In some embodiments, X 1 OCH 2 CH 3 .

[0896] In some embodiments of the compounds of motif (III') or (III'-a), X 2 -OH, -SH, -O - , -S - 、-NH 2 、-NHCH 3 、-NH(C(=O)CH 3 ),-CH 3 、-CH 2 CH3 、-CH 2 CH 2 CH 3 、-CH(CH 3 ) 2 、-OCH 3 or -OCH 2 CH 3 In some embodiments, X 2 In some embodiments, X 2 In some embodiments, X 2 -O - In some embodiments, X 2 For -S - In some embodiments, X 2 For -NH 2 In some embodiments, X 2 -NHCH 3 In some embodiments, X 2 -NH(C(=O)CH 3 ). In some embodiments, X 2 -CH 3 In some embodiments, X 2 -CH 2 CH 3 In some embodiments, X 2 -CH 2 CH 2 CH 3 In some embodiments, X 2 -CH(CH 3 ) 2 In some embodiments, X 2 OCH 3 In some embodiments, X 2 OCH 2 CH 3 .

[0897] In some embodiments of the compounds of motif (III') or (III'-a), X 3 -OH, -SH, -O - , -S - 、-NH 2 、-NHCH 3 、-NH(C(=O)CH 3 ),-CH 3 、-CH 2 CH 3 、-CH 2 CH 2 CH3 、-CH(CH 3 ) 2 、-OCH 3 or -OCH 2 CH 3 In some embodiments, X 3 In some embodiments, X 3 In some embodiments, X 3 -O - In some embodiments, X 3 For -S - In some embodiments, X 3 For -NH 2 In some embodiments, X 3 -NHCH 3 In some embodiments, X 3 -NH(C(=O)CH 3 ). In some embodiments, X 3 -CH 3 In some embodiments, X 3 -CH 2 CH 3 In some embodiments, X 3 -CH 2 CH 2 CH 3 In some embodiments, X 3 -CH(CH 3 ) 2 In some embodiments, X 3 OCH 3 In some embodiments, X 3 OCH 2 CH 3 .

[0898] In some embodiments of the compounds of motif (III') or (III'-a), X 4 -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 、-OCH3 or -OCH 2 CH 3 In some embodiments, X 4 In some embodiments, X 4 In some embodiments, X 4 -O - In some embodiments, X 4 For -S - In some embodiments, X 4 For -NH 2 In some embodiments, X 4 -NHCH 3 In some embodiments, X 4 -NH(C(=O)CH 3 ). In some embodiments, X 4 -CH 3 In some embodiments, X 4 -CH 2 CH 3 In some embodiments, X 4 -CH 2 CH 2 CH 3 In some embodiments, X 4 -CH(CH 3 ) 2 In some embodiments, X 4 OCH 3 In some embodiments, X 4 OCH 2 CH 3 .

[0899] In some embodiments of the compounds of motif (III') or (III'-a), X n -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 3In some embodiments, X n In some embodiments, X n In some embodiments, X n -O - In some embodiments, X n For -S - In some embodiments, X n For -NH 2 In some embodiments, X n -NHCH 3 In some embodiments, X n -NH(C(=O)CH 3 ). In some embodiments, X n -CH 3 In some embodiments, X n -CH 2 CH 3 In some embodiments, X n -CH 2 CH 2 CH 3 In some embodiments, X n -CH(CH 3 ) 2 In some embodiments, X n OCH 3 In some embodiments, X n OCH 2 CH 3 .

[0900] In some embodiments of the compounds of motif (III') or (III'-a), Y 1 =O, =S, =NH or =NCH 3 In some embodiments, Y 1 =0. In some embodiments, Y 1 =S. In some embodiments, Y 1 =NH. In some embodiments, Y 1 =NHCH 3 .

[0901] In some embodiments of the compounds of motif (III') or (III'-a), Y 2 =O, =S, =NH or =NCH 3 In some embodiments, Y 2 =0. In some embodiments, Y 2 =S. In some embodiments, Y 2=NH. In some embodiments, Y 2 =NHCH 3 .

[0902] In some embodiments of the compounds of motif (III') or (III'-a), Y 3 =O, =S, =NH or =NCH 3 In some embodiments, Y 3 =0. In some embodiments, Y 3 =S. In some embodiments, Y 3 =NH. In some embodiments, Y 3 =NHCH 3 .

[0903] In some embodiments of the compounds of motif (III') or (III'-a), Y 4 =O, =S, =NH or =NCH 3 In some embodiments, Y 4 =0. In some embodiments, Y 4 =S. In some embodiments, Y 4 =NH. In some embodiments, Y 4 =NHCH 3 .

[0904] In some embodiments of the compounds of motif (III') or (III'-a), Y n =O, =S, =NH or =NCH 3 In some embodiments, Y n =0. In some embodiments, Y n =S. In some embodiments, Y n =NH. In some embodiments, Y n =NHCH 3 .

[0905] In some embodiments of the compounds of motif (III') or (III'-a), A is -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A is -O-. In some embodiments, A is -S-. In some embodiments, A is -CH 2 -. In some embodiments, A is -NH-. In some embodiments, A is -N(CH 3 In some embodiments, A is -N(C(=O)CH 3)-.

[0906] In some embodiments of the compounds of motif (III') or (III'-a), A 1 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 1 In some embodiments, A 1 In some embodiments, A 1 -CH 2 -. In some embodiments, A 1 In some embodiments, A 1 -N(CH 3 )-. In some embodiments, A 1 -N(C(=O)CH 3 )-.

[0907] In some embodiments of the compounds of motif (III') or (III'-a), A 2 -O-, -S-, -CH 2 -、-NH-、-N(CH 3 )-or-N(C(=O)CH 3 )-. In some embodiments, A 2 In some embodiments, A 2 In some embodiments, A 2 -CH 2 -. In some embodiments, A 2 In some embodiments, A 2 -N(CH 3 )-. In some embodiments, A 2 -N(C(=O)CH 3 )-.

[0908] 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.

[0909] In some embodiments of compounds of motif (III) or (III'-a), Z 1 , Z 2 , Z3 , Z 4 and Z n At least one of them is -OCH 3 In some embodiments, Z 3 OCH 3 In some embodiments, Z 3 and Z 1 OCH 3 In some embodiments of the compounds of motif (I'), Z 1 , Z 2 , Z 3 , Z 4 and Z n In some embodiments, at least one of Z is -OH. 1 , Z 2 and Z 4 In some embodiments, Z 2 and Z 4 It is -OH.

[0910] In some embodiments of the compounds of motif (III') or (III'-a), Q 1 , Q 2 , Q 3 and Q 4 At least one of them is -OCH 3 In some embodiments of the compound of Formula (I), Q 1 , Q 2 , Q 3 and Q 4 In some embodiments, at least one of Q 1 and Q 4 OCH 3 In some embodiments, Q 2 and Q 3 It is -O-.

[0911] In some embodiments of the 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 is =O. 1 , Y 2 , Y 3 and Y 4 =0. In some embodiments, Y 1 , Y 2 , Y 3 , Y 4 and Y n=S. In some embodiments, Y 2 =S. In some embodiments, Y 4 is =S.

[0912] In some embodiments of compounds of motif (III') or (III'-a), X 1 , X 2 , X 3 , X 4 and X n At least one of -O - In some embodiments, X 1 , X 2 , X 3 and X 4 -O - In some embodiments, X 1 For -S - In some embodiments, X 2 For -S - In some embodiments, X 3 For -S - In some embodiments, X 4 For -S - .

[0913] In some embodiments of the compounds of motif (III') or (III'-a), 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 It is -O-.

[0914] In certain embodiments, the sequence initiator compound is as described in Table 1.

[0915] Table 1.

[0916]

[0917]

[0918]

[0919]

[0920]

[0921]

[0922]

[0923]

[0924]

[0925]

[0926]

[0927] In some embodiments, disclosed herein is a pharmaceutically acceptable salt or pharmaceutically acceptable solvate or pharmaceutical composition comprising one or more mRNAs produced / manufactured by one or more sequence initiator compounds selected from Table 1, wherein the mRNA encodes one or more pharmaceutically active proteins.

[0928] In certain embodiments, the sequence initiator compound is as described in Table 2.

[0929] Table 2

[0930]

[0931]

[0932]

[0933]

[0934]

[0935]

[0936] In some embodiments, disclosed herein is a pharmaceutically acceptable salt or pharmaceutically acceptable solvate or pharmaceutical composition comprising one or more mRNAs produced / manufactured by one or more sequence initiator compounds selected from Table 2, wherein the mRNA encodes one or more pharmaceutically active proteins.

[0937] In certain embodiments, the sequence initiator compound is as described in Table 3.

[0938] Table 3

[0939]

[0940]

[0941]

[0942]

[0943]

[0944]

[0945]

[0946]

[0947]

[0948]

[0949]

[0950]

[0951]

[0952]

[0953]

[0954]

[0955]

[0956]

[0957]

[0958]

[0959]

[0960]

[0961]

[0962]

[0963]

[0964]

[0965]

[0966]

[0967]

[0968]

[0969]

[0970]

[0971]

[0972]

[0973]

[0974]

[0975]

[0976]

[0977]

[0978]

[0979]

[0980]

[0981]

[0982]

[0983]

[0984]

[0985]

[0986]

[0987]

[0988]

[0989]

[0990]

[0991]

[0992]

[0993]

[0994]

[0995]

[0996]

[0997]

[0998]

[0999]

[1000]

[1001]

[1002]

[1003]

[1004]

[1005]

[1006]

[1007]

[1008]

[1009]

[1010]

[1011]

[1012]

[1013]

[1014]

[1015]

[1016]

[1017]

[1018]

[1019]

[1020]

[1021]

[1022]

[1023]

[1024]

[1025]

[1026]

[1027]

[1028]

[1029]

[1030]

[1031]

[1032]

[1033]

[1034]

[1035]

[1036]

[1037]

[1038]

[1039]

[1040]

[1041]

[1042]

[1043]

[1044]

[1045]

[1046]

[1047]

[1048]

[1049]

[1050]

[1051]

[1052]

[1053]

[1054]

[1055]

[1056]

[1057] In some embodiments, disclosed herein is a pharmaceutically acceptable salt or pharmaceutically acceptable solvate or pharmaceutical composition comprising one or more mRNAs produced / manufactured by one or more sequence initiator compounds selected from Table 3.

[1058] 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, wherein (a) the DNA template comprises a promoter region, the promoter region comprising 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) wherein the IVT mRNA sequence initiator hybridizes to the DNA template at least at nucleotide positions +1, +2, and +3.

[1059] 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, wherein (a) the DNA template comprises a promoter region, the promoter region comprising a transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and (b) wherein the IVT mRNA sequence initiator hybridizes to the DNA template at least at nucleotide positions +1 and +2.

[1060] Pharmaceutical composition

[1061] In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. Pharmaceutical compositions are prepared in a conventional manner using one or more pharmaceutically acceptable inactive ingredients, which assist in processing the active compound into a pharmaceutically usable preparation. Suitable formulations depend on the selected route of administration. An overview of the pharmaceutical compositions described herein can be found in, for example, Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, Pennsylvania: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, New York, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition (Lippincott Williams & Wilkins 1999), such disclosures are incorporated herein by reference.

[1062] The pharmaceutical composition can be a mixture of the sequence initiator compound described herein and one or more other chemical components (i.e., pharmaceutically acceptable ingredients), such as carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, wetting agents, defoamers, antioxidants, preservatives, or one or more combinations thereof. The pharmaceutical composition facilitates administration of the compound to an organism.

[1063] The compositions described herein can be applied to the subject in a variety of ways, including parenteral, intravenous, intradermal, intramuscular, colonic, rectal or intraperitoneal administration. In some embodiments, the sequence initiator compound described herein or a pharmaceutically acceptable salt thereof is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection or intravenous injection of the subject. In some embodiments, the pharmaceutical composition can be administered parenterally, intravenously, intramuscularly or orally. The oral medicament comprising the sequence initiator compound described herein can be any suitable form for oral administration, such as liquid, tablet, capsule, etc. Oral formulations can also be coated or treated to prevent or reduce dissolution in the stomach. Any suitable method known in the art can be used to administer the composition of the present disclosure to the subject. Suitable preparations and delivery methods for the present disclosure are generally well known in the art. For example, the sequence initiator compound described herein can be formulated into a pharmaceutical composition together with a pharmaceutically acceptable diluent, carrier or excipient. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, including pH adjusters and buffers, tonicity adjusters, wetting agents, etc., such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[1064] The pharmaceutical formulations described herein can be administered to a subject in a variety of ways by a variety of 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. The 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 melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release formulations and controlled release formulations.

[1065] In some embodiments, the pharmaceutical preparation is in the form of a tablet. In other embodiments, the pharmaceutical preparation containing the sequence initiator compound described herein is in the form of a capsule. In one aspect, the liquid dosage form for oral administration is in the form of an aqueous suspension or solution selected from, but not limited to, an aqueous oral dispersion, an emulsion, a solution, an elixir, a gel, and a syrup.

[1066] For administration by inhalation, the sequence initiator compounds described herein can be formulated for use as aerosols, mists or powders. For buccal or sublingual administration, the composition can be in the form of tablets, lozenges or gels prepared in a conventional manner. In some embodiments, the sequence initiator compounds described herein can be prepared into 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 topically applied, and can be formulated into a variety of topically applied 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.

[1067] In one aspect, described herein is a pharmaceutical composition comprising an RNA molecule and one or more pharmaceutically acceptable excipients, wherein the RNA molecule comprises an IVT mRNA sequence initiator disclosed herein. In some embodiments, the pharmaceutical composition comprises a lipid nanoparticle. In some embodiments, the RNA is encapsulated in the lipid nanoparticle.

[1068] Transcription

[1069] In eukaryotes, transcription of messenger RNA (mRNA) is accomplished by RNA polymerase II. This is a complex multi-subunit enzyme with complex regulation. In order to perform large-scale transcription in vitro, studies generally use single-subunit phage polymerases from T7, T3, SP6, K1-5, K1E, K1F or K11 phages. This polymerase family has a simple, minimal promoter sequence of about 17 nucleotides, which does not require auxiliary proteins and has minimal restrictions on the starting nucleotide sequence. Although the present application focuses on T7 RNA polymerase (T7 RNAP), it will be appreciated by those skilled in the art that the present disclosure can be implemented with other RNA polymerases.

[1070] T7 RNAP exists in at least two protein states. The first is called "abortive complex" and is related to transcription initiation. The second is a very progressive conformation, called "elongation complex". In vitro transcription can be divided into six steps: 1) binding of RNA polymerase to promoter sequence, 2) initiation of transcription, 3) non-progressive extension known as abortive transcription, during which polymerase often releases DNA template and short abortive transcripts, 4) conversion of open complexes to closed complexes, 5) progressive extension and 6) transcription termination. A large amount of RNA produced during transcription consists of short abortive fragments of about 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 synthesizing about 10-14 bases, RNA polymerase escapes from the futile cycle, loses sequence-specific contacts with the promoter DNA, and forms 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)).

[1071] The consensus sequence of the most active class III T7 promoter encompasses a 17bp sequence upstream of the transcription start site and a 6bp sequence downstream (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 second transcribed nucleotide is referred to as the +2 transcript nucleotide, and so on (Table 2). During transcription, the two chains melt to form a transcription bubble, and the bottom strand of the duplex (shown in Table 4 3' to 5') is the template for transcription. For transcript nucleotides +3 and above, the template strand primarily defines the properties of the transcribed nucleotides 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.

[1072]

[1073] Unlike DNA polymerase, T7 RNAP initiates RNA synthesis without primers. The first step of initiation is called de novo RNA synthesis, in which RNA polymerase recognizes a specific sequence on a DNA template, selects a first pair of nucleotide triphosphates complementary to the template residues at positions +1 and +2, and catalyzes the formation of phosphodiester bonds to form dinucleotides. The affinity of the starting nucleotide to the polymerase is lower than those used during extension. For the first initiation of NTP, the Kd value is 2mM, and for the second initiation of NTP, the Kd value is 80 μM, while for the NTP during extension, the Kd is about 5 μM (J.Mol.Biol. (2007) 370, 256-268). It has been found that de novo synthesis is the rate-limiting step during transcription. T7 RNAP shows a strong bias towards GTP as the initiating nucleotide (J.Biol.Chem.248:2235-2244 (1973)). Of the 17 T7 promoters in the genome, 15 are initiated by GTP (and 13 by ppGpG), with no apparent preference for NTPs during transcription elongation (J. Mol. Biol. 370: 256-268 (2007)). T7 RNA polymerase initiates poorly at the promoter encoding an A at position +1; instead, transcription is primarily initiated by the G encoded at position +2 (J. Biol. Chem. 278: 2819-2823 (2003)).

[1074] During de novo RNA synthesis, incorporation of the initiating nucleotide is primarily achieved through free energy generated by base stacking, specific interactions between polymerase residues, the guanine moiety of the initiating nucleotide, and base complementarity interactions (J. Mol. Biol. 370:256-268 (2007)).

[1075] It is known that T7 RNAP can also be initiated by short oligonucleotide primers. For example, 13 promoters in the T7 genome are known to be initiated by pppGpG (J.Mol.Biol.370:256-268 (2007)). Several groups of studies have shown that T7 RNAP can be initiated from dinucleotide primers (Biochemistry 24:5716-5723 (1985)). Axelrod et al. showed that uncapped GpA dinucleotides can be initiated from +1 and +2 template nucleotides (2'-deoxycytidine and 2'-deoxythymidine, respectively) ("CT" templates). The reaction conditions are 200 micromolar (μM) dimers and 100 μM ATP, CTP, GTP and UTP. Their reaction mixture also contains 100 μM 3'dATP, 3'dCTP, 3'dUTP or 50 μM 3'dGTP. They only observed RNA initiated by GpA, and did not observe a mixture of RNA initiated by GpA and 5'-triphosphate RNA initiated from GTP. This may be due to the reaction conditions used. 100 μM of GTP is far below the 2mM Kd of T7 polymerase for the first starting guanosine (J. Mol. Biol. (2007) 370, 256-268). Since GTP competes with the starting oligonucleotide for initiation, the use of low GTP concentration is conducive to GpA initiation, but leads to low transcription yields (estimated maximum calculated yield is <150ug / mL reaction). When using ApG, CpG, UpG or GpG to initiate transcription on the "CT" template, they observed the formation of RNA transcripts containing additional untemplated 5' nucleotides (A, C, U or G, respectively).

[1076] The methods and compositions for preparing 5'-capped RNA provided herein include but are not limited to mRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small cajal body specific RNA (scaRNA). These methods involve the use of mRNA sequence initiators, such as oligonucleotide primers containing caps, and nucleoside 5'-triphosphates (NTPs) and RNA polymerases for DNA-templated and promoter-controlled RNA synthesis. In certain aspects, the method uses an initial capping oligonucleotide primer that provides utility in RNA synthesis, particularly the synthesis of capped mRNA. Exemplary initial oligonucleotide primers have structures similar to cap 0, cap 1, cap 2 or TMG-caps of natural RNA molecules, which contain 2'-O-methylated nucleoside units at the penultimate cap 1 position of RNA and near the penultimate cap 2 5' position of RNA. The natural cap 0 structure does not have a 2'-O-methylated nucleoside unit.

[1077] Methods and compositions for preparing RNA include, but are not limited to, mRNA, snRNA, snoRNA, scaRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), and transfer messenger RNA (tmRNA), which carry modifications at or near the 5' end of the molecule. These methods involve the use of initiators, such as initiator oligonucleotide primers with or without caps, nucleoside 5'-triphosphates (NTPs), and RNA polymerases for DNA-templated and promoter-controlled RNA synthesis. In certain aspects, the methods use modified initiator oligonucleotide primers carrying structural modifications that provide utility in RNA synthesis; particularly, in 5'-modified RNA synthesis.

[1078] Exemplary starting capping oligonucleotide primers have an open 3'-OH group, which allows RNA polymerase-mediated RNA synthesis on a DNA template to be initiated by adding nucleotide units to the 3' end of the primer. The starting capping oligonucleotide primer is substantially complementary to the template DNA sequence at the transcription start site (i.e., the start site is located near the 3' end of the promoter sequence and can overlap with the promoter sequence). In certain embodiments, the starting capping oligonucleotide primer mainly guides the synthesis of RNA in one direction ("forward") from the 3' end of the primer. In certain aspects and embodiments, the starting capping oligonucleotide primer is superior to any nucleoside 5'-triphosphate for initiating RNA synthesis, thereby maximizing the generation of RNA starting with the starting capping oligonucleotide primer, and minimizing the generation of RNA starting with 5'-triphosphate nucleosides (usually GTP).

[1079] Making mRNA by in vitro transcription utilizes highly active bacteriophage RNA polymerases (T3, T7, SP6, and others). The RNA polymerase works under the control of a specific promoter that is incorporated into the DNA plasmid construct in front of the template nucleotide sequence. The transcription process usually begins with a purine nucleoside 5'-triphosphate (usually GTP) and continues until the RNA polymerase encounters a termination sequence or completes the DNA template.

[1080] As described above, the mCAP dinucleotide analog containing cap 0 7mG(5′)ppp(5′)N has been used to initiate in vitro transcription (e.g., RNA 1:957-967 (1995)). Capped RNA molecules produced using these dinucleotide analogs contain cap 0. However, only about 50% of the synthesized capped RNA molecules have the correct "forward" orientation of cap 0. In order to convert RNA with cap 0 to RNA with cap 1, an additional enzymatic reaction must be performed using a (nucleoside-2'-O) methyltransferase. However, this conversion may not be quantitative; it is not easy to control and it is difficult to separate the remaining cap 0 RNA from the...

Claims

1. An in vitro transcribed (IVT) mRNA sequence initiator comprising a compound of formula (I) or a salt or solvate thereof: in B 1 for B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase; Z 1 and Z 2 Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3 or NHC(=O)CH3; Z 3 , Z 4 and Z n Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3; Q 1 and Q 4 Each is independently -CH2-, -CH=CH-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-; Q 2 and Q 3 Each is independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; 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; Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is independently =O, =S, =NH or =NCH3; A. A 1 and A 2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and p = 0, 1, 2, 3, 4, 5 or 6, The condition is that the compound of formula (I) satisfies one or more of the following restrictive conditions (i) to (iii): (i)X 1 , X 2 , X 3 , X 4 and X n At least one of -SH or -S - (ii) Y 1 , Y 2 , Y 3 , Y 4 and Y n At least one of is =S; and (iii) A, A 1 and A 2 At least one of them is -S-.

2. The IVT mRNA sequence initiator according to claim 1, wherein X 1 , X 2 , X 3 , X 4 and X n At least one of -SH or -S - ; Y 1 , Y 2 , Y 3 , Y 4 and Y n At least one of =S; or A, A 1 and A 2 At least one of them is -S-.

3. An in vitro transcribed (IVT) mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof: in B 1 for B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase; Z 1 and Z' are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3) or -SCH3; Z 2 and Z" are each independently fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3 or NHC(=O)CH3; Z'' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3 or -OCH2CH3; Z 3 , Z 4 and Z n Each is independently hydrogen, fluorine, -OH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3; Q 1 and Q 4 Each is independently -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-; Q 2 and Q 3 Each is independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; 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; Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is independently =O, =S, =NH or =NCH3; A. A 1 and A 2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and p=0, 1, 2, 3, 4, 5 or 6.

4. An in vitro transcribed (IVT) mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof: in B 1 for B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase; Z' and Z" are each 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; Z 1 and Z 2 Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3 or NHC(=O)CH3; Z 3 , Z 4 and Z n Each is independently hydrogen, fluorine, -OH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3; Q 1 and Q 4 Each is independently -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-; Q 2 and Q 3 Each is independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; 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; Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is independently =O, =S, =NH or =NCH3; A. A 1 and A 2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and p=0, 1, 2, 3, 4, 5 or 6.

5. An in vitro transcribed (IVT) mRNA sequence initiator comprising a compound of formula (II) or a salt or solvate thereof: in B 1 for B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase; Z' and Z" are each 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; Z 1 and Z 2 Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3 or NHC(=O)CH3; Z 3 , Z 4 and Z n Each is independently hydrogen, fluorine, -OH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3; Q 1 and Q 4 Each is independently -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-; Q 2 and Q 3 Each is independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; 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; Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is independently =O, =S, =NH or =NCH3; A. A 1 and A 2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and p=0, 1, 2, 3, 4, 5 or 6.

6. An in vitro transcribed (IVT) mRNA sequence initiator comprising a compound of formula (III) or a salt or solvate thereof: in B 1 for B 2 is a modified or non-natural nucleobase; B 3 and B n each independently a natural, modified or unnatural nucleobase; Z 1 and Z 2 Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3 or NHC(=O)CH3; Z 3 , Z 4 and Z n Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3; Q 1 and Q 4 Each is independently -CH2-, -CH=CH-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-; Q 2 and Q 3 Each is independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; 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; Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is independently =O, =S, =NH or =NCH3; A. A 1 and A 2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and p=0, 1, 2, 3, 4, 5 or 6.

7. The IVT mRNA sequence initiator according to claim 1, wherein the initiator has a structure of formula (Ia):

8. The IVT mRNA sequence initiator according to claim 1, wherein the initiator has the structure of formula (Ib):

9. The IVT mRNA sequence initiator according to claim 1, wherein the initiator has a structure of formula (Ic):

10. The IVT mRNA sequence initiator according to claim 1, wherein the initiator has the structure of formula (Id):

11. The IVT mRNA sequence initiator according to claim 6, wherein the initiator has a structure of formula (III-a):

12. The IVT mRNA sequence initiator according to any one of claims 1 to 11, wherein Z 3 It is hydrogen, fluorine, -OH, -OCH3, - or -OCH2CH3.

13. The IVT mRNA sequence initiator according to claim 12, wherein Z 3 It is -OCH3.

14. The IVT mRNA sequence initiator according to claim 12, wherein Z 4 and Z n Each is independently -OH or -OCH3.

15. The IVT mRNA sequence initiator according to any one of claims 1 to 2, wherein Z 3 , Z 4 and Z n Each is independently -OH or -OCH3.

16. The IVT mRNA sequence initiator according to any one of claims 1 to 15, wherein Y 2 , Y 4 and Y n Each is independently =O or =S.

17. The IVT mRNA sequence initiator according to any one of claims 1 to 16, wherein X 2 and X 3 Each independently is -O - or -S - .

18. The IVT mRNA sequence initiator according to claim 6 or 11, wherein B2 is 19. The IVT mRNA sequence initiator according to any one of claims 6 or 11, wherein the compound is 20. The IVT mRNA sequence initiator according to claim 5, wherein the compound is 21. The IVT mRNA sequence initiator according to any one of claims 1, 2 or 10, wherein the compound is 22. The IVT mRNA sequence initiator according to any one of claims 1 to 18, wherein the compound comprises a phosphorothioate.

23. The IVT mRNA sequence initiator of claim 22, wherein the phosphorothioate comprises a chiral phosphorus center.

24. The IVT mRNA sequence initiator according to claims 1 to 2, 10, 21, wherein the compound is 25. The IVT mRNA sequence initiator according to claim 1 to 2, 10 or 21, wherein the compound is 26. A composition comprising the IVT mRNA sequence initiator according to claim 22 or 23, wherein the composition comprises a racemic mixture of the R diastereomer and the S diastereomer of the compound.

27. A composition comprising the IVT mRNA sequence initiator according to claim 24 or 25, wherein the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 60%.

28. A composition comprising the IVT mRNA sequence initiator according to claim 24 or 25, wherein the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 80%.

29. A composition comprising the IVT mRNA sequence initiator according to claim 24 or 25, wherein the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 90%.

30. A composition comprising the IVT mRNA sequence initiator according to claim 24 or 25, wherein the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 95%.

31. A composition comprising the IVT mRNA sequence initiator according to claim 24 or 25, wherein the stereochemical purity of the IVT mRNA sequence initiator in the composition is at least 98%.

32. An mRNA sequence having a 5' terminal region motif (I'): in B 1 for B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase; Z 1 and Z 2 Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3 or NHC(=O)CH3; Z 3 , Z 4 and Z n Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3; Q 1 and Q 4 Each is independently -CH2-, -CH=CH-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-; Q 2 and Q 3 Each is independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; 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; Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is independently =O, =S, =NH or =NCH3; A. A 1 and A 2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and p = 0, 1, 2, 3, 4, 5 or 6, The condition is that the compound of formula (I) satisfies one or more of the following restrictive conditions (i) to (iii): (i)X 1 , X 2 , X 3 , X 4 and X n At least one of -SH or -S - (ii) Y 1 , Y 2 , Y 3 , Y 4 and Y n At least one of is =S; and (iii) A, A 1 and A 2 At least one of them is -S-.

33. according to claim 16, there is the mRNA sequence of 5 ' end region motif, wherein X 1 , X 2 , X 3 , X 4 and X n At least one of -SH or -S - ; Y 1 , Y 2 , Y 3 , Y 4 and Y n At least one of =S; or A, A 1 and A 2 At least one of them is -S-.

34. An mRNA sequence having a 5' terminal region motif (II'): in B 1 for B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase; Z 1 and Z' are each independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH(CH3), -NH2, -NH(C(=O)CH3) or -SCH3; Z 2 and Z" are each independently fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3 or NHC(=O)CH3; Z'' is hydrogen, fluorine, -CH3, -CH2CH3, -OCH3 or -OCH2CH3; Z 3 , Z 4 and Z n Each is independently hydrogen, fluorine, -OH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3; Q 1 and Q 4 Each is independently -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-; Q 2 and Q 3 Each is independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; 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; Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is independently =O, =S, =NH or =NCH3; A. A 1 and A 2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and p=0, 1, 2, 3, 4, 5 or 6.

35. An mRNA sequence having a 5' terminal region motif (II'): in B 1 for B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase; Z' and Z" are each 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; Z 1 and Z 2 Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3 or NHC(=O)CH3; Z 3 , Z 4 and Z n Each is independently hydrogen, fluorine, -OH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3; Q 1 and Q 4 Each is independently -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-; Q 2 and Q 3 Each is independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; 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; Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is independently =O, =S, =NH or =NCH3; A. A 1 and A 2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and p=0, 1, 2, 3, 4, 5 or 6.

36. An mRNA sequence having a 5' terminal region motif (II'): in B 1 for B 2 , B 3 and B n each independently a natural, modified or unnatural nucleobase; Z' and Z" are each 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; Z 1 and Z 2 Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -SCH3, -OCH2CH3, -NH2, NHCH3 or NHC(=O)CH3; Z 3 , Z 4 and Z n Each is independently hydrogen, fluorine, -OH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3; Q 1 and Q 4 Each is independently -CH=CH-, -CH2-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-; Q 2 and Q 3 Each is independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; 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; Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is independently =O, =S, =NH or =NCH3; A. A 1 and A 2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and p=0, 1, 2, 3, 4, 5 or 6.

37. An mRNA sequence having a 5' terminal region motif (III'): in B 1 for B 2 is a modified or non-natural nucleobase; B 3 and B n each independently a natural, modified or unnatural nucleobase; Z 1 and Z 2 Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -OCH2CH3, -SCH3, -NH2, NHCH3 or NHC(=O)CH3; Z 3 , Z 4 and Z n Each is independently hydrogen, fluorine, -OH, -SH, -CH3, -CH2CH3, -OCH3, -NH2, -NHCH3, -NH(C(=O)CH3), -OCH2CH3, -OCH2OCH3, -OCH2CH2CH3, -OCH(CH3)2, -SCH3 or -OCH2CH2OCH3; Q 1 and Q 4 Each is independently -CH2-, -CH=CH-, -CH2O-, -CH2S-, -CH2CH2-, -CH2CF2-, -CH2NH2-, -CH2NH(CH3)- or -CH2N(C(=O)CH3)-; Q 2 and Q 3 Each is independently -O-, -S-, -CH2-, -CF2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; 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; Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is independently =O, =S, =NH or =NCH3; A. A 1 and A 2 are each independently -O-, -S-, -CH2-, -NH-, -N(CH3)- or -N(C(=O)CH3)-; and p=0, 1, 2, 3, 4, 5 or 6.

38. according to claim 32, there is the mRNA sequence of 5' terminal region motif (I'-a), wherein said motif has the structure of motif (I'-a):

39. according to claim 32, there is the mRNA sequence of 5' terminal region motif (I'-b), wherein said motif has the structure of motif (I'-b):

40. according to claim 32, there is the mRNA sequence of 5' terminal region motif (I'-c), wherein said motif has the structure of motif (I'-c):

41. according to claim 32, there is the mRNA sequence of 5' end region motif (I'-d), wherein said motif has the structure of motif (I'-d):

42. according to claim 32, there is the mRNA sequence of 5' terminal region motif (I'-e), wherein said motif has the structure of motif (I'-e):

43. according to claim 32, there is the mRNA sequence of 5' end region motif (I'-f), wherein said motif has the structure of motif (I'-f):

44. according to claim 32, there is the mRNA sequence of 5' terminal region motif (I'-g), wherein said motif has the structure of motif (I'-g):

45. according to claim 32, there is the mRNA sequence of 5' terminal region motif (I'-h), wherein said motif has the structure of motif (I'-h):

46. ​​according to any one of claims 34 to 36, there is a 5' end region motif (II ') mRNA sequence, wherein the motif has the structure of motif (II '-a):

47. according to any one of claims 34 to 36, there is a 5' end region motif (II '-b) of the mRNA sequence, wherein the motif has the structure of motif (II '-b):

48. according to any one of claims 34 to 36, there is a 5' end region motif (II '-c) of the mRNA sequence, wherein the motif has the structure of motif (II '-c):

49. according to any one of claims 34 to 36, there is a 5' end region motif (II '-d) mRNA sequence, wherein the motif has the structure of motif (II '-d):

50. according to any one of claims 34 to 36, there is a 5' end region motif (II '-e) of the mRNA sequence, wherein the motif has the structure of motif (II '-e):

51. according to any one of claims 34 to 36, there is a 5' end region motif (II '-f) mRNA sequence, wherein the motif has the structure of motif (II '-f):

52. according to any one of claims 34 to 36, there is a 5' end region motif (II '-g) mRNA sequence, wherein the motif has the structure of motif (II '-g):

53. according to any one of claims 34 to 36, there is a 5' end region motif (II-h) mRNA sequence, wherein the motif has the structure of motif (II'-h):

54. according to any one of claims 34 to 36, there is a 5' end region motif (II '-i) of the mRNA sequence, wherein the motif has the structure of motif (II '-i):

55. according to any one of claims 34 to 36, there is a 5' end region motif (II '-j) mRNA sequence, wherein the motif has the structure of motif (II '-j):

56. according to any one of claims 34 to 36, there is a 5' end region motif (II '-k) mRNA sequence, wherein the motif has the structure of motif (II '-k):

57. according to the mRNA sequence with 5' end region motif (II'-1) described in any one in claim 34 to 36, wherein said motif has the structure of motif (II'-1):

58. according to any one of claims 34 to 36, there is a 5' end region motif (II '-m) mRNA sequence, wherein the motif has the structure of motif (II '-m):

59. according to claim 35, there is the mRNA sequence of 5' terminal region motif (II'-n), wherein said motif has the structure of motif (II'-n):

60. The mRNA sequence with the 5' end region motif (II'-o) according to claim 35, wherein the motif has the structure of motif (II'-o):

61. according to claim 35, there is the mRNA sequence of 5' terminal region motif (II'-p), wherein said motif has the structure of motif (II'-p):

62. according to claim 35, there is the mRNA sequence of 5' terminal region motif (II'-q), wherein said motif has the structure of motif (II'-q):

63. according to claim 0, there is the mRNA sequence of 5' end region motif (II'-r), wherein said motif has the structure of motif (II'-r):

64. according to claim 35, there is the mRNA sequence of 5' terminal region motif (II'-s), wherein said motif has the structure of motif (II'-s):

65. according to claim 35, there is the mRNA sequence of 5' terminal region motif (II'-t), wherein said motif has the structure of motif (II'-t):

66. according to claim 35, there is the mRNA sequence of 5' terminal region motif (II'-u), wherein said motif has the structure of motif (II'-u):

67. according to claim 37, there is the mRNA sequence of 5' terminal region motif (III'-a), wherein said motif has the structure of motif (III'-a):

68. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 37, wherein Z 3 It is hydrogen, fluorine, -OH, -OCH3, - or -OCH2CH3.

69. according to the mRNA sequence with 5 ' end region motif described in claim 68, wherein Z 3 It is -OCH3.

70. according to the mRNA sequence with 5 ' end region motif described in claim 68, wherein Z 4 and Z n Each is independently -OH or -OCH3.

71. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 37, wherein Z 3 , Z 4 and Z n Each is independently -OH or -OCH3.

72. according to the mRNA sequence with 5 ' end region motif described in any one in claim 35 or 44 to 71, wherein B 1 for 73. according to the mRNA sequence with 5 ' end region motif described in any one in claim 35 or 44 to 71, wherein B 1 for 74. according to the mRNA sequence with 5 ' end region motif described in any one in claim 35 or 44 to 71, wherein B 1 for 75. according to the mRNA sequence with 5 ' end region motif according to claim 36, wherein B 1 for 76. according to the mRNA sequence with 5 ' end region motif according to claim 37, wherein B 2 for 77. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 37, wherein Z 1 It is fluorine, -OH or -OCH3.

78. according to the mRNA sequence with 5 ' end region motif according to claim 77, wherein Z 1 For fluorine.

79. according to the mRNA sequence with 5 ' end region motif according to claim 77, wherein Z 1 It is -OH.

80. according to the mRNA sequence with 5 ' end region motif described in claim 77, wherein Z 1 It is -OCH3.

81. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 37, wherein Z 2 It is fluorine, -OH or -OCH3.

82. according to the mRNA sequence with 5 ' end region motif described in claim 81, wherein Z 2 For fluorine.

83. according to the mRNA sequence with 5 ' end region motif described in claim 81, wherein Z 2 It is -OH.

84. according to the mRNA sequence with 5 ' end region motif described in claim 81, wherein Z 2 It is -OCH3.

85. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 84, wherein Q 1 and Q 4 It is -CH2O-.

86. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 85, wherein Q 2 and Q 3 Each is -O-.

87. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 86, wherein Y 1 and Y 3 Each is =0.

88. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 87, wherein Y 2 , Y 4 and Y n Each is independently =O or =S.

89. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 86, wherein Y 1 , Y 2 , Y 3 , Y 4 and Y n One or more of them is =S.

90. according to the mRNA sequence with 5 ' end region motif according to claim 89, wherein Y 2 is =S.

91. according to the mRNA sequence with 5 ' end region motif according to claim 89, wherein Y 4 is =S.

92. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 86, wherein Y 1 , Y 2 , Y 3 , Y 4 and Y n Each is =0.

93. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 92, wherein X 1 , X 4 and X n Each is -O - .

94. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 93, wherein X 2 and X 3 Each independently is -O - or -S - .

95. according to the mRNA sequence with 5 ' end region motif described in claim 94, wherein X 3 -O - .

96. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 92, wherein X 1 , X 2 , X 3 , X 4 and X n One or more of -S - .

97. according to the mRNA sequence with 5 ' end region motif described in claim 96, wherein X 2 For -S - .

98. according to the mRNA sequence with 5 ' end region motif described in claim 96, wherein X 4 -S - .

99. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 92, wherein X 1 , X 2 , X 3 , X 4 and X n Each is -O - .

100. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 99, wherein A, A 1 and A 2 Each is -O-.

101. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 99, wherein A, A 1 and A 2 One or more of them is -S-.

102. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 99 or 100, wherein A is-S-, and A 1 and A 2 It is -O-.

103. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 99 or 100, wherein A 2 is -S-, and A and A 1 It is -O-.

104. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 99, wherein A, A 1 and A 2 For -O-.

105. The mRNA sequence with a 5' end region motif according to any one of claims 32, 34, 37 or 68 to 103, wherein p is 0.

106. An mRNA sequence with a 5' end region motif according to any one of claims 32, 34, 37 or 50 to 103, wherein p is 1.

107. An mRNA sequence with a 5' end region motif according to any one of claims 32, 34, 37 or 50 to 103, wherein p is 2.

108. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 107, wherein B 2 , B 3 and B n Each is independently adenine, cytosine, guanine, uracil, thymine, hypoxanthine or purine.

109. according to the mRNA sequence with 5 ' end region motif described in claim 108, wherein B 2 is adenine, and B 3 For guanine.

110. according to the mRNA sequence with 5 ' end region motif described in claim 109, wherein B 2 is guanine, and B 3 Adenine.

111. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 to 107, wherein B 2 is a modified adenine, a modified cytosine, a modified guanine, a modified uracil, a modified thymine, a modified hypoxanthine or a modified purine, and B 3 and B n Each is independently adenine, cytosine, guanine, uracil, thymine, hypoxanthine or purine.

112. according to the mRNA sequence with 5 ' end region motif described in claim 111, wherein B 2 is a modified adenine, and B 3 For guanine.

113. according to the mRNA sequence with 5 ' end region motif described in claim 112, wherein B 2 is 2-aminoadenine, and B 3 For guanine.

114. An mRNA sequence with a 5' end region motif according to any one of claims 32 to 113, wherein protein expression is increased.

115. according to the mRNA sequence with 5' end region motif described in claim 35 to 50, wherein Q 1 and Q 4 -CH2O-; Q 2 and Q 3 -O-;X n Each independently represents -OH, -SH, O - or S - ; Y n are each independently =O or =S; and B 1 for 116. according to the mRNA sequence with 5' end region motif described in claim 35 to 50, wherein Q 1 and Q 4 -CH2O-; Q 2 and Q 3 -O-;X n Each independently represents -OH, -SH, O - or S - ; Y n are each independently =O or =S; and B 1 for 117. according to the mRNA sequence with 5' end region motif described in claim 35 to 50, wherein Q 1 and Q 4 -CH2O-; Q 2 and Q 3 -O-;X n Each independently represents -OH, -SH, O - or S - ; Y n are each independently =O or =S; and B 1 for 118. according to the mRNA sequence with 5 ' end region motif described in claim 107 to 117, wherein B 2 Adenine.

119. according to the mRNA sequence with 5 ' end region motif described in claim 107 to 117, wherein B 3 For guanine.

120. according to the mRNA sequence with 5 ' end region motif described in any one in claim 16 to 119, wherein B2 is 121. according to the mRNA sequence with 5 ' end region motif according to claim 37, wherein said motif is 122. according to the mRNA sequence with 5 ' end region motif described in any one in claim 36 or 46, wherein said motif is 123. according to the mRNA sequence with 5 ' end region motif described in any one in claim 32 or 41, wherein said motif is 124. according to claim 32 to 50 each have the mRNA sequence of 5 ' end region motif, and wherein said motif comprises thiophosphate.

125. The mRNA sequence according to claim 123 having a 5' end region motif, wherein the phosphorothioate comprises a chiral phosphorus center.

126. according to the mRNA sequence with 5 ' end region motif according to claim 122, wherein said motif is 127. according to the mRNA sequence with 5 ' end region motif according to claim 122, wherein said motif is 128. A complex comprising an mRNA sequence having a 5' terminal region motif and a DNA template, wherein the mRNA sequence having a 5' terminal region motif comprises a compound according to any one of claims 32 to 126, wherein The DNA template includes 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 with the DNA template at least at nucleotide positions +1, +2 and +3.

129. A complex comprising an mRNA sequence having a 5' terminal region motif and a DNA template, wherein the mRNA sequence having a 5' terminal region motif comprises a compound according to any one of claims 32 to 126, wherein The DNA template includes 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 The mRNA sequence having the 5' end region motif hybridizes with the DNA template at least at nucleotide positions +1 and +2.

130. An RNA molecule comprising an mRNA sequence having a 5' end region motif according to any one of claims 32 to 129.

131. The RNA of claim 129, wherein the RNA comprises a guide RNA or a nuclease mRNA.

132. The RNA of claim 130, wherein the RNA comprises mRNA.

133. A method for expressing mRNA, the method comprising introducing the mRNA according to claim 131 into a cell lysate to express the mRNA.

134. The method of claim 132, further comprising measuring the expression level of the mRNA.

135. The method of claim 133, wherein the expression level of the mRNA is at least 2-fold, 3-fold, 4-fold, 6-fold, 8-fold or 20-fold that of the corresponding mRNA without the mRNA sequence having the 5' end region motif.

136. The method of claim 132, wherein the cell is a HeLa cell.

137. The method of claim 132, wherein at most 0.1 wt% dsRNA is produced.

138. The method of claim 136, wherein at most 0.09 wt% dsRNA is produced.

139. The method of claim 137, wherein at most 0.06 wt% dsRNA is produced.

140. The method of claim 138, wherein at most 0.04 wt% dsRNA is produced.

141. A method for detecting cellular immune stimulation produced by mRNA, the method comprising (a) contacting a preparation comprising the capped mRNA of any one of claims 32 to 126 with a cellular reporter cell line, and (b) measuring RIG-I activation in the cellular reporter cell line.

142. The method of claim 140, wherein the cell reporter cell line is a HEK-Lucia RIG-I model.

143. The method of any one of claims 140 or 141, wherein cellular immune stimulation is reduced by at least 20% compared to uncapped mRNA.

144. The method of claim 142, wherein cellular immune stimulation is reduced by at least 50% compared to uncapped mRNA.

145. The method of claim 143, wherein cellular immune stimulation is reduced by at least 70% compared to uncapped mRNA.

146. The method of claim 144, wherein cellular immune stimulation is reduced by at least 100% compared to uncapped mRNA.

147. The method of claim 145, wherein cellular immune stimulation is reduced by at least 150% compared to uncapped mRNA.

148. A method for producing an mRNA sequence having a 5'-terminal region motif according to any one of claims 32 to 131 using an IVT reaction, the method comprising (a) mixing a DNA template, a polymerase, an mRNA sequence initiator comprising a phosphorothioate group, and nucleoside triphosphates (NTPs) at a specific molar ratio of the mRNA sequence initiator to the NTPs to produce a mixture, (b) incubating the mixture at a specific temperature and duration, and (c) collecting and purifying the mRNA sequence having a 5'-terminal region motif from the mixture.

149. The method of claim 147, wherein the molar ratio of the mRNA sequence initiator to the NTP is about 1:5, and wherein the method is capable of producing a yield of at least 80% and a capping efficiency of at least 80%.

150. The method of claim 147, wherein the molar ratio of the mRNA sequence initiator to the NTP is 1:2.5, and wherein the method is capable of producing a yield of at least 80% and a capping efficiency of at least 85%.

151. The method of claim 147, wherein the molar ratio of the mRNA sequence initiator to the NTP is about 1:1.67, and wherein the method is capable of producing a yield of at least 80% and a capping efficiency of at least 90%.

152. The method of claim 147, wherein the molar ratio of the mRNA sequence initiator to the NTP is about 1:1.25, and wherein the method is capable of producing a yield of at least 80% and a capping efficiency of at least 90%.

153. The method of claim 147, wherein the molar ratio of the mRNA sequence initiator to the NTP is about 1:1, and wherein the method is capable of producing a yield of at least 80% and a capping efficiency of at least 90%.

154. The method of claim 147, wherein the molar ratio of the mRNA sequence initiator to the NTP is about 1:5, and wherein the method is capable of producing a yield of at least 3 mg of mRNA per milliliter (mL) of IVT reaction and a capping efficiency of at least 80%.

155. The method of claim 147, wherein the molar ratio of the mRNA sequence initiator to the NTP is about 1:2.5, and wherein the method is capable of producing a yield of at least 3 mg mRNA per mL IVT reaction and a capping efficiency of at least 85%.

156. The method of claim 147, wherein the molar ratio of the mRNA sequence initiator to the NTP is about 1:1.67, and wherein the method is capable of producing a yield of at least 3 mg mRNA per mL IVT reaction with a capping efficiency of at least 90%.

157. The method of claim 147, wherein the molar ratio of the mRNA sequence initiator to the NTP is about 1:1.25, and wherein the method is capable of producing a yield of at least 3 mg mRNA per mL IVT reaction with a capping efficiency of at least 90%.

158. The method of claim 147, wherein the molar ratio of the mRNA sequence initiator to the NTP is about 1:1, and wherein the method is capable of producing a yield of at least 3 mg mRNA per mL IVT reaction with a capping efficiency of at least 90%.

159. The method of claim 147, wherein the NTP is GTP, ATP, CTP, UTP, or a modified NTP, or a combination thereof.

160. The method of claim 158, wherein the modified NTP is the 5'-triphosphate of N1-methylpseudouridine.

161. A cell comprising an RNA molecule comprising an mRNA sequence having a 5' end region motif according to any one of claims 32 to 126.

162. A cell containing a polypeptide or protein translated from an RNA molecule comprising an mRNA sequence having a 5' end region motif according to any one of claims 32 to 126.

163. A pharmaceutical composition comprising an RNA molecule and one or more pharmaceutically acceptable excipients, wherein the RNA molecule comprises an mRNA sequence having a 5' end region motif according to any one of claims 32 to 126.

164. The pharmaceutical composition of claim 162, further comprising one or more single guide RNAs designed to target one or more specific locations of one or more genes of interest to elicit a pharmacological effect upon administration to a mammal.

165. The pharmaceutical composition of claim 162, comprising lipid nanoparticles.

166. A pharmaceutical composition according to claim 163, wherein the RNA is encapsulated in lipid nanoparticles.

167. A method for synthesizing an RNA molecule, the method comprising: a. introducing an mRNA sequence having a 5' end region motif according to any one of claims 32 to 126 into a mixture comprising an RNA polymerase, and b. incubating the mixture for a time sufficient to allow transcription of the RNA molecule.

168. The method of claim 134, wherein the mixture further comprises a DNA template and nucleoside triphosphates.

169. A method of gene editing, comprising introducing an RNA molecule according to claim 129 or 131 or a pharmaceutical composition according to any one of claims 163 to 166 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.

170. A method for reducing the risk of coronary artery disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 163 to 166.

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