Oligonucleotide compositions and methods thereof

Abstract

This disclosure provides, in particular, oligonucleotide compositions and methods thereof. In some embodiments, this disclosure provides structural elements comprising various chemical modifications, including sugar, nucleobase, and / or nucleotide inter-linking, that can be used for adenosine editing. In some embodiments, this disclosure provides methods for preventing or treating conditions, disorders, or diseases that may benefit from adenosine editing in nucleic acids.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 586,396, filed September 28, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Oligonucleotides can be used in a variety of applications, such as therapeutic, diagnostic, and / or research applications. For example, oligonucleotides that target multiple genes can be used to treat conditions, disorders, or diseases associated with such target genes. Summary of the Invention

[0004] In particular, this disclosure provides designed oligonucleotides and compositions thereof, which contain modifications as described herein (e.g., modifications to ribosomal sugars and / or internucleotide linkages, and patterns thereof). In some embodiments, the techniques of this disclosure (compounds (e.g., oligonucleotides), compositions, methods, etc.) (e.g., oligonucleotides, oligonucleotide compositions, methods, etc.) are particularly useful for editing nucleic acids, such as site-specific editing of nucleic acids (e.g., editing of target adenosine). In some embodiments, as shown herein, the provided techniques can significantly improve the efficiency of nucleic acid editing, such as modification of one or more A residues, such as converting A to I. In some embodiments, this disclosure provides techniques for editing in RNA (e.g., for modifying A residues, such as converting A to I). In some embodiments, this disclosure provides techniques for editing in transcripts (e.g., mRNA) (e.g., for modifying A residues, such as converting A to I). In particular, the provided techniques provide the benefit of using endogenous proteins such as ADAR (adenosine deaminase acting on RNA) proteins (e.g., ADAR1 and / or ADAR2) to edit nucleic acids, such as modifying A (e.g., as a result of G to A mutations). Those skilled in the art will recognize that such utilization of endogenous proteins avoids many challenges and / or provides a variety of benefits compared to those technologies that require the delivery of exogenous components (e.g., proteins (e.g., those engineered to bind oligonucleotides (and / or their duplexes with target nucleic acids) to provide the desired activity), nucleic acids encoding proteins, viruses, etc.).

[0005] Specifically, in some embodiments, the provided oligonucleotides of the technology comprise useful sugar modifications and / or patterns thereof (e.g., the presence and / or absence of certain modifications), nucleobase modifications and / or patterns thereof (e.g., the presence and / or absence of certain modifications), internucleotide linking modifications and / or stereochemical and / or patterns thereof [e.g., type, modification, and / or configuration (Rp or Sp) of chiral linked phosphorus, etc.], which, when combined with one or more other structural elements described herein (e.g., additional chemical motifs), can provide high activity and / or a variety of desired properties, such as high nucleic acid editing efficiency, high selectivity, high stability, high cellular uptake, low immunostimulation, low toxicity, improved distribution, improved affinity, etc. In some embodiments, the provided oligonucleotides provide high stability, for example, when compared with oligonucleotides having a high percentage of native RNA sugars for adenosine editing. In some embodiments, the provided oligonucleotides provide high activity, such as adenosine editing activity. In some embodiments, the provided oligonucleotide provides high selectivity. For example, in some embodiments, the provided oligonucleotide provides selective modification of the target adenosine in the target nucleic acid relative to other adenosines in the same target nucleic acid (e.g., the modification at the target adenosine is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more than the modification at another adenosine or all other adenosines in the target nucleic acid).

[0006] In particular, this disclosure provides engineered oligonucleotides and compositions that have improved properties and / or activities compared to reference oligonucleotides and compositions (e.g., those described herein or reported in the art). For example, in some embodiments, as shown herein, the provided oligonucleotides and compositions may provide improved stability, pharmacokinetic properties, pharmacodynamic properties, and / or improved activity (e.g., for A to I editing). Various engineered oligonucleotides and compositions are described herein. For example, in some embodiments, this disclosure provides oligonucleotides and compositions thereof, including chiral-controlled oligonucleotide compositions wherein the oligonucleotide comprises several (e.g., 1, 2, 3, 4, or 5 or more; in some embodiments, 3 or more) independently containing sugar modifications (e.g., 2'-OR modifications, where R is optionally substituted C) at its 5'-end and 3'-end. 1-6Nucleosides of alkyl groups (e.g., 2'-OMe, 2'-MOE, etc.) and bicyclic sugars (e.g., LNA sugar, cEt sugar, etc.). In some embodiments, the first few (e.g., 1, 2, 3, 4, or 5 or more; in some embodiments, 3 or more) nucleosides and / or the last few (e.g., 1, 2, 3, 4, or 5 or more; in some embodiments, 3 or more) nucleosides independently contain sugar modifications. In some embodiments, the first 3 or more and the last 3 or more nucleosides independently contain sugar modifications. In some embodiments, the internucleotide linkages of one or more nucleotides bonded to such nucleosides are uncharged internucleotide linkages, such as phosphorylguanidine internucleotide linkages, like n001. In some embodiments, the first and last internucleotide linkages are both independently uncharged internucleotide linkages. In some embodiments, the first and last internucleotide linkages are both independently phosphorylguanidine internucleotide linkages. In some embodiments, the first and last internucleotide linkages are both independently n001. In some embodiments, they are both chiral controlled and are Rp. In some embodiments, the oligonucleotide comprises nucleoside NO, which contains a natural DNA sugar (two 2'-H), a natural RNA sugar, or a sugar modified with 2'-F. In some embodiments, when the oligonucleotide is used for adenosine editing, NO is a nucleoside opposite to the target adenosine. In some embodiments, the sugar of NO is a natural DNA sugar. In some embodiments, the sugar of N1 (with a "+" before the number or nothing indicating counting in the 5'- direction (5'...N1N0N)) -1 … 3')) is a 2'-F modified sugar, a natural DNA sugar, or a natural RNA sugar. In some embodiments, the sugar of N1 is a DNA sugar. In some embodiments, N -1 The sugar (number preceded by a "-" or nothing indicating counting in the 3' direction (5' ... N1N0N) -1 …3')) is a 2'-F modified sugar, a natural DNA sugar, or a natural RNA sugar. In some embodiments, N -1 The sugar is DNA sugar. In some embodiments, N -3 The sugar is a 2'-F modified sugar. In some embodiments, the oligonucleotide comprises a plurality of 2'-F modified sugars and a plurality of 2'-modified sugars (e.g., 2'-OR modified sugars, where R is optionally a substituted C) between N2 and its 5'-terminus. 1-6Alkyl groups, bicyclic sugars such as LNA sugars, cEt sugars, etc.). In some embodiments, the oligonucleotide from N2 to its 5'-end (e.g., when the first subdomain of the second domain terminates with N2 and includes N2, the first domain and the first subdomain are combined) comprises one or more (e.g., 1-20, 1-15, 1-10, 2-15, 2-10 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) 2'-F blocks and one or more (e.g., 1-20, 1-15, 1-10, 2-15, 2-10 or 1, 2, 3, 4, ... 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more separable blocks, wherein each nucleotide in the 2'-F block independently contains a 2'-F modification, each nucleotide in the separable block independently does not contain a 2'-F modification, and each block independently contains one or more (e.g., 1-20, 1-15, 1-10, 2-15, 2-10 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) nucleotides. In some embodiments, there are two or more such 2'-F blocks and two or more such separable blocks. In some embodiments, one or more or all of such separable blocks are independently bonded to two 2'-F blocks. In some embodiments, each nucleotide in one or more of the isolated blocks independently comprises a 2'-OR modification, wherein R is an optionally substituted C 1-6 The alkyl group or bicyclic sugar, such as LNA sugar, cEt sugar, etc. In some embodiments, each nucleotide in one or more or all of the separable blocks independently comprises a 2'-OR modification, wherein R is an optionally substituted C 1-6 Alkyl group. In some embodiments, each nucleoside in one or more or all of the segregated blocks independently comprises 2'-OMe or 2'-MOE modification. In some embodiments, each of such 2'-F and segregated blocks independently comprises 1, 2, 3, 4 or 5 nucleosides. In some embodiments, nucleosides close to N0, such as N2, N1, N0, N -1 N -2 The following do not contain large 2'-modifications, such as 2'-MOE. In some embodiments, N2, N1, N0, N -1 and N -2 The sugars are independently natural DNA sugars, 2'-F modified sugars, or 2'-OMe modified sugars. In some embodiments, N1, N0, N -1 The sugars are all natural DNA sugars. In some embodiments, the bonding between each chiral nucleotide is independently chiral controlled.

[0007] As demonstrated herein, and particularly, this disclosure provides useful modified sugars, modified nucleotides, and modified nucleosides that are useful at various positions within oligonucleotides, for example, for targeted adenosine editing. Specifically, in some embodiments, this disclosure provides useful modified sugars, modified nucleotides, and modified nucleosides that can be in N1, N0, or N... -1 This technology is used at the N0 site. In some embodiments, when compared with reference technologies, such as using a 2'-F modified sugar or DNA sugar at the N0 site, or using cytosine at the N0 site, the provided technology can provide improved properties, such as improved stability and / or editing activity. In some embodiments, this disclosure provides a technology containing 5'-N1N0N -1 -3' oligonucleotides, wherein N1, N0 and N -1 Each is an independent nucleoside and is linked by internucleotide bonds. In some embodiments, this disclosure provides a 5'-N1N0N... -1 -3' oligonucleotides, wherein N1, N0 and N -1 Each oligonucleotide is an independent nucleoside and is linked by an internucleotide bond, wherein the oligonucleotide is capable of binding to a target nucleic acid, and N0 is the counterpart to the target adenosine. In some embodiments, N0 comprises a modified sugar, a modified nucleobase, or a modified nucleoside. In some embodiments, N1 comprises a modified sugar, a modified nucleobase, or a modified nucleoside. In some embodiments, N -1 It contains modified sugars, modified nucleobases, or modified nucleosides.

[0008] In some embodiments, this disclosure provides an oligonucleotide comprising a first domain and a second domain, wherein the first domain comprises one or more 2'-F modifications, and the second domain comprises one or more sugars without 2'-F modifications. In some embodiments, the provided oligonucleotide comprises one or more chiral-modified nucleotide linkages.

[0009] In some embodiments, this disclosure provides oligonucleotides comprising:

[0010] (a) the first structural domain; and

[0011] (b) Second structural domain,

[0012] in:

[0013] The first domain contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more sugars containing 2'-F modification;

[0014] The second domain contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more sugars, each independently containing a 2'-OR modified sugar, where R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-OL). B -4', where L B (e.g., optionally substituted -CH2-).

[0015] This oligonucleotide contains 5'-N1N0N -1 -3', where N1, N0 and N -1 Each is an independent nucleoside and they are linked by internucleotide bonds;

[0016] This oligonucleotide is in N0, N -1 Or, the N1 position contains a modified sugar, a modified nucleobase, or a modified nucleoside; and

[0017] This oligonucleotide can bind to the target nucleic acid, where N0 is opposite to the target adenosine.

[0018] In some embodiments, this disclosure provides oligonucleotides comprising:

[0019] (a) the first structural domain; and

[0020] (b) Second structural domain,

[0021] About 20%-80% of all sugars in the first domain (e.g., about 25%-80%, 30%-80%, 35%-80%, 40%-80%, 40%-70%, 40%-60%, 50%-80%, 50%-75%, 50%-60%, 55%-80%, 60%-80%, or about 50%, 55%, 60%, 65%, 70%, 75% or 80%) contain 2'-F modification;

[0022] The second domain contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more modified sugars that are not modified with 2'-F, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of all sugars in the second domain are not modified with 2'-F;

[0023] This oligonucleotide contains 5'-N1N0N -1 -3', where N1, N0 and N -1 Each is an independent nucleoside and they are linked by internucleotide bonds;

[0024] This oligonucleotide is in N0, N-1 Or, the N1 position contains a modified sugar, a modified nucleobase, or a modified nucleoside; and

[0025] This oligonucleotide can bind to the target nucleic acid, where N0 is opposite to the target adenosine.

[0026] In some embodiments, this disclosure provides oligonucleotides comprising:

[0027] (a) the first structural domain; and

[0028] (b) Second structural domain,

[0029] The first domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more sugars containing 2'-F modification and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more sugars each independently containing 2'-OR modification, wherein R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-OL). B -4', where L B (Optionally substituted -CH2-, etc.); and

[0030] The second domain contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more sugars, each independently containing a 2'-OR modified sugar, wherein R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-OL). B -4', where L B (Optionally substituted -CH2- etc.).

[0031] In some embodiments, this disclosure provides oligonucleotides comprising:

[0032] (a) the first structural domain; and

[0033] (b) Second structural domain,

[0034] Approximately 20%-80% of all sugars in the first domain (e.g., approximately 25%-80%, 30%-80%, 35%-80%, 40%-80%, 40%-70%, 40%-60%, 50%-80%, 50%-75%, 50%-60%, 55%-80%, 60%-80%, or approximately 50%, 55%, 60%, 65%, 70%, 75%, or 80%) contain a 2'-F modification, and approximately 20%-70% of all sugars in the first domain (e.g., approximately 20%-60%, 20%-50%, 30%-60%, 30%-50%, 40%-50%, or approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%) independently contain a 2'-OR modification, wherein R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-OL). B -4', where L B (Optionally substituted -CH2-, etc.); and

[0035] The second domain contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more modified sugars that are not 2'-F modified, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of all sugars in the second domain are not 2'-F modified.

[0036] In some embodiments, the second domain comprises, or is composed of, a first subdomain, a second subdomain, and a third subdomain as described herein. In some embodiments, the first subdomain comprises one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars each independently containing a 2'-OR modified sugar, wherein R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-OL). B -4', where L B (Optionally substituted -CH2-, etc.). In some embodiments, more of this sugar is present in the first subdomain than in sugars modified with 2'-F. In some embodiments, the sugars in the second subdomain are all free of any 2'-OR modification, where R is an optionally substituted C. 1-6 Aliphatic group, or 2'-OL B-4'. In some embodiments, each sugar in the second subdomain is independently a natural DNA sugar, a natural RNA sugar, or a 2'-F modified sugar. In some embodiments, each sugar in the second subdomain is independently a natural DNA sugar or a natural RNA sugar. In some embodiments, each sugar in the second subdomain is independently a natural DNA sugar or a 2'-F modified sugar. In some embodiments, each sugar in the second subdomain is independently a natural DNA sugar. In some embodiments, three nucleotides are present in the second subdomain. In some embodiments, when bound to a target, the second nucleotide of the three nucleotides is opposite to the target adenosine. In some embodiments, the sugar of the second nucleotide does not contain any 2'-OR modification as described herein (e.g., 2'-OMe, 2'-MOE, etc.). In some embodiments, such a sugar is a natural DNA sugar. In some embodiments, it is a natural RNA sugar. In some embodiments, it is a 2'-F modified sugar. In some embodiments, the third subdomain comprises one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars that each independently contain a 2'-OR modified sugar, wherein R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-OL). B -4', where L B (Optionally substituted -CH2-, etc.). In some embodiments, more of this sugar is present in the third subdomain than in sugars modified with 2'-F.

[0037] In some embodiments, the second domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more modified sugars that independently contain 2'-OR modification, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars in the second domain contain 2'-OR modification, wherein R is optionally a substituted C 1-6 Aliphatic group. In some embodiments, R is methyl. In some embodiments, R is -CH2CH2OCH3. As described herein, other sugar modifications may also be utilized according to this disclosure, optionally having the base modifications and / or internucleotide linking modifications described herein.

[0038] In some embodiments, the oligonucleotide comprises or has a 5'-first domain-second domain-3' structure. In some embodiments, the second domain comprises or has a 5'-first subdomain-second subdomain-third subdomain-3' structure. In some embodiments, the oligonucleotide comprises or has a 5'-first domain-first subdomain-second subdomain-third domain-3' structure. In some embodiments, the oligonucleotide is conjugated to additional portions, such as various additional chemical portions as described herein. In some embodiments, the oligonucleotide comprises additional portions, such as additional portions as described herein. In some embodiments, the additional chemical portions are or comprise small molecule portions, carbohydrate portions (e.g., GalNAc portions), nucleic acid portions (e.g., oligonucleotide portions, nucleic acid portions that can provide and / or regulate one or more properties and / or activities, etc. (e.g., portions of RNase H-dependent oligonucleotides, RNAi oligonucleotides, aptamers, gRNA, etc.)), and / or peptide portions.

[0039] In some embodiments, the base sequence of the provided oligonucleotide is substantially complementary to the base sequence of the target nucleic acid containing the target adenosine. In some embodiments, the provided oligonucleotide contains one or more mismatches (non-Watson-Crick base pairs) when aligned with the target nucleic acid. In some embodiments, the provided oligonucleotide contains one or more wobbles (e.g., GU, IA, GA, IU, IC, etc.) when aligned with the target nucleic acid. In some embodiments, mismatches and / or wobbles may help one or more proteins (e.g., ADAR1, ADAR2, etc.) recognize the duplex formed by the provided oligonucleotide and the target nucleic acid. In some embodiments, the provided oligonucleotide forms a duplex with the target nucleic acid. In some embodiments, the ADAR protein recognizes and binds to such a duplex. In some embodiments, a nucleoside opposite to the target adenosine is located in the middle of the provided oligonucleotide, for example, 5-50 nucleosides on the 5' side and 1-50 nucleosides on its 3' side. In some embodiments, the 5' side has more nucleosides than the 3' side. In some embodiments, the 5' side has fewer nucleosides than the 3' side. In some embodiments, the 5' side has the same number of nucleosides as the 3' side. In some embodiments, the provided oligonucleotide comprises 15-40 (e.g., 15, 20, 25, 30, etc.) consecutive bases of the oligonucleotides described in the table. In some embodiments, the base sequence of the provided oligonucleotide is or comprises the base sequence of the oligonucleotides described in the table.

[0040] In some embodiments, by utilizing a variety of structural elements (e.g., various modifications, stereochemistry, and patterns thereof), the present disclosure can achieve the desired properties and high activity with short oligonucleotides (e.g., those oligonucleotides with a length of about 20-40, 25-40, 25-35, 26-32, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides).

[0041] In some embodiments, the provided oligonucleotide comprises modified nucleosides. In some embodiments, the modified nucleosides promote the modification of target adenosine. In some embodiments, the nucleosides opposite to the target adenosine maintain interaction with enzymes such as ADAR compared to the presence of U, while the interaction with the target adenosine is less strong than with U (e.g., fewer hydrogen bonds are formed). In some embodiments, the opposing nucleosides and / or their associated sugars provide a degree of flexibility (e.g., compared to U) to facilitate the modification of target adenosine by enzymes (e.g., ADAR1, ADAR2, etc.). In some embodiments, the 5' or 3' nucleosides immediately adjacent to (the target adenosine) the opposing nucleosides (e.g., I and its derivatives) enhance the modification of target adenosine. In particular, this disclosure recognizes that such nucleosides can result in less steric hindrance than G when the provided oligonucleotide and its target nucleic acid duplex interact with modifying enzymes (e.g., ADAR1 or ADAR2). In some embodiments, the base sequence of an oligonucleotide is selected (e.g., when several adenosine residues are suitable targets) and / or designed (e.g., by utilizing a variety of nucleobases as described herein) such that steric hindrance can be reduced or eliminated (e.g., no G is adjacent to the opposite nucleoside of target A).

[0042] According to this disclosure, various internucleotide linkages can be used in oligonucleotides. In some embodiments, the oligonucleotide contains one or more types of internucleotide linkages. In some embodiments, the oligonucleotide contains two or more types of internucleotide linkages. In some embodiments, the oligonucleotide contains at least three types of internucleotide linkages. In some embodiments, the linkage contains a linkage phosphorus atom bonded to an oxygen atom that is not bonded to or is not part of the skeletal sugar (“PO linkage,” e.g., a native phosphate linkage). In some embodiments, the linkage contains a linkage phosphorus atom bonded to a sulfur atom that is not bonded to or is not part of the skeletal sugar (“PS linkage,” e.g., a thiophosphate linkage). In some embodiments, the linkage contains a linkage phosphorus atom bonded to a nitrogen atom that is not bonded to or is not part of the skeletal sugar (“PN linkage,” e.g., n001). In some embodiments, the oligonucleotide contains one or more PS linkages. In some embodiments, the oligonucleotide contains one or more PO linkages. In some embodiments, the oligonucleotide contains one or more PN linkages. In some embodiments, the oligonucleotide contains one or more PS linkages and one or more PO linkages. In some embodiments, the oligonucleotide contains one or more PS linkages and one or more PN linkages. In some embodiments, the oligonucleotide comprises one or more PS links, one or more PN links, and one or more PO links. In some embodiments, the PS links are phosphate thioester links. In some embodiments, each PS link is independently a phosphate thioester link. In some embodiments, the PO links are native phosphate ester links. In some embodiments, each PO link is independently a native phosphate ester link. In some embodiments, the PN links are phosphorylguanidine links. In some embodiments, each PN link is independently a phosphorylguanidine link.

[0043] In some embodiments, the first domain comprises one or more PO bonds, one or more PS bonds, and one or more PN bonds. In some embodiments, the first subdomain comprises one or more PO bonds, one or more PS bonds, and / or one or more PN bonds. In some embodiments, the first subdomain comprises one or more PO bonds. In some embodiments, the first subdomain comprises one or more natural phosphate bonds. In some embodiments, the second subdomain comprises one or more modified internucleotide bonds. In some embodiments, each internucleotide bonded to the nucleoside of the second subdomain is independently a modified internucleotide bond. In some embodiments, each internucleotide bonded to the nucleoside of the second subdomain is independently a PS or PN bond. In some embodiments, the third subdomain comprises one or more PO bonds, one or more PS bonds, and / or one or more PN bonds. In some embodiments, the third subdomain comprises one or more PO bonds. In some embodiments, the third subdomain comprises one or more natural phosphate bonds. In some embodiments, the third subdomain comprises one or more PS bonds. In some embodiments, the third subdomain comprises one or more PN bonds. In some embodiments, the third subdomain comprises one or more PO bonds, one or more PS bonds, and one or more PN bonds. In some embodiments, the first nucleotide linking of the first domain or oligonucleotide is a PN linking. In some embodiments, the last nucleotide linking of the third subdomain or oligonucleotide is a PN linking. In some embodiments, natural DNA sugars are linked to modified nucleotides. In some embodiments, natural DNA sugars are linked to PN or PS nucleotides. In some embodiments, each natural DNA sugar in an oligonucleotide or a portion thereof (e.g., the first domain, the first subdomain, the second subdomain, the third subdomain, etc.) is independently linked to a modified nucleotide. In some embodiments, each natural DNA sugar is independently linked to a PN or PS nucleotide. In some embodiments, natural RNA sugars are linked to modified nucleotides. In some embodiments, natural RNA sugars are linked to PN or PS nucleotides. In some embodiments, each natural RNA sugar in an oligonucleotide or a portion thereof (e.g., the first domain, the first subdomain, the second subdomain, the third subdomain, etc.) is independently linked to a modified nucleotide. In some embodiments, each natural RNA sugar is independently linked to a PN or PS nucleotide.

[0044] In some embodiments, a 2'-F modified sugar is inter-linked with a modified nucleotide. In some embodiments, a 2'-F modified sugar is inter-linked with a PN or PS nucleotide. In some embodiments, each 2'-F modified sugar in an oligonucleotide or a portion thereof (e.g., a first domain, a first subdomain, a second subdomain, a third subdomain, etc.) is independently inter-linked with a modified nucleotide. In some embodiments, each 2'-F modified sugar is independently inter-linked with a PN or PS nucleotide. In some embodiments, each PO link is independently a native phosphate ester link. In some embodiments, each PS link is independently an inter-linked phosphate thioester nucleotide. In some embodiments, one or more PN links are independently inter-linked nucleotides without a negative charge. In some embodiments, one or more PN links are independently inter-linked neutral nucleotides. In some embodiments, one or more PN links are independently phosphorylguanidine links. In some embodiments, each PN link is independently a phosphorylguanidine link. In some embodiments, one or more PN links are independently n001. In some embodiments, each PN link is independently n001.

[0045] In some embodiments, the oligonucleotides of this disclosure provide modified internucleotide linkages (i.e., internucleotide linkages that are not naturally phosphate-linked). In some embodiments, the linking phosphorus of the modified internucleotide linkage (e.g., chiral internucleotide linkage) is chiral and can be present in different configurations (Rp and Sp). In particular, this disclosure demonstrates that the incorporation of modified internucleotide linkages, especially with stereochemical control over the linking phosphorus center (such that a configuration is enriched at this control center compared to stereorandom oligonucleotide preparations), can significantly improve properties (e.g., stability) and / or activities (e.g., adenosine modification activity (e.g., conversion of adenosine to inosine)). In some embodiments, the provided oligonucleotides have significantly higher stereochemical purity than stereorandom preparations. In some embodiments, the provided oligonucleotides are chirally controlled.

[0046] In some embodiments, the oligonucleotides disclosed herein comprise one or more chiral nucleotide linkages, wherein the linking phosphorus is chiral (e.g., phosphate thioester linkages). In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides are linked, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all nucleotides are linked (e.g., 50%-100%, 60%-100%, 70%-100%, 75%-100%, 80%-100%, 90%-100%, 95%-100%, 60%-95%, 70%-95%, 75%-95%, 80%-95%, 85%-95%, 90%-95%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, etc.), or all nucleotides are chiral nucleotide links. In some embodiments, at least one nucleotide link is a chiral nucleotide link. In some embodiments, at least one nucleotide link is a natural phosphate link. In some embodiments, each nucleotide link is independently a chiral nucleotide link. In some embodiments, at least one chiral nucleotide link is a phosphate thioester nucleotide link. In some embodiments, each is a phosphate thioester nucleotide link. In some embodiments, one or more chiral nucleotide links are independently uncharged nucleotide links or neutral nucleotide links. In some embodiments, one or more chiral nucleotide links are independently phosphorylguanidine nucleotide links. In some embodiments, one or more chiral nucleotide links are independently chiral controlled. In some embodiments, each chiral nucleotide link is independently chiral controlled. In some embodiments, one or more chiral nucleotide links are not chiral controlled. In some embodiments, each phosphate thioester nucleotide link is independently chiral controlled. In some embodiments, each modified nucleotide link is independently a phosphate thioester or uncharged nucleotide link. In some embodiments, each modified nucleotide link is independently a phosphate thioester or neutral nucleotide link. In some embodiments, each modified nucleotide link is independently a phosphate thioester or neutral nucleotide link. In some embodiments, each modified nucleotide link is independently a phosphate thioester or phosphorylguanidine nucleotide link. In some embodiments, the phosphorylguanidine nucleotide link is n001. In some embodiments, each phosphorylguanidine nucleotide link is n001. In some embodiments, each uncharged nucleotide link is n001. In some embodiments, each neutral nucleotide link is n001. In some embodiments, the modified nucleotide link is n002. In some embodiments, it is n006.In some embodiments, it is n020. In some embodiments, it is n004. In some embodiments, it is n008. In some embodiments, it is n025. In some embodiments, it is n026. Various modified internucleotide linkages are described herein. The linking phosphorus can be Rp or Sp. In some embodiments, at least one linking phosphorus is Rp. In some embodiments, at least one linking phosphorus is Sp. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chiral nucleotides are linked, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all chiral nucleotides are linked (e.g., 50%-100%, 60%-100%, 70%-100%, 75%-100%, 80%-100%, 90%-100%, 95%-100%, 60%-95%, 70%-95%, 75%-95%, 80%-95%, 85%-95%, 90%-95%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, etc.), or all chiral nucleotides are sp. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 phosphate-thioester nucleotides are linked together, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all phosphate-thioester nucleotides are linked together (e.g., 50%-100%, 60%-100%). The percentages of all phosphate-thioester nucleotides are Sp (70%-100%, 75%-100%, 80%-100%, 90%-100%, 95%-100%, 60%-95%, 70%-95%, 75%-95%, 80%-95%, 85%-95%, 90%-95%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, etc.), or all phosphate-thioester nucleotides are Sp. In some embodiments, at least 50% of all phosphate-thioester nucleotides are Sp. In some embodiments, at least 60% of all phosphate-thioester nucleotides are Sp. In some embodiments, at least 70% of all phosphate-thioester nucleotides are Sp. In some embodiments, at least 75% of all phosphate-thioester nucleotides are Sp. In some embodiments, at least 80% of all phosphate-thioester nucleotides are Sp. In some embodiments, at least 85% of all phosphate-thioester nucleotides are Sp. In some embodiments, at least 90% of all phosphate thioester nucleotide linkages are sp. In some embodiments, at least 95% of all phosphate thioester nucleotide linkages are sp.In some embodiments, at least 96% of all phosphate-thionucleotide (PTN) linkages are sp. In some embodiments, at least 97% of all PTN linkages are sp. In some embodiments, at least 98% of all PTN linkages are sp. In some embodiments, all PTN linkages are sp. In some embodiments, no more than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive PTN linkages are Rp. In some embodiments, no more than 3 consecutive PTN linkages are Rp. In some embodiments, no more than 4 consecutive PTN linkages are Rp. In some embodiments, no more than 5 consecutive PTN linkages are Rp. In some embodiments, no more than 6 consecutive PTN linkages are Rp. In some embodiments, no more than 7 consecutive PTN linkages are Rp. In some embodiments, no more than 8 consecutive PTN linkages are Rp. In some embodiments, no more than 9 consecutive PTN linkages are Rp. In some embodiments, no more than 10 consecutive phosphate-thioester nucleotides are Rp. In some embodiments, consecutive Rp phosphate-thioester nucleotides are not used in portions where most (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) or all of the sugar is native DNA and / or RNA and / or a 2'-F modified sugar. In some embodiments, when consecutive Rp phosphate-thioester nucleotides are used, one or more or most (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) or all of such nucleotides are independently bonded to the sugar, which can improve stability. In some embodiments, when using sequential Rp phosphate thioester nucleotide linkages, one or more (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) or all such nucleotide linkages are independently linked to a bicyclic sugar or a 2'-OR modified sugar (where R is optionally a substituted C). 1-6 Aliphatic groups are bonded. In some embodiments, when using sequential Rp phosphate thioester nucleotide linkages, one or more or most (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) or all such nucleotide linkages are independently linked to a 2'-OR modified sugar (where R is optionally a substituted C). 1-6Aliphatic group) bonding. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe modified sugar or a 2'-MOE modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe modified sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE modified sugar.

[0047] In some embodiments, the stereochemistry of one or more chiral phosphates of the provided oligonucleotides is controlled in the composition. In some embodiments, this disclosure provides compositions comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides share a common base sequence and are independently linked between one or more chiral nucleotides (“chiral-controlled internucleotide linkages”) (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 1, 2, 3). The oligonucleotides share the same linking phosphorus configuration (e.g., all Rp or all Sp for chiral linking phosphorus) at at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the inter-chiral linking phosphorus. In some embodiments, they share the same stereochemistry at each chiral linking phosphorus. In some embodiments, the multiple oligonucleotides share the same composition. In some embodiments, the multiple oligonucleotides are structurally identical except for the inter-nucleotide links. In some embodiments, the multiple oligonucleotides are structurally identical. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in the composition, or all oligonucleotides sharing a common base sequence, share the skeletal chiral center pattern of the plurality of oligonucleotides. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in the composition, or all oligonucleotides sharing a common base sequence, are the plurality of oligonucleotides.

[0048] In some embodiments, this disclosure provides a chiral-controlled oligonucleotide composition, wherein at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in the composition, or all oligonucleotides having the same oligonucleotide base sequence, or all oligonucleotides having the same base sequence and sugar and base modifications, or all oligonucleotides having the same composition, are independently linked between one or more chiral nucleotides of the oligonucleotide (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1...). -10, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more chiral nucleotides are linked together, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of all chiral nucleotides are linked together, sharing the same linking phosphorus configuration (e.g., for chiral linking phosphorus, all are Rp or all are Sp). In some embodiments, this disclosure provides a chiral-controlled oligonucleotide composition, wherein at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in the composition, or all oligonucleotides having the same base sequence, or all oligonucleotides having the same base sequence and sugar and base modifications, or all oligonucleotides having the same composition, are one or more forms of oligonucleotides (e.g., acidic forms, salt forms (e.g., pharmaceutically acceptable salt forms; other salt forms of the corresponding acidic or basic forms of oligonucleotides, as understood by those skilled in the art, where the oligonucleotide is a salt) etc.).

[0049] In some embodiments, as shown herein, chiral-controlled oligonucleotide compositions offer numerous advantages over corresponding stereorandom oligonucleotide compositions, such as higher stability, activity, etc. In some embodiments, it has been observed that chiral-controlled oligonucleotide compositions provide high levels of adenosine modification (e.g., conversion of A to I) activity when employing various isoforms of the ADAR protein (e.g., p150 and p110 forms of ADAR1), while corresponding stereorandom compositions only provide high levels of adenosine modification (e.g., conversion of A to I) activity when employing certain isoforms of the ADAR protein (e.g., p150 isoform of ADAR1).

[0050] In some embodiments, the provided oligonucleotide includes an additional portion, such as a targeting portion, a carbohydrate portion, etc. In some embodiments, the additional portion is or includes a ligand for a desialylate glycoprotein receptor. In some embodiments, the additional portion is or includes GalNAc or a derivative thereof. In particular, the additional portion may facilitate delivery to certain target sites, such as cells, tissues, organs, etc. (e.g., sites containing receptors that interact with the additional portion). In some embodiments, the additional portion facilitates delivery to the liver.

[0051] In some embodiments, this disclosure provides techniques for preparing oligonucleotides and compositions thereof, particularly chiral-controlled oligonucleotide compositions. In some embodiments, the provided oligonucleotides and compositions thereof are of high purity. In some embodiments, the oligonucleotides of this disclosure are stereochemically pure at the linking phosphorus sites between chiral nucleotides, being at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the oligonucleotides of this disclosure are stereoselectively prepared and substantially free of stereoisomers. In some embodiments, in a provided composition comprising a plurality of oligonucleotides sharing the same base sequence with a shared chiral linking phosphorus stereochemistry (e.g., comprising one or more of Rp and / or Sp, wherein each chiral linking phosphorus is independently Rp or Sp), at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition sharing the same base sequence with the plurality of oligonucleotides share the same chiral linking phosphorus stereochemistry pattern or the plurality of oligonucleotides. In some embodiments, in the provided composition comprising a plurality of oligonucleotides sharing the same base sequence with the same chiral phosphorus stereochemical pattern, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition sharing the same composition with the plurality of oligonucleotides share the same chiral phosphorus stereochemical pattern or the plurality of oligonucleotides.

[0052] In some embodiments, this disclosure describes useful techniques for evaluating oligonucleotides and compositions thereof. For example, various techniques of this disclosure can be used to evaluate adenosine modifications. As will be understood by those skilled in the art, in some embodiments, adenosine modification / editing can be assessed by sequencing, mass spectrometry, evaluation of the products (e.g., RNA, protein, etc.) of the modified nucleic acid (e.g., where the adenosine of the target nucleic acid is converted to inosine), optionally taking into account other components present in the modification system (e.g., in vitro system, ex vivo system, cell, tissue, organ, organism, subject, etc.) (e.g., ADAR protein). Those skilled in the art will understand that oligonucleotides providing adenosine modification of the target nucleic acid can also provide modified nucleic acids (e.g., where the target adenosine is converted to I) and one or more of their products (e.g., mRNA, protein, etc.). Some useful techniques are described in the examples.

[0053] As described herein, the oligonucleotides and compositions disclosed herein can be provided / utilized in various forms. In some embodiments, this disclosure provides compositions comprising one or more forms of oligonucleotides, such as acidic forms (e.g., where native phosphate esters are bonded in the form of –O(P(O)(OH)-O- and thiophosphate nucleotides are bonded in the form of –O(P(O)(SH)-O-), base forms, and salt forms (e.g., where native phosphate esters are bonded in the form of salts (e.g., sodium salts –O(P(O)(O)-O-). - Na + (-O-) exists, and the thiophosphate nucleotides are linked in salt form (e.g., sodium salt (–O(P(O)(S))). - Na + Oligonucleotides can exist in various salt forms, including pharmaceutically acceptable salts, as will be understood by those skilled in the art, and in solutions (e.g., various aqueous buffer systems), cations can dissociate from anions. In some embodiments, this disclosure provides pharmaceutical compositions comprising the provided oligonucleotide and / or one or more pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is a chiral-controlled oligonucleotide composition.

[0054] The provided technology can be used for a variety of purposes. For example, those skilled in the art will understand that the provided technology can be used for many purposes involving adenosine modification, such as correcting G to A mutations, regulating the levels of certain nucleic acids and / or products encoded by them (e.g., reducing protein levels by introducing A to G / I modifications), regulating splicing, regulating translation (e.g., regulating translation initiation and / or termination sites by introducing A to G / I modifications), regulating RNA / protein interactions, etc.

[0055] In some embodiments, this disclosure provides techniques for preventing or treating conditions, disorders, or diseases suitable for adenosine modification (e.g., converting A to I or G). As understood by those skilled in the art, I can perform one or more functions of G, such as base pairing, translation, etc. In some embodiments, G-to-A mutations can be corrected by A-to-I conversion, enabling the production of one or more products, such as proteins, of nucleic acids in the form of G. In some embodiments, this disclosure provides techniques for preventing or treating conditions, disorders, or diseases associated with mutations, including administering a provided oligonucleotide or composition thereof to a subject susceptible to or suffering from the condition, disorder, or disease, the oligonucleotide or composition being capable of editing the mutation. In some embodiments, this disclosure provides techniques for preventing or treating conditions, disorders, or diseases associated with G-to-A mutations, including administering a provided oligonucleotide or composition thereof to a subject susceptible to or suffering from the condition, disorder, or disease, the oligonucleotide or composition being capable of modifying A. In some embodiments, the provided techniques modify A in transcripts (e.g., RNA transcripts). In some embodiments, A is converted to I. In some embodiments, during translation, a protein synthesis machine reads I as G. In some embodiments, the A form encodes one or more proteins having one or more higher desired activities and / or one or more better desired properties compared to proteins encoded by their corresponding G forms. In some embodiments, the A form provides a higher level of one or more proteins having one or more higher desired activities and / or one or more better desired properties compared to their corresponding G forms. In some embodiments, the product encoded by the A form is structurally different from the product encoded by its corresponding G form (e.g., longer, in some embodiments a full-length protein). In some embodiments, the A form provides a structurally identical product (e.g., a protein) compared to its corresponding G form.

[0056] In some embodiments, this disclosure provides techniques for modulating RNA / protein interactions by editing target adenosine, for example, using oligonucleotides and compositions described herein. In some embodiments, this disclosure provides techniques for modulating the levels of RNA and / or products encoded therewith by, for example, editing target adenosine. In some embodiments, this disclosure provides techniques for increasing the levels of RNA and / or products encoded therewith (e.g., peptides) by, for example, editing target adenosine. In some embodiments, target adenosine is present in an RNA motif, such as an AU-enriched element (ARE) motif. In some embodiments, the RNA motif is or contains a regulatory element. In some embodiments, the RNA motif is or contains a cis-regulatory element. In some embodiments, the motif, such as the ARE motif, contains one or more AUUUAs within A and U-enriched regions. In some embodiments, the ARE motif contains an AUUUA core sequence within a U-enriched sequence (e.g., WWWU(AUUUA)UUUW, where W is A or U). In some embodiments, the AUUUA element is repeated. In some embodiments, the ARE motif contains dispersed AUUUA motifs within or near U-enriched regions. In some embodiments, the ARE motif contains overlapping AUUUA motifs within or near U-enriched regions. In some embodiments, the ARE motif comprises one or more of WUUUW, WWUUUWW, WWWUUUWWW, WWWWUUUWWWW, WWWWWUUUWWWWW and / or AWUAAA, where W is U or A. ARE motifs can have different lengths. In some embodiments, the ARE motif is or comprises a sequence of about 50-150 bases. As understood by those skilled in the art, various RNA motifs, such as the ARE motif, can facilitate the binding of various RNA-binding peptides or proteins. In some embodiments, editing adenosine in RNA regulates the properties, structure, function, etc., of RNA. In some embodiments, editing adenosine in RNA regulates RNA processing, stability, transport, etc. In some embodiments, editing adenosine in RNA regulates the interaction of RNA with other entities (e.g., RNA-binding peptides, such as ARE-binding peptides, miRNAs, etc.). Many peptides (e.g., HuA, HuB, HuC, HuD, HuR, etc.) have been reported to bind to ARE motifs and stabilize mRNA, while others (e.g., AUF1, TTP, BRF1, TIA-1, TIAR, KSRP, etc.) have been reported to bind to ARE motifs and destabilize mRNA. In some embodiments, adenosine editing enhances binding to ARE motifs that stabilize mRNA. In some embodiments, adenosine editing reduces binding to ARE motifs that destabilize mRNA. In some embodiments, adenosine editing enhances binding to ARE motifs that stabilize mRNA and reduces binding to ARE motifs that destabilize mRNA.In some embodiments, adenosine editing reduces binding to ARE motifs that stabilize mRNA. In some embodiments, adenosine editing enhances binding to ARE motifs that destabilize mRNA. In some embodiments, adenosine editing reduces binding to ARE motifs that stabilize mRNA and enhances binding to ARE motifs that destabilize mRNA. In some embodiments, editing adenosine in RNA regulates RNA levels. In some embodiments, editing adenosine in RNA regulates levels of products encoded by it (e.g., peptides). In some embodiments, editing adenosine in RNA increases RNA levels. In some embodiments, editing adenosine in RNA increases levels of products encoded by it (e.g., peptides). In some embodiments, RNA is a transcript. In some embodiments, RNA is mRNA. In some embodiments, adenosine is in a UTR region. In some embodiments, adenosine is in a 5'-UTR region. In some embodiments, adenosine is in a 3'-UTR region. In some embodiments, adenosine is in an RNA motif. In some embodiments, the RNA motif is an ARE motif. In some embodiments, this disclosure provides techniques for preventing or treating conditions, disorders, or diseases associated with levels of transcripts and / or products encoded therein. These techniques include administering a provided oligonucleotide or composition thereof to a subject susceptible to or suffering from a condition, disorder, or disease, the oligonucleotide or composition of which may modify a target adenosine in the transcript. In some embodiments, this disclosure provides techniques for preventing or treating conditions, disorders, or diseases, these techniques include administering a provided oligonucleotide or composition thereof to a subject susceptible to or suffering from a condition, disorder, or disease, the oligonucleotide or composition of which may modify a target adenosine in RNA, thereby regulating (e.g., increasing) the levels of RNA and / or products encoded therein. In some embodiments, the RNA is a transcript. In some embodiments, the transcript is a PKD1 transcript, such as PKD1 mRNA. In some embodiments, the product encoded therein is a PKD1 polypeptide, such as PKD1 protein. In some embodiments, the transcript is an LDLR transcript, such as LDLR mRNA. In some embodiments, the product encoded therein is an LDLR polypeptide, such as LDLR protein. In some embodiments, the transcript is a KLOTHO transcript, such as KLOTHO mRNA. In some embodiments, the product encoded therefrom is a KLOTHO polypeptide, such as KLOTHO protein. In some embodiments, the transcript is a CTLA4 transcript, such as CTLA4 mRNA. In some embodiments, the product encoded therefrom is a CTLA4 polypeptide, such as CTLA4 protein. In some embodiments, the transcript is an LCAT transcript, such as LCAT mRNA. In some embodiments, the product encoded therefrom is an LCAT polypeptide, such as LCAT protein. In some embodiments, the target adenosine is in the RNA motif. In some embodiments, the RNA motif is an ARE motif.In some embodiments, the RNA motif is in the PKD1 transcript, for example, in the 5' UTR of PKD1 mRNA. In some embodiments, the RNA motif is in the PKD1 transcript, for example, in the 3' UTR of PKD1 mRNA. In some embodiments, the RNA motif is in the LDLR transcript, for example, in the 5' UTR of LDLR mRNA. In some embodiments, the RNA motif is in the LDLR transcript, for example, in the 3' UTR of LDLR mRNA. In some embodiments, the RNA motif is in the KLOTHO transcript, for example, in the 5' UTR of KLOTHO mRNA. In some embodiments, the RNA motif is in the KLOTHO transcript, for example, in the 3' UTR of KLOTHO mRNA. In some embodiments, the RNA motif is in the CTLA4 transcript, for example, in the 5' UTR of CTLA4 mRNA. In some embodiments, the RNA motif is in the CTLA4 transcript, for example, in the 3' UTR of CTLA4 mRNA. In some embodiments, the RNA motif is in the LCAT transcript, for example, in the 5' UTR of the LCAT mRNA. In some embodiments, the RNA motif is in the LCAT transcript, for example, in the 3' UTR of the LCAT mRNA. Various target adenosines and targeting regions are described herein, such as those targeted by oligonucleotides in the relevant tables.

[0057] As those skilled in the art will understand, many conditions, disorders, or diseases are associated with mutations that can be modified by the provided techniques and can be prevented and / or treated using the provided techniques. For example, more than 20,000 conditions, disorders, or diseases have been reported to be associated with G to A mutations and can benefit from A to I editing. Attached Figure Description

[0058] Figure 1 The provided technology enables the editing of target transcripts. Oligonucleotides containing various modifications such as base modifications (e.g., [3nU]), linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, etc.), and stereochemistry and their patterns were designed and evaluated. Primary mouse hepatocytes were nakedly administered with (a) 3 uM or (b) 0.3 uM oligonucleotides targeting various target adenosines in UGP2. Cells were harvested after 96 hours, and RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent the standard error of the mean (SEM).

[0059] Figure 2The provided technology enables the editing of target transcripts. Oligonucleotides were designed and evaluated to incorporate various modifications such as base modifications (e.g., [3nU]), linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, etc.), and stereochemistry and their patterns. Primary mouse hepatocytes were administered naked with an indicator oligonucleotide targeting UGP2 at indicated concentrations. All oligonucleotides were GalNAc conjugated. Cells were harvested after 72 hours, and RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent SEM.

[0060] Figure 3 The provided technology enables in vivo editing of target transcripts. Oligonucleotides containing various modifications such as base modifications (e.g., [3nU]), linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, etc.), and stereochemistry and their patterns were designed and evaluated. Transgenic hADAR-p110 mice were subcutaneously administered 10 mg / kg on days 0, 2, and 4, using an indicator oligonucleotide composition targeting UGP2 or ACTB or PBS as a negative control. All oligonucleotides were GalNAc conjugated. On day 7, the animals were sacrificed and the livers were collected. RNA was isolated from the livers and transcribed into cDNA. (a) Quantification of editing was performed by Sanger sequencing. (b) Quantification of the accumulation of indicator oligonucleotides in the livers. Error bars represent SEM. N=5 / group.

[0061] Figure 4 The provided technology enables the editing of target transcripts. Oligonucleotides containing various modifications, such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe, homologous DNA sugars, etc.), and stereochemistry and their patterns, are designed and evaluated. Primary human hepatocytes are nakedly administered with 1 uM of an indicator oligonucleotide targeting UGP2. Cells are harvested after 48 hours, and RNA is collected and transcribed into cDNA. Editing is quantified by Sanger sequencing. Error bars represent SEM.

[0062] Figure 5The provided technology enables the editing of target transcripts. Oligonucleotides containing various modifications, such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, LNA sugars, etc.), and stereochemistry and their patterns, are designed and evaluated. Primary human hepatocytes are administered naked with 1 uM of an indicator oligonucleotide targeting UGP2. Cells are harvested after 48 hours, and RNA is collected and transcribed into cDNA. Editing is quantified by Sanger sequencing. Error bars represent SEM.

[0063] Figure 6 The provided technology enables the editing of target transcripts. Oligonucleotides containing various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, etc.), and stereochemistry and their patterns were designed and evaluated. Primary human hepatocytes were nakedly administered with 1 uM of an indicator oligonucleotide targeting UGP2. Cells were harvested after 48 hours, and RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent SEM. This indicates that no data exists.

[0064] Figure 7 The provided technology enables editing. Oligonucleotides were designed and evaluated to incorporate various modifications such as base modifications (e.g., [3nU], [isoG], [c7A]), linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, LNA, etc.) and stereochemistry and their patterns. Primary human hepatocytes were administered naked with indicator oligonucleotides targeting PAH at indicated concentrations (0.125 uM, 0.042 uM, 0.014 uM, 0.005 uM). All oligonucleotides were GalNAc conjugated. Cells were harvested after 48 hours, and RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent SEM. N=2. Two-way ANOVA with multiple comparison correction (Dunnett) was performed relative to the control oligonucleotide (ADR-0107337). p ≤ 0.05; p ≤ 0.01; p ≤ 0.001; p ≤ 0.0001.

[0065] Figure 8 The provided technology enables editing. Oligonucleotides were designed and evaluated to incorporate various modifications such as base modifications (e.g., [3nU], [isoG], [c7A]), linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, LNA, etc.) and stereochemistry and their patterns. Primary human hepatocytes were administered naked with indicator oligonucleotides targeting PAH at indicated concentrations (0.125 uM, 0.042 uM, 0.014 uM, 0.005 uM). All oligonucleotides were GalNAc conjugated. Cells were harvested after 48 hours, and RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent SEM. N=2. Two-way ANOVA with multiple comparison correction (Dunnett) was performed relative to the control oligonucleotide (ADR-0107333). p ≤ 0.05; p ≤ 0.01; p ≤ 0.001; p ≤ 0.0001.

[0066] Figure 9 The provided technology enables editing. Oligonucleotides were designed and evaluated to incorporate various modifications such as base modifications (e.g., [3nU]), linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe, homologous DNA), and stereochemistry and their patterns. Primary human hepatocytes were administered naked with indicator oligonucleotides targeting PAHs at indicated concentrations. All oligonucleotides were GalNAc conjugated. Cells were harvested after 48 hours, and RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent SEM. N=2.

[0067] Figure 10The provided technology enables editing. Oligonucleotides were designed and evaluated to incorporate various modifications such as base modifications (e.g., [3nU]), linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe, homologous DNA), and stereochemistry and their patterns. Primary human hepatocytes were administered naked with indicator oligonucleotides targeting PAHs at indicated concentrations. All oligonucleotides were GalNAc conjugated. Cells were harvested after 48 hours, and RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent SEM. N=2.

[0068] Figure 11 The provided technology enables in vivo editing. Oligonucleotides containing various modifications such as base modifications (e.g., [3nU]), linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001) etc.), sugar modifications (e.g., 2'-F, 2'-OMe), and stereochemistry and their patterns were designed and evaluated. C57BL / 6 mice or transgenic huADAR-p110 mice were subcutaneously administered 10 mg / kg on days 0, 2, and 4, using PAH-targeting indicator oligonucleotides or PBS as negative controls. All oligonucleotides were GalNAc conjugated. On day 7, animals were sacrificed and livers were collected. RNA was isolated from the livers and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent SEM. N = 4 mice / group. Two-way ANOVA with multiple comparison correction (Dunnett) was performed. ns, not significant; p ≤ 0.0001.

[0069] Figure 12 The provided technology enables editing. Oligonucleotides containing various modifications such as base modifications (e.g., [3nU]), linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, 2'-MOE), and stereochemistry and their patterns were designed and evaluated. HEK293T cells were transfected with plasmids expressing human ADAR1-p110 and PAH R261Q. Cells were then reverse-transfected with 25 nM of PAH-targeting indicator oligonucleotides. Cells were harvested after 48 hours, and RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent SEM. N=3.

[0070] Figure 13The provided technology can enable the editing of target transcripts and the upregulation of peptides encoded by them. Oligonucleotides containing various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns were designed and evaluated. Primary human hepatocytes were nakedly administered with 10 uM of an indicator oligonucleotide targeting LDLR. Cells were harvested after 48 hours. (a) RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. (b) Proteins were collected and quantified by Bradford assay (for total protein) and LDLR ELISA. Error bars represent standard deviations. For (a), N=4; for (b), N=14. NT = simulation treated with PBS.

[0071] Figure 14 The provided technology can provide upregulation of target transcripts. Oligonucleotides containing various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns were designed and evaluated. Primary human renal tubular epithelial cells were transfected with 100 nM of the PKD1-targeting indicator oligonucleotide. Cells were harvested and RNA collected after 48 hours. The fold change in PKD1 mRNA levels was quantified by qPCR. Error bars represent standard deviation. N=4. NTC = Treatment with a control oligonucleotide that does not target PKD1.

[0072] Figure 15 The provided technology enables the editing of target transcripts. Oligonucleotides containing various modifications, such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns, were designed and evaluated. HEK293T cells were transfected with plasmids encoding ADAR-p110 and CFTR (WT, G542X, or W1282X). After 24 hours, cells were transfected with 25 nM of an indicator oligonucleotide targeting CFTR or UGP2 (control; ADR-0102506; showing data for UGP2 editing). After another 48 hours, cells were harvested, and RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent standard deviation. N=2.

[0073] Figure 16The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the composition shown at a 10 μM dose via naked uptake, as illustrated. After 5 days of treatment (n=2 biological replicates), A to G edits were measured via amplicon sequencing (as understood by those skilled in the art, inosine is read as G in many biological contexts). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0074] Figure 17 The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0075] Figure 18 The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, homologous DNA, arabinose), base modifications (e.g., [3nU]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0076] Figure 19The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, homologous DNA, arabinose), base modifications (e.g., [3nU]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0077] Figure 20 The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0078] Figure 21 The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, homologous DNA, FANA), base modifications (e.g., [3nU]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0079] Figure 22The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, homologous DNA, FANA), base modifications (e.g., [3nU]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0080] Figure 23 The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0081] Figure 24 The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0082] Figure 25The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, acyclic sugars), base modifications (e.g., [3nU], [mi5C]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0083] Figure 26 The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU], hypoxanthine), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0084] Figure 27 The provided technology can enable the editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe, 2'-MOE), base modifications (e.g., [3nU], hypoxanthine), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the composition shown at a 10 μM dose via naked uptake, as indicated. A- to G editing (n = 2 biological replicates) was measured by amplicon sequencing 5 days after treatment. (Indicates data not included). The X-axis represents oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean. NT = Untested.

[0085] Figure 28The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to incorporate various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, 2'-MOE), base modifications (e.g., [3nU]), and stereochemistry and their patterns. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Patient-derived (MECP2) R168X Cortical neurons were treated with the indicated composition at a 10 μM dose via naked uptake, as shown. Editing from A to G was measured by amplicon sequencing 5 days after treatment (n = 2 biological replicates). The X-axis represents the oligonucleotide composition, while the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0086] Figure 29 The provided technology can enable the editing of target transcripts. Oligonucleotides containing various modifications, such as bonding modifications (e.g., PS, PN (e.g., phosphorylguanidine bonding, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns, are designed and evaluated. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence, with the control oligonucleotide composition (ADR-0102535) targeting the UGP2 coding sequence. From hMECP2 R168X Primary cortical neurons isolated from knock-in mice were treated with an indicated dose of the naked uptake of the indicated composition. Five days after treatment, A to G edits at the MECP2 target site were measured by amplicon sequencing (n=2 biological replicates). The X-axis represents the dose (µM), and the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0087] Figure 30 The provided technology enables the editing of target transcripts and the expression of the MECP2 protein. Oligonucleotides containing various modifications, such as bonding modifications (e.g., PS, PN (e.g., phosphorylguanidine bonding, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns, are designed and evaluated. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence, with the control oligonucleotide composition (ADR-0102535) targeting the UGP2 coding sequence. [The text then abruptly shifts to a description of hMECP2, mentioning its inclusion in the original text.] R168X Knock-in mouse primary neurons were treated with a 30 uM naked uptake indicator composition. Fluorescence microscopy imaging showed cellular expression of the MECP2 protein.

[0088] Figure 31The provided technology can enable the editing of target transcripts. Oligonucleotides containing various modifications, such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns, are designed and evaluated. All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence, using a control oligonucleotide composition (ADR-0102535). [The text abruptly ends here, likely due to an incomplete sentence or missing information.] R168X Primary mouse neurons with knock-in and hADAR P110 were treated with an indicated dose of the indicated composition via naked uptake. A- to G edits (n = 2 biological replicates) were measured by amplicon sequencing 5 days after treatment. The X-axis represents oligonucleotide composition, and the Y-axis represents the percentage of edits. Error bars represent the standard deviation of the mean.

[0089] Figure 32 The provided technology can enable the editing of target transcripts. Oligonucleotides containing various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns, including sugar modifications at the N0 position (e.g., [m1d], [c1C], [25d3r], [ld], [Ald], [23tfu], and / or [dma6]). Primary mouse hepatocytes are administered naked with an indicated concentration of the indicator oligonucleotide targeting UGP2.

[0090] Figure 33 The provided technology can enable the editing of target transcripts. It designs and evaluates various modifications, including bonded modifications (e.g., PS, PN (e.g., phosphorylguanidine bonds, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns, including N... -1 or N +1 Oligonucleotides with positional sugar modifications (e.g., homologous DNA, [B5thpyr], [R3thpyr], [oxa], [m1d], [c1C], [Ld], [ALd], [23tfu]) and / or base modifications (e.g., [isoC], [mi5C], [purine]). Primary mouse hepatocytes were administered naked with an indicated concentration of the indicator oligonucleotide targeting UGP2.

[0091] Figure 34The provided technology can enable the editing of target transcripts. Oligonucleotides containing various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, LNA), base modifications (e.g., [3nU]), and stereochemistry and their patterns are designed and evaluated. Primary mouse hepatocytes are administered naked with an indicated concentration of the indicator oligonucleotide targeting UGP2.

[0092] Figure 35 The provided technology can provide editing of target transcripts. Oligonucleotides are designed and evaluated to include various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe), base modifications (e.g., [3nU]), and stereochemistry and their patterns, including sugars at the N0 position (e.g., native DNA sugars), sugar modifications (e.g., 2'-OMe), and / or base modifications (e.g., [n3U]). All oligonucleotide compositions target the early TGA stop codon within the MECP2 coding sequence. Primary mouse MECP2 is targeted with indicator oligonucleotides at indicated concentrations. R168X Neurons were administered the drug naked.

[0093] Figure 36 The provided technology can enable the editing of target transcripts. Oligonucleotides containing various modifications, such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, [ld]), base modifications (e.g., [3nU]), and stereochemistry and their patterns, are designed and evaluated. Naked administration of 3 uM of the CFTR-targeting indicator oligonucleotide to human bronchial epithelial cells expressing the CFTR-W1282X allele is performed.

[0094] Figure 37 The provided technology can enable the editing of target transcripts. Oligonucleotides containing various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, [ld]), base modifications (e.g., [3nU]), and stereochemistry and their patterns are designed and evaluated. Primary mouse hepatocytes are administered naked with 0.1 uM of the indicator oligonucleotide targeting UGP2.

[0095] Figure 38 The provided technology can enable the editing of target transcripts. It designs and evaluates various modifications, including bonded modifications (e.g., PS, PN (e.g., phosphorylguanidine bonds, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe, [ld]), base modifications (e.g., [3nU]), and stereochemistry and their patterns, including N...-1 , N0 or N +1 Oligonucleotides with positional sugar modifications (e.g., [ld]) and / or base modifications (e.g., [n3U]). Primary mouse hepatocytes were administered naked with 0.1 uM of the indicator oligonucleotide targeting UGP2.

[0096] Figure 39 The provided technology can enable the editing of target transcripts. Oligonucleotides are designed and evaluated to include various modifications such as linking modifications (e.g., PS, PN (e.g., phosphorylguanidine linking, such as n001)), sugar modifications (e.g., 2'-F, 2'-OMe, [ld]), base modifications (e.g., [3nU]), and stereochemistry and their patterns, including sugars at the N0 position (e.g., native DNA sugars), sugar modifications (e.g., 2'-F, 2'-OMe, LNA), and / or base modifications (e.g., [n3U]). Primary mouse hepatocytes are administered naked with 0.3 uM of the indicator oligonucleotide targeting UGP2.

[0097] Figure 40 The provided technology can enable the editing of target transcripts. It designs and evaluates various modifications, including bond modifications (e.g., PS, PN (e.g., phosphoryl guanidine bonds, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe, [ld]), base modifications (e.g., [3nU]), and stereochemistry and their patterns, including N... -1 , N0 or N +1 Oligonucleotides with positional sugar modifications (e.g., [s4d], [As4d], [Lh23pyr], [S6Lh23pyr], [Lthpyr], [thpyr], [h23pyr]) and / or base modifications (e.g., [n3U]). Primary mouse hepatocytes were administered naked with 0.3 uM of the indicator oligonucleotide targeting UGP2.

[0098] Figure 41 The technology provided allows for in vivo editing. (Transgenic hMECP2) R168X hADAR1-p110 mice and transgenic male hADAR1-p110 (MECP2+) / y Mice were treated with a 10 μg ICV injection containing a composition of ADR-0105153. Cortical, hippocampal, and brainstem tissues were collected six weeks after injection, and the percentage of MECP2 editing was assessed.

[0099] Figure 42 The technology provided allows for in vivo editing. (Transgenic hMECP2) R168X hADAR1-p110 mice and transgenic hADAR1-p110 males (MECP2)+ / y ) and females (MECP2) + / x Mice were treated with a 10 μg ICV injection containing a composition of ADR-0105153. Brain tissue was collected six weeks after injection, and the percentage of MECP2 protein expression in the whole brain was assessed (left panel). The number of cells expressing MECP2 was also measured (right panel).

[0100] Figure 43 The technology provided allows for in vivo editing. (Transgenic hMECP2) R168X hADAR1-p110 mice were treated with 10 μg ICV injection of a composition containing ADR-0105153. Six weeks post-injection, tissues from the cortex, striatum, hippocampus, thalamus and hypothalamus, midbrain, cerebellum and pons and medulla oblongata were collected, and the percentage of MECP2 protein expression was assessed. The number of cells expressing MECP2 was also measured.

[0101] Figure 44 The technology provided allows for in vivo editing. (Transgenic hMECP2) R168X hADAR1-p110 mice and transgenic male hADAR1-p110 (MECP2+) / y Mice were treated with a composition containing ADR-0105153 by injection of 10 μg ICV. Mice were weighed at 28 days and 42 days of age (corresponding to six weeks after injection). Detailed Implementation

[0102] The technology of this disclosure can be more readily understood by referring to the following detailed description of certain embodiments.

[0103] definition

[0104] Unless otherwise indicated, the following definitions shall apply as used herein. For the purposes of this disclosure, chemical elements are identified according to the periodic table, CAS version, Handbook of Chemistry and Physics, 75th edition. Furthermore, the general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5th edition, edited by Smith, MB, and March, J., John Wiley & Sons, New York: 2001.

[0105] As used herein, unless the context clearly specifies otherwise, in this disclosure, (i) the terms “a” or “an” are to be understood as “at least one”; (ii) the term “or” is to be understood as “and / or”; (iii) the terms “comprising” and “comprise” (whether or not used with “but not limited to”) are to be understood as covering each of the components or steps listed individually or together with one or more other components or steps; (iv) the term “another” is to be understood as at least another / second or more; (v) the terms “about” and “approximately” are to be understood as allowing standard deviation, as would be understood by one of ordinary skill in the art; and (vi) wherever a range is provided, the endpoints are included.

[0106] Unless otherwise stated, the description of oligonucleotides and their elements (e.g., base sequence, sugar modification, internucleotide linkages, linkage phosphorus stereochemistry, their patterns, etc.) is carried out from 5' to 3'. As those skilled in the art will understand, in some embodiments, oligonucleotides may be provided and / or used as salt forms, particularly pharmaceutically acceptable salt forms (e.g., sodium salts). As those skilled in the art will also understand, in some embodiments, individual oligonucleotides in a composition may be considered to have the same composition and / or structure, even in such compositions (e.g., liquid compositions), particularly, such oligonucleotides may be in different one or more salt forms at a given time (and, for example, in liquid compositions, they may be soluble and the oligonucleotide chain may be present in anionic form). For example, those skilled in the art will understand that, at a given pH, internucleotide linkages along an oligonucleotide chain may be in acid (H) form or one of a variety of possible salt forms (e.g., sodium salts or salts of different cations, depending on which ions may be present in the preparation or composition), and will understand that as long as their acid form (e.g., with H) is acceptable, + If all cations (if any) have the same composition and / or structure, such a single oligonucleotide can be properly considered to have the same composition and / or structure.

[0107] Aliphatic: As used herein, "aliphatic" means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated (but non-aromatic) units, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more unsaturated (but non-aromatic) units, or a combination thereof. In some embodiments, the aliphatic group contains 1-50 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-20 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-9 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-8 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-7 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms. In still other embodiments, the aliphatic group contains 1-5 aliphatic carbon atoms, and in yet another embodiment, the aliphatic group contains 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, straight-chain or branched substituted or unsubstituted alkyl, alkenyl, alkynyl groups and mixtures thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0108] Alkenyl: As used herein, the term “alkenyl” refers to an aliphatic group as defined herein that has one or more double bonds.

[0109] Alkyl: As used herein, the term "alkyl" has its common meaning in the art and may comprise a saturated aliphatic group, including straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic group), alkyl-substituted cycloalkyl, and cycloalkyl-substituted alkyl. In some embodiments, the alkyl group has 1-100 carbon atoms. In some embodiments, the straight-chain or branched alkyl group has about 1-20 carbon atoms in its main chain (e.g., for a straight chain of C1-C1). 20 For branches of C2-C 20 ), and alternatively about 1-10. In some embodiments, the cycloalkyl ring has about 3-10 carbon atoms in its ring structure when such a ring is monocyclic, bicyclic or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, the alkyl group may be a lower alkyl group, wherein the lower alkyl group comprises 1-4 carbon atoms (e.g., C1-C4 for straight-chain lower alkyl groups).

[0110] Alkyne group: As used herein, the term "alkynyl group" refers to an aliphatic group as defined herein that has one or more triple bonds.

[0111] Analog: The term "analog" includes any chemical part that is structurally different from a reference chemical part or class of parts, but is capable of performing at least one function of such a reference chemical part or class of parts. As non-limiting examples, nucleotide analogs are structurally different from nucleotides but perform at least one function of nucleotides; nucleobase analogs are structurally different from nucleosides but perform at least one function of nucleosides; and so on.

[0112] Animal: As used herein, the term "animal" means any member of the animal kingdom. In some embodiments, "animal" means a human being at any developmental stage. In some embodiments, "animal" means a non-human animal at any developmental stage. In some embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, an animal includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0113] Aryl: As used herein, the term "aryl" alone or as part of a larger portion of "aralkyl," "ararylalkoxy," or "aryloxyalkyl" refers to a monocyclic, bicyclic, or polycyclic system having a total of five to thirty ring members, wherein at least one ring in the system is aromatic. In some embodiments, an aryl is a monocyclic, bicyclic, or polycyclic cyclic system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, each monocyclic unit is aromatic. In some embodiments, an aryl is a biaryl. The term "aryl" may be used interchangeably with the term "aryl ring." In some embodiments of this disclosure, "aryl" refers to an aromatic cyclic system including, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracene, etc., which may have one or more substituents. As used herein, the term "aryl" also includes groups fused with an aromatic ring to one or more non-aromatic rings, such as indanyl, phthalimide, naphthalimide, phenanthridine, or tetrahydronaphthyl.

[0114] Characteristic portion: As used herein, the term "characteristic portion" in the broadest sense refers to a portion of a substance whose presence (or absence) is related to the presence (or absence) of a particular characteristic, property, or activity of that substance. In some embodiments, a characteristic portion of a substance is a portion found in the substance and in related substances that share the particular characteristic, property, or activity, but not in those substances that do not share the particular characteristic, property, or activity. In some embodiments, the characteristic portion shares at least one functional property with the whole substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is a portion containing a continuous segment or a collection of continuous segments of amino acids that together are characteristic of the protein or polypeptide. In some embodiments, each such continuous segment typically contains at least 2, 5, 10, 15, 20, 50, or more amino acids. Typically, a characteristic portion of a substance (e.g., a characteristic portion of a protein, antibody, etc.) is a portion that, in addition to the sequence and / or structural identity specified above, shares at least one functional property with the associated whole substance. In some embodiments, the characteristic portion may be biologically active.

[0115] Chiral control: As used herein, “chiral control” refers to the control of the stereochemical name of the chiral phosphorus in the chiral internucleotide link within an oligonucleotide. As used herein, a chiral internucleotide link is an internucleotide link in which the linking phosphorus is chiral. In some embodiments, control is achieved by chiral elements not present in the sugar and base moieties of the oligonucleotide, for example, in some embodiments, by using one or more chiral auxiliaries during oligonucleotide preparation, which are typically part of a chiral phosphorusamide used during oligonucleotide preparation. In contrast to chiral control, those skilled in the art will recognize that if conventional oligonucleotide synthesis is used to form chiral internucleotide links, such conventional oligonucleotide synthesis without the use of chiral auxiliaries cannot control the stereochemistry at the chiral internucleotide link. In some embodiments, the stereochemical designation of each chiral phosphorus in each chiral internucleotide link within the oligonucleotide is controlled.

[0116] Chiral controlled oligonucleotide compositions: As used herein, the terms “chiral controlled oligonucleotide composition”, “chiral controlled nucleic acid composition”, etc., refer to compositions comprising multiple oligonucleotides (or nucleic acids) that share a common base sequence, wherein these multiple oligonucleotides (or nucleic acids) share the same bonded phosphorus stereochemistry (a chiral or stereodefined internucleotide bond in which the chiral phosphorus is Rp or Sp in the composition (“stereodefined”), rather than a random mixture of Rp and Sp as an achiral controlled internucleotide bond). In some embodiments, the chiral-controlled oligonucleotide composition comprises a plurality of oligonucleotides (or nucleic acids) that share: 1) a common base sequence, 2) a common backbone linking pattern, and 3) a common backbone phosphorus modification pattern, wherein the plurality of oligonucleotides (or nucleic acids) share the same linking phosphorus stereochemistry at one or more chiral nucleotide links (chiral-controlled or stereodefined nucleotide links whose chiral linking phosphorus is Rp or Sp in the composition (“stereodefined”), rather than a random mixture of Rp and Sp as in achiral-controlled nucleotide links). The levels of the plurality of oligonucleotides (or nucleic acids) in the chiral-controlled oligonucleotide composition are predetermined / controlled or enriched (e.g., stereoselectively forming one or more chiral nucleotide links by chiral-controlled oligonucleotide preparation) compared to random levels in achiral-controlled oligonucleotide compositions. In some embodiments, the chiral-controlled oligonucleotide composition comprises about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 5%, 10%, 20%, 3%) of all oligonucleotides. 0%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) are the oligonucleotides.In some embodiments, the chiral-controlled oligonucleotide composition comprises approximately 1%-100% (e.g., approximately 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%) of all oligonucleotides sharing a common base sequence, a common backbone bonding pattern, and a common backbone phosphorus modification pattern. Or approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) are the multiple oligonucleotides. In some embodiments, the level is approximately 1%-100% (e.g., approximately 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%) of all oligonucleotides in the composition; or all oligonucleotides in the composition that share a common base sequence (e.g., multiple oligonucleotides or a base sequence of one type of oligonucleotide); or all oligonucleotides in the composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linking type pattern, and / or a common internucleotide linking modification pattern. 0%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).In some embodiments, the plurality of oligonucleotides share the same stereochemistry at the bonding sites between about 1 to 50 (e.g., about 1 to 10, 1 to 20, 5 to 10, 5 to 20, 10 to 15, 10 to 20, 10 to 25, 10 to 30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral nucleotides. In some embodiments, the plurality of oligonucleotides are present in amounts ranging from about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, about 5%, 10%, 15%, 20%, 25%, 30%). The chiral nucleotides (35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) share the same stereochemistry at their inter-nucleotide linkages. In some embodiments, the multiple oligonucleotides (or nucleic acids) share the same sugar and / or nucleobase modification patterns. In some embodiments, the multiple oligonucleotides (or nucleic acids) are multiple forms of the same oligonucleotide (e.g., acids and / or multiple salts of the same oligonucleotide). In some embodiments, the multiple oligonucleotides (or nucleic acids) have the same composition. In some embodiments, the level of the plurality of oligonucleotides (or nucleic acids) is about 1%-100% of all oligonucleotides (or nucleic acids) in the composition having the same composition as the plurality of oligonucleotides (or nucleic acids) (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%). -90%, or approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).In some embodiments, each chiral nucleotide link is a chiral-controlled nucleotide link, and the composition is a fully chiral oligonucleotide composition. In some embodiments, the plurality of oligonucleotides (or nucleic acids) are structurally identical. In some embodiments, the chiral-controlled nucleotide links have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%, typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% diastereomeric purity. In some embodiments, the chiral-controlled nucleotide links have at least 95% diastereomeric purity. In some embodiments, the chiral-controlled nucleotide links have at least 96% diastereomeric purity. In some embodiments, the chiral-controlled nucleotide links have at least 97% diastereomeric purity. In some embodiments, the chiral-controlled nucleotide links have at least 98% diastereomeric purity. In some embodiments, the chiral-controlled internucleotide linkages have at least 99% diastereomeric purity. In some embodiments, the percentage of the level is or is at least (DS). nc , where DS is the diastereomeric purity as described in this disclosure (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or higher), and nc is the number of chiral-linked phosphorus groups as described in this disclosure (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, the percentage of the level is or is at least (DS). nc Where DS is the diastereomeric purity as described in this disclosure (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or higher), and nc is the number of chiral-controlled internucleotide bonds as described in this disclosure (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 5-50, 5-40, 5-30, 5-25, 5-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more). In some embodiments, the percentage of the level is or is at least (DS). nc Where DS is 95%-100%. For example, when DS is 99% and nc is 10, the percentage is or is at least 90% (99%). 10≈ 0.90 = 90%). In some embodiments, the level of the plurality of oligonucleotides in the composition is expressed as the product of the diastereomeric purity of each chiral-linked phosphorus. In some embodiments, the level of the plurality of oligonucleotides in the composition is expressed as the product of the diastereomeric purity of each chiral-controlled internucleotide linking strand in the oligonucleotide. In some embodiments, the diastereomeric purity of the internucleotide linking two nucleosides in the oligonucleotide (or nucleic acid) is expressed as the diastereomeric purity of the internucleotide linking dimer of the same two nucleosides, wherein comparable conditions are used, and in some cases, the same synthetic cycle conditions are used to prepare the dimer (e.g., for the linking of Nx and Ny in oligonucleotides.......NxNy......, the dimer is NxNy). In some embodiments, not all chiral internucleotide links are chiral-controlled internucleotide links, and the composition is a partially chiral-controlled oligonucleotide composition. In some embodiments, as commonly observed in stereorandom oligonucleotide compositions (e.g., from conventional oligonucleotide synthesis, such as phosphoramidite methods, as understood by those skilled in the art), the non-chiral controlled internucleotide linkages have diastereomeric purities of less than about 80%, 75%, 70%, 65%, 60%, 55%, or about 50%. In some embodiments, multiple oligonucleotides (or nucleic acids) have the same type. In some embodiments, the chiral controlled oligonucleotide composition comprises various oligonucleotide types or nucleic acid types at a non-random or controlled level. For example, in some embodiments, the chiral controlled oligonucleotide composition comprises one and no more than one oligonucleotide type. In some embodiments, the chiral controlled oligonucleotide composition comprises more than one oligonucleotide type. In some embodiments, the chiral controlled oligonucleotide composition comprises multiple oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide composition is a composition of oligonucleotides of one oligonucleotide type comprising multiple oligonucleotides of that oligonucleotide type at a non-random or controlled level.

[0117] Similarly, the term "comparable" is used herein to describe conditions or circumstances in which two groups (or more groups) are sufficiently similar to each other to allow for comparison of results obtained or observed phenomena. In some embodiments, a group of comparable conditions or circumstances is characterized by a plurality of substantially identical features and one or a few varying features. Those skilled in the art will understand that groups of conditions are comparable to each other when characterized by a sufficient number and type of substantially identical features to guarantee a reasonable conclusion that differences in results obtained or observed phenomena in different groups of conditions or circumstances are caused by or indicated by variations in those varying features.

[0118] Cycloaliphatic group: The terms “cycloaliphatic group,” “carbocyclic,” “carbocyclic group,” “carbocyclic,” and “carbocyclic” are used interchangeably and, as used herein, refer to a saturated or partially unsaturated but non-aromatic cycloaliphatic monocyclic, bicyclic, or polycyclic ring system as described herein, having 3 to 30 ring members unless otherwise specified. Cycloaliphatic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornel, adamantyl, and cyclooctadienyl. In some embodiments, the cycloaliphatic group has 3–6 carbons. In some embodiments, the cycloaliphatic group is saturated and is cycloalkyl. The term “cycloaliphatic” may also include an aliphatic ring fused with one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, the cycloaliphatic group is bicyclic. In some embodiments, the cycloaliphatic group is tricyclic. In some embodiments, the cycloaliphatic group is polycyclic. In some embodiments, "cycloaliphatic" refers to a fully saturated or non-aromatic C3-C6 monocyclic hydrocarbon or C8-C6 monocyclic hydrocarbon containing one or more unsaturated units. 10 Bicyclic or polycyclic hydrocarbons having a single attachment point extending to the rest of the molecule, or referring to C9-C hydrocarbons that are fully saturated or contain one or more unsaturated units but are not aromatic. 16 Polycyclic hydrocarbons have a single attachment point that extends to the rest of the molecule.

[0119] Heteroaliphatic group: As used herein, the term "heteroaliphatic group" has its common meaning in the art and refers to an aliphatic group as described herein in which one or more carbon atoms are independently replaced by one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In some embodiments, one or more units selected from C, CH, CH2, and CH3 are independently replaced by one or more heteroatoms (including their oxidized and / or substituted forms). In some embodiments, the heteroaliphatic group is a heteroalkyl group. In some embodiments, the heteroaliphatic group is a heteroalkenyl group.

[0120] Heteroalkyl: As used herein, the term “heteroalkyl” is given in its ordinary sense in the art and refers to an alkyl group in which one or more carbon atoms are independently replaced by one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

[0121] Heteroaryl: As used herein, the terms “heteroaryl” and “heteroaryl-”, used alone or as part of a larger portion such as “heteroarylalkyl” or “heteroarylalkoxy”, refer to a monocyclic, bicyclic, or polycyclic cyclic system having a total of five to thirty ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, a heteroaryl is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), having 5, 6, 9, or 10 ring atoms in some embodiments. In some embodiments, each monocyclic unit is aromatic. In some embodiments, a heteroaryl has 6, 10, or 14 π electrons shared in the cyclic array; and has one to five heteroatoms in addition to carbon atoms. Heteroaryl groups include, but are not limited to, thiophene, furanyl, pyrrole, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indoleazinyl, purine, naphridinyl, and pteridinyl. In some embodiments, the heteroaryl group is a heterobiaryl group, such as bipyridinyl. As used herein, the terms “heteroaryl” and “heteroaryl-” also include groups fused to one or more aryl, alicyclic, or heterocyclic rings, wherein the group or connecting point is located on the heteroaryl ring. Non-limiting examples include indolyl, isoindolyl, benzothiophenyl, benzofuranyl, dibenzofuranyl, indazoleyl, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, cyclolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinazinyl, carbazoleyl, acridineyl, phenazinyl, phenothiazinyl, phenotoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3–b]–1,4–oxazin–3(4H)–one. Heteroaryl groups can be monocyclic, bicyclic, or polycyclic. The term “heteroaryl” is used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which contains an optionally substituted ring. The term "heteroaryl" refers to an alkyl group substituted with a heteroaryl group, wherein the alkyl moiety and the heteroaryl moiety are optionally substituted independently.

[0122] Heteroatom: As used herein, the term "heteroatom" means an atom that is not carbon or hydrogen. In some embodiments, a heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or silicon (including oxidized forms of nitrogen, sulfur, phosphorus, or silicon; charged forms of nitrogen (e.g., quaternized forms, imine groups, etc.), phosphorus, sulfur, oxygen; etc.). In some embodiments, a heteroatom is silicon, phosphorus, oxygen, sulfur, or nitrogen. In some embodiments, a heteroatom is silicon, oxygen, sulfur, or nitrogen. In some embodiments, a heteroatom is oxygen, sulfur, or nitrogen.

[0123] Heterocycle: As used herein, the terms “heterocycle,” “heterocyclic group,” “heterocyclic group,” and “heterocycle” are used interchangeably as used herein and refer to a monocyclic, bicyclic, or polycyclic ring portion (e.g., 3 to 30 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, the heterocyclic group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic portion that is saturated or partially unsaturated and has one or more, preferably one to four, heteroatoms as defined above, in addition to a carbon atom. When used with respect to the ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, and nitrogen, nitrogen may be N (e.g., in 3,4-dihydro-2H-pyrrole), NH (e.g., in pyrrolealkyl), or + NR (as in N-substituted pyrrolidinyl groups). Heterocycles can be attached to their side groups at any heteroatom or carbon atom to produce a stable structure, and any ring atom can optionally be substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolyl, piperazine, dioxalyl, dioxopentyl, diazazolyl, oxonitrileyl, thioazazolyl, morpholinyl, and quininecycloyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical” are used interchangeably herein and also include groups in which the heterocyclic ring is fused with one or more aryl, heteroaryl, or cycloaliphatic rings, such as indololinyl, 3H-indolyl, benzodihydropyranyl, phenanthridineyl, or tetrahydroquinolinyl. Heterocyclic groups can be monocyclic, bicyclic, or polycyclic. The term “heterocyclic alkyl” refers to an alkyl group substituted with a heterocyclic group, wherein the alkyl and heterocyclic moiety are optionally substituted independently.

[0124] Identity: As used herein, the term "identity" refers to the overall relevance between polymer molecules (e.g., between nucleic acid molecules (e.g., oligonucleotides, DNA, RNA, etc.) and / or between polypeptide molecules). In some embodiments, polymer molecules are considered "substantially identical" if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. For example, the percentage of identity between two nucleic acid or polypeptide sequences can be calculated by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second sequences to achieve optimal alignment, and dissimilar sequences can be ignored for comparison purposes). In some embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. Nucleotides at the corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., a nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. Taking into account the number of gaps and the length of each gap (which needs to be introduced to achieve optimal alignment of the two sequences), the percentage of identity between two sequences is a function of the number of identical positions shared by the sequences. Sequence comparison and determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. For example, the Meyers and Miller algorithm (CABIOS, 1989, 4: 11-17) can be used to determine the percentage of identity between two nucleotide sequences, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons using the ALIGN program employ a PAM120 weighted residual table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the NWSgapdna.CMP matrix can be used, and the GAP program in the GCG software package can be used to determine the percentage of identity between two nucleotide sequences.

[0125] Nucleotide bond: As used herein, the phrase “nucleotide bond” generally refers to a bond that links nucleoside units of oligonucleotides or nucleic acids. In some embodiments, the nucleotide bond is a phosphodiester bond, as widely found in naturally occurring DNA and RNA molecules (the native phosphodiester bond (-OP(=O)(OH)O-), which, as understood by those skilled in the art, can exist in salt form). In some embodiments, the nucleotide bond is a modified nucleotide bond (not a native phosphodiester bond). In some embodiments, the nucleotide bond is a “modified nucleotide bond” in which at least one oxygen atom or -OH of the phosphodiester bond is replaced by a different organic or inorganic moiety. In some embodiments, such an organic or inorganic moiety is selected from =S, =Se, =NR', –SR', –SeR', –N(R')2, B(R')3, –S–, –Se–, and –N(R')–, wherein each R' is independently as defined and described in this disclosure. In some embodiments, the internucleotide linkage is a phosphotriester linkage, a thiophosphate linkage (or a thiophosphate diester linkage, i.e., -OP(=O)(SH)O-, which, as understood by those skilled in the art, may exist in salt form), or a thiophosphate triester linkage. In some embodiments, the modified internucleotide linkage is a thiophosphate linkage. In some embodiments, the internucleotide linkage is, for example, one of a PNA (peptide nucleic acid) or PMO (diaminophosphate morpholino oligomer) linkage. In some embodiments, the modified internucleotide linkage is a negatively charged internucleotide linkage. In some embodiments, the modified internucleotide linkage is a neutral internucleotide linkage (e.g., n001 in some of the provided oligonucleotides). Those skilled in the art will understand that, due to the presence of an acidic or basic moiety in the linkage, the internucleotide linkage can exist as an anion or a cation at a given pH. In some embodiments, the modified nucleotide linkages are modified nucleotide linkages named s, s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17, and s18, as described in WO 2017 / 210647.

[0126] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, etc., rather than within an organism (e.g., an animal, plant, and / or microorganism).

[0127] In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., an animal, plant, and / or microorganism).

[0128] Linked phosphorus: As defined herein, the phrase “linked phosphorus” is used to indicate that the specific phosphorus atom referred to is a phosphorus atom present in an internucleotide bond corresponding to the phosphorus atom of a phosphodiester nucleotide bond naturally present in DNA and RNA. In some embodiments, the linking phosphorus atom is located in a modified internucleotide bond, wherein each oxygen atom of the phosphodiester bond is optionally and independently replaced by an organic or inorganic portion. In some embodiments, the linking phosphorus atom is chiral (e.g., as in thiophosphate nucleotide bonds). In some embodiments, the linking phosphorus atom is achiral (e.g., as in native phosphate bonds).

[0129] Modified nucleobase: The terms "modified nucleobase," "modified base," etc., refer to a chemical portion that is chemically different from a nucleobase but capable of performing at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase comprising a modification. In some embodiments, a modified nucleobase is capable of having at least one function of a nucleobase, for example, forming a portion in a polymer capable of pairing with a nucleic acid base comprising at least a complementary base sequence. In some embodiments, a modified nucleobase is a substituted A, T, C, G, or U, or a substituted tautomer of A, T, C, G, or U. In some embodiments, in the context of oligonucleotides, a modified nucleobase refers to a nucleobase that is not A, T, C, G, or U.

[0130] Modified nucleosides: "Modified nucleosides" refer to those derived from natural nucleosides or chemically similar moieties, but containing chemical modifications that distinguish them from natural nucleosides. Non-limiting examples of modified nucleosides include those containing modifications at the bases and / or sugars. Non-limiting examples of modified nucleosides include those having a 2' modification at the sugar. Non-limiting examples of modified nucleosides also include baseless nucleosides (which lack a nucleobase). In some embodiments, modified nucleosides are capable of having at least one function of a nucleoside, for example, forming a moiety in a polymer capable of pairing with nucleic acid bases comprising at least a complementary base sequence.

[0131] Modified nucleotides: The term "modified nucleotide" includes any chemical portion that differs structurally from a natural nucleotide but is capable of performing at least one function of a natural nucleotide. In some embodiments, modified nucleotides comprise modifications at sugar, base, and / or internucleotide links. In some embodiments, modified nucleotides comprise modified sugars, modified nucleobases, and / or modified internucleotide links. In some embodiments, modified nucleotides are capable of having at least one function of a nucleotide, for example, forming subunits in polymers capable of pairing with nucleic acid bases comprising at least a complementary base sequence.

[0132] Modified sugars: The term "modified sugar" refers to a portion that can replace a sugar. Modified sugars mimic the spatial arrangement, electronic properties, or some other physicochemical properties of sugars. In some embodiments, as described in this disclosure, the modified sugar is a substituted ribose or deoxyribose. In some embodiments, the modified sugar contains a 2'-modification. Examples of useful 2'-modifications are widely used in the art and described herein. In some embodiments, the 2'-modification is 2'-F. In some embodiments, the 2'-modification is 2'-OR, where R is an optionally substituted C. 1-10 Aliphatic group. In some embodiments, the 2'-modification is 2'-OMe. In some embodiments, the 2'-modification is 2'-MOE. In some embodiments, the modified sugar is a bicyclic sugar (e.g., the sugar used in LNA, BNA, etc.). In some embodiments, in the case of oligonucleotides, the modified sugar is a sugar of ribose or deoxyribose that is not typically found in natural RNA or DNA.

[0133] Nucleic Acids: As used herein, the term “nucleic acid” includes any nucleotide and its polymers. As used herein, the term “polynucleotide” refers to a polymer of nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA) or combinations thereof) of any length. These terms refer to the primary structure of a molecule and include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. These terms include RNA or DNA analogs as equivalents, which contain modified nucleotides and / or modified polynucleotides (such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides). These terms cover polynucleotides or oligonucleotides (RNA) and polydeoxynucleotides or oligodeoxynucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleotides and / or modified nucleotides; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate ester bridges and / or modified internucleotide bonds. This term covers nucleic acids containing any combination of nucleosides, modified nucleosides, sugars, modified sugars, phosphate bridges, or modified internucleotide bonds. Examples include, but are not limited to, nucleic acids containing a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both a ribose and a deoxyribose moiety, and nucleic acids containing both a ribose moiety and a modified ribose moiety. Unless otherwise specified, the prefix "poly-" refers to nucleic acids containing 2 to approximately 10,000 nucleotide monomer units, and the prefix "oligo-" refers to nucleic acids containing 2 to approximately 200 nucleotide monomer units.

[0134] Nucleotides: The term "nucleotide" refers to the portion of a nucleic acid that participates in the formation of hydrogen bonds, which bind one nucleic acid strand to another complementary strand in a sequence-specific manner. The most common naturally occurring nucleotides are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, naturally occurring nucleotides are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, naturally occurring nucleotides are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, nucleotides comprise a heteroaryl ring, wherein the ring atom is nitrogen, and when in a nucleoside, the nitrogen is partially bonded to the sugar. In some embodiments, nucleotides comprise a heterocycle, wherein the ring atom is nitrogen, and when in a nucleoside, the nitrogen is partially bonded to the sugar. In some embodiments, the nucleotide is a "modified nucleotide," that is, a nucleotide other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleotide is a substituted A, T, C, G, or U. In some embodiments, the modified nucleotide is a substituted tautomer of A, T, C, G, or U. In some embodiments, the modified nucleotide is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleotide mimics the spatial arrangement, electronic properties, or some other physicochemical properties of nucleotides and retains the hydrogen-bonded, sequence-specific binding of one nucleic acid strand to another. In some embodiments, the modified nucleotide can pair with all five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting unwinding behavior, recognition by intracellular enzymes, or activity of oligonucleotide duplexes. As used herein, the term "nucleobase" also encompasses structural analogs of naturally occurring nucleotides or nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, a nucleobase is an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U. In some embodiments, "nucleobase" refers to a nucleobase unit (e.g., A, T, C, G, or U) in an oligonucleotide or nucleic acid.

[0135] Nucleoside: The term "nucleoside" refers to the portion in which a nucleobase or a modified nucleobase is covalently bonded to a sugar or a modified sugar. In some embodiments, the nucleoside is a natural nucleoside, such as adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, the nucleoside is a modified nucleoside, such as a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, the nucleoside is a modified nucleoside, such as a substituted tautomer of a natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, "nucleoside" refers to a nucleoside unit in an oligonucleotide or nucleic acid.

[0136] Nucleotide: As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a nucleobase, a sugar, and one or more internucleotide bonds (e.g., phosphate ester bonds in native DNA and RNA). Naturally occurring bases [guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)] are derivatives of purines or pyrimidines, but it should be understood that naturally occurring and non-naturally occurring base analogues are also included. Naturally occurring sugars are pentose sugars, namely deoxyribose (which forms DNA) or ribose (which forms RNA), but it should be understood that naturally occurring and non-naturally occurring sugar analogues are also included. Nucleotides are linked by internucleotide bonds to form nucleic acids, or polynucleotides. Many internucleotide bonds are known in the art (e.g., but not limited to phosphate esters, thiophosphate esters, borane phosphate esters, etc.). Artificial nucleic acids include PNA (peptide nucleic acid), phosphate triesters, thiophosphates, H-phosphonates, aminophosphates, boron phosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates, and other variants of the phosphate ester backbone of natural nucleic acids, as described herein. In some embodiments, a natural nucleotide comprises naturally occurring bases, sugars, and nucleotide-to-nucleotide bonds. As used herein, the term "nucleotide" also encompasses structural analogs used in place of natural nucleotides or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs. In some embodiments, "nucleotide" refers to an oligonucleotide or a nucleotide unit in a nucleic acid.

[0137] Oligonucleotides: The term “oligonucleotide” refers to a polymer or oligomer of nucleotides and can contain any combination of natural and non-natural nucleobases, sugars, and internucleotide bonds.

[0138] Oligonucleotides can be single-stranded or double-stranded. Single-stranded oligonucleotides can have double-stranded regions (formed by the two parts of a single-stranded oligonucleotide), and double-stranded oligonucleotides containing two oligonucleotide chains can have single-stranded regions, such as regions where the two oligonucleotide chains are not complementary to each other. Examples of oligonucleotides include, but are not limited to, structural genes, genes containing control and termination regions, self-replicating systems (such as viral DNA or plasmid DNA), single-stranded and double-stranded RNAi reagents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermir, aptamers, antimir, antagomir, Ul adaptors, triple-stranded oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0139] The oligonucleotides disclosed herein can have various lengths. In certain embodiments, the length of the oligonucleotide can be from about 2 to about 200 nucleotides. In several related embodiments, the length of the (single-stranded, double-stranded, or triple-stranded) oligonucleotide can range from about 4 to about 10 nucleotides, from about 10 to about 50 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, and from about 20 to about 30 nucleotides. In some embodiments, the length of the oligonucleotide is from about 9 to about 39 nucleotides. In some embodiments, the length of the oligonucleotide is from about 25 to about 70 nucleotides. In some embodiments, the length of the oligonucleotide is from about 26 to about 70 nucleotides. In some embodiments, the length of the oligonucleotide is from about 27 to about 70 nucleotides. In some embodiments, the length of the oligonucleotide is from about 28 to about 70 nucleotides. In some embodiments, the length of the oligonucleotide is from about 29 to about 70 nucleotides. In some embodiments, the length of the oligonucleotide is from about 30 to about 70 nucleotides. In some embodiments, the length of the oligonucleotide is from about 31 to about 70 nucleotides. In some embodiments, the oligonucleotide has a length of from about 32 to about 70 nucleotides. In some embodiments, the oligonucleotide has a length of from about 25 to about 60 nucleotides. In some embodiments, the oligonucleotide has a length of from about 25 to about 50 nucleotides. In some embodiments, the oligonucleotide has a length of from about 25 to about 40 nucleotides. In some embodiments, the oligonucleotide has a length of from about 30 to about 40 nucleotides. In some embodiments, the oligonucleotide has a length of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the oligonucleotide has a length of at least 4 nucleotides. In some embodiments, the oligonucleotide has a length of at least 5 nucleotides. In some embodiments, the oligonucleotide has a length of at least 6 nucleotides. In some embodiments, the oligonucleotide has a length of at least 7 nucleotides. In some embodiments, the oligonucleotide has a length of at least 8 nucleotides. In some embodiments, the oligonucleotide has a length of at least 9 nucleotides. In some embodiments, the oligonucleotide has a length of at least 10 nucleotides. In some embodiments, the oligonucleotide has a length of at least 11 nucleotides. In some embodiments, the oligonucleotide has a length of at least 12 nucleotides. In some embodiments, the oligonucleotide has a length of at least 15 nucleotides. In some embodiments, the oligonucleotide has a length of at least 15 nucleotides. In some embodiments, the oligonucleotide has a length of at least 16 nucleotides. In some embodiments, the oligonucleotide has a length of at least 17 nucleotides. In some embodiments, the oligonucleotide has a length of at least 18 nucleotides. In some embodiments, the oligonucleotide has a length of at least 19 nucleotides. In some embodiments, the oligonucleotide has a length of at least 20 nucleotides. In some embodiments, the oligonucleotide has a length of at least 25 nucleotides.In some embodiments, the oligonucleotide has a length of at least 26 nucleotides. In some embodiments, the oligonucleotide has a length of at least 27 nucleotides. In some embodiments, the oligonucleotide has a length of at least 28 nucleotides. In some embodiments, the oligonucleotide has a length of at least 29 nucleotides. In some embodiments, the oligonucleotide has a length of at least 30 nucleotides. In some embodiments, the oligonucleotide has a length of at least 31 nucleotides. In some embodiments, the oligonucleotide has a length of at least 32 nucleotides. In some embodiments, the oligonucleotide has a length of at least 33 nucleotides. In some embodiments, the oligonucleotide has a length of at least 34 nucleotides. In some embodiments, the oligonucleotide has a length of at least 35 nucleotides. In some embodiments, the oligonucleotide has a length of at least 36 nucleotides. In some embodiments, the oligonucleotide has a length of at least 37 nucleotides. In some embodiments, the oligonucleotide has a length of at least 38 nucleotides. In some embodiments, the oligonucleotide has a length of at least 39 nucleotides. In some embodiments, the oligonucleotide has a length of at least 40 nucleotides. In some embodiments, the oligonucleotide has a length of 25 nucleotides. In some embodiments, the oligonucleotide has a length of 26 nucleotides. In some embodiments, the oligonucleotide has a length of 27 nucleotides. In some embodiments, the oligonucleotide has a length of 28 nucleotides. In some embodiments, the oligonucleotide is 29 nucleotides long. In some embodiments, the oligonucleotide is 30 nucleotides long. In some embodiments, the oligonucleotide is 31 nucleotides long. In some embodiments, the oligonucleotide is 32 nucleotides long. In some embodiments, the oligonucleotide is 33 nucleotides long. In some embodiments, the oligonucleotide is 34 nucleotides long. In some embodiments, the oligonucleotide is 35 nucleotides long. In some embodiments, the oligonucleotide is 36 nucleotides long. In some embodiments, the oligonucleotide is 37 nucleotides long. In some embodiments, the oligonucleotide is 38 nucleotides long. In some embodiments, the oligonucleotide is 39 nucleotides long. In some embodiments, the oligonucleotide is 40 nucleotides long. In some embodiments, each nucleotide counted in the length of the oligonucleotide independently contains a nucleobase that contains a ring having at least one nitrogen ring atom. In some embodiments, each nucleotide counted in the length of the oligonucleotide independently contains A, T, C, G, or U, or optionally substituted A, T, C, G, or U, or optionally substituted tautomers of A, T, C, G, or U.

[0140] Oligonucleotide type: As used herein, the phrase “oligonucleotide type” is used to define oligonucleotides having a specific base sequence, backbone linkage pattern (i.e., internucleotide linkage pattern, such as phosphate ester, thiophosphate ester, trithiophosphate ester, etc.), backbone chiral center pattern [i.e., phosphorus-linked stereochemistry (Rp / Sp)], and backbone phosphorus modification pattern. In some embodiments, oligonucleotides with a common designated “type” are structurally identical to each other.

[0141] Those skilled in the art will understand that the synthetic methods of this disclosure provide a degree of control during the synthesis of oligonucleotide chains, allowing for the prior design and / or selection of each nucleotide unit of the oligonucleotide chain to have a specific stereochemistry at the phosphorus-linked site and / or to have a specific modification at the phosphorus-linked site, and / or to have a specific base, and / or to have a specific sugar. In some embodiments, the oligonucleotide chain is designed and / or selected in advance to have a specific combination of stereocenters at the phosphorus-linked site. In some embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the phosphorus-linked site. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of having one or more structural features. In some embodiments, this disclosure provides compositions comprising a plurality of oligonucleotide molecules or compositions thereof (e.g., chiral-controlled oligonucleotide compositions). In some embodiments, all such molecules belong to the same type (i.e., are structurally identical to each other). However, in some embodiments, the provided compositions comprise a plurality of oligonucleotides of different types (typically in predetermined relative amounts).

[0142] Optionally Substituted: As described herein, compounds of this disclosure may contain optionally substituted, substituted, and / or unsubstituted moieties. Generally, the term “substituted” means that one or more hydrogens of the specified moieties are independently replaced by substituents. Unless otherwise indicated, an “optionally substituted” group may have a substituent independently at each substituted position of the group, and when more than one position in any given structure is substituted by two or more substituents, the substituents may be the same or different at each position. In some embodiments, the optionally substituted group is unsubstituted. In some embodiments, the optionally substituted group is substituted. Various substituents are described below.

[0143] The monovalent substituent is independently a halogen; –(CH2) 0–4 R°;–(CH2) 0–4 OR°;-O(CH2) 0-4 R o –O–(CH2) 0–4 C(O)OR°; –(CH2) 0–4CH(OR°)2;–(CH2) 0–4 Ph, which can be replaced by R°; -(CH2) 0–4 O(CH2) 0–1 Ph, which can be substituted by R°; –CH=CHPh, which can be substituted by R°; –(CH2) 0–4 O(CH2) 0–1 -pyridyl group, which can be substituted by R°; –NO2; -CN; –N3; -(CH2) 0–4 N(R°)2;–(CH2) 0–4 N(R°)C(O)R°; –N(R°)C(S)R°; –(CH2) 0–4 N(R°)C(O)N(R°)2;-N(R°)C(S)N(R°)2;–(CH2) 0–4 N(R°)C(O)OR°; –N(R°)N(R°)C(O)R°; –N(R°)N(R°)C(O)N(R°)2; –N(R°)N(R°)C(O)OR°; –(CH2) 0–4 C(O)R°; –C(S)R°; –(CH2) 0–4 C(O)OR°;–(CH2) 0–4 C(O)SR°;-(CH2) 0–4 C(O)OSi(R°)3;–(CH2) 0–4 OC(O)R°;–OC(O)(CH2) 0–4 SR°, -SC(S)SR°; -(CH2) 0–4 SC(O)R°;–(CH2) 0–4 C(O)N(R°)2; –C(S)N(R°)2; –C(S)SR°; -SC(S)SR°, -(CH2) 0–4 OC(O)N(R°)2;-C(O)N(OR°)R°; –C(O)C(O)R°; –C(O)CH2C(O)R°; –C(NOR°)R°; –(CH2) 0–4 SSR°;–(CH2) 0–4 S(O)2R°;–(CH2) 0–4 S(O)₂OR°;–(CH₂) 0–4 OS(O)2R°;-S(O)2N(R°)2;-(CH2) 0–4S(O)R°; –N(R°)S(O)2N(R°)2; –N(R°)S(O)2R°; –N(OR°)R°; –C(NH)N(R°)2; –Si(R°)3; –OSi(R°)3; –P(R°)2; –P(OR °)2; -OP(R°)2; -OP(OR°)2; -N(R°)P(R°)2; -B(R°)2; -OB(R°)2; -P(O)(R°)2; -OP(O)(R°)2; -N(R°)P(O)(R°)2; –(C 1-4 (linear or branched alkylene)O–N(R°)2; or –(C 1-4 (linear or branched alkylene)C(O)O–N(R°)2; wherein each R° may be independently substituted as defined below and independently be hydrogen, C 1-10 (For example, C) 1-6 C 1-4 (etc.) aliphatic groups, C 1-10 (For example, C) 1-6 C 1-4 (etc.) having 1-5 heteroaliphatic groups independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, C 6-10 (For example, C6, C) 10 aryl groups, 5-10 (e.g., 5-9, 5-6, 5, 6, 9, 10, etc.) heteroaryl groups having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -CH2-(C 6-10 (For example, C6, C) 10 Aryl group, -O(CH2) 0-1 (C 6-10 (For example, C6, C) 10 (etc.) aryl), -CH2- (5-10 (e.g., 5-9, 5-6, 5, 6, 9, 10, etc.) heteroaryl groups having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur), -O(CH2). 0-1(5-10 (e.g., 5-9, 5-6, 5, 6, 9, 10, etc.) heteroaryl groups having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur), 3-10 (e.g., 3-6, 5-6, 3, 4, 5, 6, 7, 8, 9, 10, etc.) monocyclic, bicyclic, or polycyclic, saturated or partially unsaturated rings having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, or, despite the above definition, two independently occurring R° Together with the atoms between them, they form 3-10 (e.g., 3-6, 5-6, 3, 4, 5, 6, 7, 8, 9, 10, etc.) monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aromatic rings (for aromatic rings, 5-10 (e.g., 5-9, 5-6, 5, 6, 9, 10, etc.) elements) which can be substituted as defined below.

[0144] The monovalent substituents on R° (or the ring formed by two independently occurring R° and the atoms in between) are independently halogens, –(CH2). 0–2 R ● –(halogenated R) ● ), –(CH2) 0–2 OH, –(CH2) 0–2 OR ● –(CH2) 0–2 CH(OR ● )2; –O(halogenated R ● –CN, –N3, –(CH2) 0–2 C(O)R ● –(CH2) 0–2 C(O)OH, –(CH2) 0–2 C(O)OR ● –(CH2) 0–2 SR ● –(CH2) 0–2 SH, –(CH2) 0–2 NH2、–(CH2) 0–2 NHR ● –(CH2) 0–2 NR ● 2. –NO2, –SiR ● 3. –OSiR ● 3. -C(O)SR ● 、 –(C 1–4 (straight-chain or branched alkylene)C(O)OR ● Or –SSR ● , where each R ●It is either unsubstituted or preceded by "halogenated" and is substituted by only one or more halogens, and is independently selected from C. 1–4 Aliphatic groups, –CH2Ph, –O(CH2) 0–1 Ph, or a 3-6 (e.g., 3-5, 5-6, etc.) saturated, partially unsaturated, or aromatic ring (5- or 6-membered for aromatic rings) having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. The divalent substituents on the saturated carbon atom of R° are independently =O or =S.

[0145] The divalent substituents are independently the following: =O, =S, =NNR 2、=NNHC(O)R =NNHC(O)OR =NNHS(O)2R =NR =NOR -O(C(R) 2)) 2–3 O- or -S(C(R) 2)) 2–3 S-, where R Each time it appears independently, it is selected from hydrogen, and the substituted C can be defined as follows: 1-6 Aliphatic group or unsubstituted 3-6 (e.g., 3-5, 5-6, etc.) saturated, partially unsaturated, or aromatic ring (5- or 6-membered for aromatic rings) having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. The divalent substituent bonded to the adjacent substituted carbon atom of the "optionally substituted" group is independently -O(CR). 2) 2–3 O-, where R Each time it appears independently, it is selected from hydrogen, and the substituted C can be defined as follows: 1-6 Aliphatic group, or unsubstituted 3-6 (e.g., 3-5, 5-6, etc.) saturated, partially unsaturated or aromatic rings (5- or 6-membered for aromatic rings) having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0146] R The substituents on the aliphatic groups are independently halogens, –R ● -(halogenated R) ● –OH, -OR ● –O (halogenated R) ● ), –CN, –C(O)OH, –C(O)OR ● –NH2, –NHR ● –NR ● 2 or –NO2, where each R ●It is either unsubstituted or, in the case of a preceding "halogenation", substituted by only one or more halogens, and independently C. 1–4 Aliphatic groups, –CH2Ph, –O(CH2) 0–1 Ph, or a 3-6 (e.g., 3-5, 5-6, etc.) saturated, partially unsaturated, or aromatic ring (5- or 6-membered for aromatic rings) having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0147] The substituents on the substituted nitrogen are independently –R † -NR † 2. -C(O)R † –C(O)OR † –C(O)C(O)R † –C(O)CH2C(O)R † –S(O)2R † –S(O)2NR † 2. -C(S)NR † 2. –C(NH)NR † 2 or –N(R) † )S(O)2R † ; where each R † Independently, for hydrogen, the substituted C can be defined as follows: 1–6 Aliphatic group, unsubstituted –OPh or unsubstituted 3-6 (e.g. 3-5, 5-6, etc.) saturated, partially unsaturated or aromatic ring (5- or 6-membered for aromatic rings) having 0-4 independent heteroatoms selected from nitrogen, oxygen and sulfur, or, despite the above definition, two independently occurring R groups. † Together with the atoms between them, they form unsubstituted 3-12 (e.g., 3-10, 3-6, 5-10, 5-6, 3, 4, 5, 6, 7, 8, 9, 10, etc.) saturated, partially unsaturated, or aromatic monocyclic or bicyclic rings with 0-4 independent heteroatoms selected from nitrogen, oxygen, and sulfur.

[0148] R † The substituents on the aliphatic groups are independently halogens, -R ● -(halogenated R) ● -OH, -OR ● –O (halogenated R) ● ), –CN, –C(O)OH, –C(O)OR ● –NH2, –NHR ● –NR ● 2 or –NO2, where each R ● It is either unsubstituted or, in the case of a preceding "halogenation", substituted by only one or more halogens, and independently C. 1–4Aliphatic groups, –CH2Ph, –O(CH2) 0–1 Ph, or a 3-6 (e.g., 3-5, 5-6, etc.) saturated, partially unsaturated, or aromatic ring (5- or 6-membered for aromatic rings) having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0149] P-modification: As used herein, the term "P-modification" refers to any modification at the bond phosphorus site other than stereochemical modification. In some embodiments, P-modification includes adding, substituting, or removing a side group portion covalently attached to the bonded phosphorus.

[0150] Partially Unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety containing at least one double or triple bond. As defined herein, the term “partially unsaturated” is intended to cover rings having multiple unsaturated sites, but not to include aryl or heteroaryl moieties.

[0151] Pharmaceutical Composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen, showing a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including those suitable for: oral administration, such as enemas (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for oral, sublingual, and systemic absorption), pellets, powders, granules, or pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, for example, as a sterile solution or suspension or sustained-release formulation; topical administration, such as as a cream, ointment, sustained-release patch, or spray applied to the skin, lungs, or mouth; vaginal or rectal administration, such as as a suppository, cream, or foam; sublingual; ocular; transdermal; or nasal, pulmonary, and other mucosal surfaces.

[0152] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” means compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues to the extent of reasonable medical judgment without excessive toxicity, irritation, allergic reactions or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0153] Pharmaceutically acceptable carriers: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or mediator, such as a liquid or solid filler, diluent, excipient, or solvent encapsulation material, which participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; astragalus powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic and compatible substances used in pharmaceutical formulations.

[0154] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salt" means a salt of such compounds suitable for use in a pharmaceutical context, i.e., suitable for contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., within the bounds of reasonable medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts, which are amino-containing salts formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or using organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucohepanoate, glycerophosphate, glucuronate, hemisulfate, heptaate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. In some embodiments, the provided compound (e.g., an oligonucleotide) comprises one or more acidic groups, and the pharmaceutically acceptable salt is an alkali metal salt, an alkaline earth metal salt, or an ammonium salt (e.g., an ammonium salt of N(R)3, wherein each R is independently defined and described in this disclosure). Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt. In some embodiments, the pharmaceutically acceptable salt is a calcium salt. In some embodiments, the pharmaceutically acceptable salt suitably includes non-toxic ammonium, quaternary ammonium, and amine cations formed using balancing ions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, alkyl groups having 1 to 6 carbon atoms, sulfonates, and arylsulfonates.In some embodiments, the provided compounds comprise more than one acidic group; for example, oligonucleotides may comprise two or more acidic groups (e.g., natural phosphate ester linkages and / or modified nucleotide linkages). In some embodiments, pharmaceutically acceptable salts (or generally, salts) of such compounds comprise two or more cations that may be identical or different. In some embodiments, in pharmaceutically acceptable salts (or generally, salts), all ionizable hydrogen atoms in the acidic groups (e.g., in aqueous solutions with pKa not exceeding about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2; in some embodiments, not exceeding about 7; in some embodiments, not exceeding about 6; in some embodiments, not exceeding about 5; in some embodiments, not exceeding about 4; in some embodiments, not exceeding about 3) are replaced by cations. In some embodiments, each thiophosphate and phosphate ester group exists independently in its salt form (e.g., if a sodium salt, -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively). In some embodiments, each thiophosphate and phosphate ester nucleotide linker exists independently in its salt form (e.g., -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively, if sodium salts). In some embodiments, pharmaceutically acceptable salts are sodium salts of oligonucleotides. In some embodiments, pharmaceutically acceptable salts are sodium salts of oligonucleotides, wherein each acidic phosphate and modified phosphate ester group (e.g., thiophosphate, phosphate, etc.) (if any) exists in salt form (all in sodium salt form).

[0155] Predetermined: Predetermined (or predetermined) means intentionally selected, non-random, or controlled, as opposed to random occurrence, random or uncontrolled implementation. Those skilled in the art who read this specification will understand that this disclosure provides techniques that allow selection of specific chemical and / or stereochemical features to be incorporated into an oligonucleotide composition and further allow controlled preparation of oligonucleotide compositions having such chemical and / or stereochemical features. Such provided compositions are “predetermined” as described herein. Because certain oligonucleotides may be accidentally produced by processes that are not controlled to intentionally produce specific chemical and / or stereochemical features, compositions containing these oligonucleotides may not be “predetermined” compositions. In some embodiments, a predetermined composition is a composition that can be intentionally reproduced (e.g., by repetition of a controlled process). In some embodiments, a predetermined level of a plurality of oligonucleotides in a composition means that the absolute and / or relative amounts (ratios, percentages, etc.) of the plurality of oligonucleotides in the composition are controlled. In some embodiments, the predetermined level of a plurality of oligonucleotides in a composition is obtained by chiral-controlled oligonucleotide preparation.

[0156] Protecting Group: As used herein, the term “protecting group” is those well known in the art and included in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd ed., John Wiley & Sons, 1999 (the entire contents of which are incorporated herein by reference). It also includes those protecting groups particularly applicable to the nucleoside and nucleotide chemistry described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al., 06 / 2012, Chapter 2 (the entire contents of which are incorporated herein by reference). Suitable amino protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenyl methyl carbamate (Fmoc), 9-(2-sulfonyl)fluorenyl methyl carbamate, 9-(2,7-dibromo)fluorenyl methyl carbamate, 2,7-di-tert-butyl carbamate-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl ester (DBD-Tmoc), 4-methoxybenzoyl methyl carbamate (Phenoc), and carbamate. 2,2,2-Trichloroethyl ester (Troc), 2-trimethylsilyl ethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), ammonia 1-Methyl-1-(4-biphenyl)ethyl carbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylformamido)ethyl carbamate, tert-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate Alloc, 1-Isopropyl Allyl Carbamate (Ipaoc), Cinnamyl Carbamate (Coc), 4-Nitrocinnamyl Carbamate (Noc), 8-Quinolinyl Carbamate, N-Hydroxypiperidinyl Carbamate, Alkyl Dithiocarbamate, Benzyl Carbamate (Cbz), p-Methoxybenzyl Carbamate (Moz), p-Nitrobenzyl Carbamate, p-Bromobenzyl Carbamate, p-Chlorobenzyl Carbamate, 2,4-Dichlorobenzyl ester, 4-methylsulfinyl benzyl ester of carbamate (Msz), 9-anthraylmethyl ester of carbamate, diphenylmethyl ester of carbamate, 2-methylthioethyl ester of carbamate, 2-methylsulfonylethyl ester of carbamate, 2-(p-toluenesulfonyl)ethyl ester of carbamate, [2-(1,3-dithiaalkyl)]methyl ester of carbamate (Dmoc), 4-methylbenzenethio ester of carbamate (Mtpc), 2,4-dimethylbenzenethio ester of carbamate (Bmpc), 2-phosphonoethyl ester of carbamate (Peoc), 2-triphenylphosphonoisopropyl ester of carbamate (Ppoc), 1,1-dimethyl-2-carbamate - Cyanoethyl ester, m-chloro-p-acyloxybenzyl ester of carbamate, p-(dihydroxyboryl)benzyl ester of carbamate, 5-benzisoxazolyl methyl ester of carbamate, 2-(trifluoromethyl)-6-chromone methyl ester of carbamate (Tcroc), m-nitrophenyl ester of carbamate, 3,5-dimethoxybenzyl ester of carbamate, o-nitrobenzyl ester of carbamate, 3,4-dimethoxy-6-nitrobenzyl ester of carbamate, phenyl(o-nitrophenyl)methyl ester of carbamate, phenothiazinyl-(10)-carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N'-phenylaminothiocarbonyl derivative, tert-amyl ester of carbamate, S-thiocarbamate Benzyl ester, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decoxybenzyl carbamate, 2,2-dimethoxycarbonyl vinyl carbamate, o-(N,N-dimethylformamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylformamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinamide carbamate p-(p'-methoxyphenylazo)benzyl ester, 1-methylcyclobutyl ester, 1-methylcyclohexyl ester, 1-methyl-1-cyclopropylmethyl ester, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl ester, 1-methyl-1-(p-phenylazophenyl)ethyl ester, 1-methyl-1-phenylethyl ester, 1-methyl-1-(4-pyridyl)ethyl ester, phenyl ester, p-(benzoazo)benzyl ester, 2,4,6-tri-tert-butylphenyl ester, 4-(trimethylammonium)benzyl ester, 2,4,6-carbamate6-Trimethylbenzyl ester, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, pyridine amide, 3-pyridylformamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetylacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2-methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylazophenoxy)propionamide, 4-chlorobutyryl Amines, 3-methyl-3-nitrobutamide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazoline-2-one, N-phthalimide, N-dithiosuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldimethylsilylazopentanide adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexane-2-one, 5-substituted 1,3-dibenzyl -1,3,5-Triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridinone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrololin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzocycloheptylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N- 2,7-Dichloro-9-fluorenylmethyleneamine, N-ferroceneylmethylamino (Fcm), N-2-pyridinemethylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylamine, N-p-methoxybenzylamine, N-diphenylmethyleneamine, N-[(2-pyridyl)trimethylmethyl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, N-p-nitrobenzylamine, N-salicylamine, N-5-chlorosalicylamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylamine, N-(5,5-Dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylboronic acid derivatives, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosoamines, amine N-oxides, diphenylphosphamide (Dpp), dimethylthiophosphamide (Mpt), diphenylthiophosphamide (Ppt), dialkylaminophosphate, dibenzylaminophosphate, diphenylaminophosphate, benzenesulfinamide, o-nitrobenzenesulfinamide (Nps), 2,4-dinitrobenzenesulfinamide, pentachlorobenzenesulfinamide, 2-nitro-4-methoxybenzenesulfinamide, triphenylmethylsulfinamide, 3-nitropyridinesulfinamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6- Trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylbenzodihydropyran-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilyl ethyl sulfonamide (SES), 9-anthracitesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and benzoylmethylsulfonamide.

[0157] Suitable protected carboxylic acids further include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butylbiphenylsilyl, triisopropylsilyl, etc. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, triphenylmethyl, tert-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl groups (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2-pyridinemethyl and 4-pyridinemethyl.

[0158] Suitable hydroxyl protecting groups include methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyl dimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, di(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3- Bromotetrahydropyranyl, Tetrahydrothiaranyl, 1-Methoxycyclohexyl, 4-Methoxytetrahydropyranyl (MTHP), 4-Methoxytetrahydrothiaranyl, 4-Methoxytetrahydrothiaranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxane-2-yl, Tetrahydrofuranyl, Tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-bridged methylenebenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1- Methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylhydroselenoyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridinemethyl, 4-pyridinemethyl, 3-methyl-2-pyridinemethyl N-oxide, diphenylmethyl, p,p'-dinitrodiphenylmethyl, 5-dibenzocycloheptyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenyl methyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromobenzoyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidephenyl)methyl, 4,4',4''-tris(acetylpropionyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)di(4',4''-dimethoxyphenyl)methyl, 1,1-di(4-methoxyphenyl)-1'-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)tonyl, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodithio-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyltert-hexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate Phenoxyacetic acid ester, p-chlorophenoxyacetic acid ester, 3-phenylpropionate, 4-oxovalerate (acetylpropionate), 4,4-(ethylidene dithio)valerate (acetylpropionyl dithioacetal), neovalerate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec) 2-(triphenylphosphonium)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoic acid Esters, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, dichlorophenylacetic acid ester, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, p-(methoxycarbonyl)benzoate, α-naphthylcarbamate, nitrates, alkyl N,N,N',N'-tetramethyldiaminophosphate, alkyl N-phenylcarbamate, borates, dimethylphosphothio, alkyl 2,4-dinitrophenyl sulfenate, sulfates, methanesulfonate, benzyl sulfonate, and toluenesulfonate (Ts). To protect 1,2-Diol or 1,3-Diol, with protecting groups including methylene acetal, ethylene acetal, 1-tert-butyl ethylene ketal, 1-phenyl ethylene ketal, (4-methoxyphenyl)ethylene acetal, 2,2,2-trichloroethylene acetal, acetal ketal, cyclopentylene ketal, cyclohexylene ketal, cycloheptylene ketal, benzylene acetal, p-methoxybenzylene acetal, 2,4-dimethoxybenzylene ketal, 3,4-dimethoxybenzylene acetal, 2-nitrobenzylene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylene orthocyanin. Esters, 1-ethoxyethylidene orthoesters, 1,2-dimethoxyethylidene orthoesters, α-methoxybenzyl orthoesters, 1-(N,N-dimethylamino)ethylene derivatives, α-(N,N'-dimethylamino)benzyl derivatives, 2-oxacyclopentyl orthoesters, di-tert-butylsilyl group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxane derivatives) (TIPDS), tetra-tert-butoxydisiloxane-1,3-dialkyl derivatives (TBDS), carbonates, cycloboronic esters, ethylboronic esters, and phenylboronic esters.

[0159] In some embodiments, the hydroxyl protecting group is acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, biphenylmethyl, p-nitrobenzyl, triphenylmethyl (triphenylmethyl), 4,4'-dimethoxytriphenylmethyl, trimethyl Silyl methyl ... 4''-Trimethoxytriphenylmethyl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethyl The hydroxyl protecting group is selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butylbiphenylsilyl, and 4,4'-dimethoxytriphenylmethyl. In some embodiments, the hydroxyl protecting group is selected from the group consisting of triphenylmethyl, monomethoxytriphenylmethyl, and 4,4'-dimethoxytriphenylmethyl. In some embodiments, the phosphorus-linked protecting group is a group attached to a phosphorus-linked (e.g., internucleotide link) connection throughout the oligonucleotide synthesis. In some embodiments, the protecting group is attached to the sulfur atom of a thiophosphate group. In some embodiments, the protecting group is attached to the oxygen atom of an internucleotide thiophosphate link. In some embodiments, the protecting group is attached to the oxygen atom of an internucleotide phosphate link.In some embodiments, the protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarbamoyl)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, or 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

[0160] Subject: As used herein, the term “subject” or “test subject” means any organism to which a compound or composition is administered according to this disclosure, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, the subject is a human. In some embodiments, the subject may have and / or be susceptible to diseases, disorders, and / or conditions.

[0161] Substantially: As used herein, the term "substantially" refers to qualitative conditions that exhibit all or nearly all of the features or properties of interest. A base sequence substantially identical or complementary to a second sequence is not exactly identical or complementary to the second sequence, but is substantially or nearly identical or complementary. In some embodiments, an oligonucleotide having a sequence substantially complementary to another oligonucleotide or nucleic acid forms a duplex with that oligonucleotide or nucleic acid in a manner similar to that of an oligonucleotide having a completely complementary sequence. Furthermore, those skilled in the art of biology and / or chemistry will understand that biological and / or chemical phenomena, if any, rarely reach completion and / or proceed to completion or achieve or avoid absolute results. Therefore, the term "substantially" is used herein to capture the potential incompleteness inherent in many biological and / or chemical phenomena.

[0162] Sugar: The term "sugar" refers to a monosaccharide or polysaccharide in closed and / or open forms. In some embodiments, a sugar is a monosaccharide. In some embodiments, a sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexapyranose moieties. As used herein, the term "sugar" also encompasses structural analogs used to replace conventional sugar molecules, such as diols, polymers forming the backbone of nucleic acid analogs, diol nucleic acids ("GNAs"), etc. As used herein, the term "sugar" also encompasses structural analogs used to replace natural nucleotides or naturally occurring nucleotides, such as modified sugars and nucleotide sugars. In some embodiments, a sugar is an RNA or DNA sugar (ribose or deoxyribose). In some embodiments, a sugar is a modified ribose or deoxyribose, for example, 2'-modified, 5'-modified, etc. As described herein, in some embodiments, when used with oligonucleotides and / or nucleic acids, modified sugars can provide one or more desired properties, activities, etc. In some embodiments, a sugar is an optionally substituted ribose or deoxyribose. In some embodiments, "sugar" refers to a sugar unit in an oligonucleotide or nucleic acid.

[0163] Susceptible: An individual "susceptible" to a disease, disorder, and / or condition is one whose risk of developing such a disease, disorder, and / or condition is higher than that of the general public. In some embodiments, an individual susceptible to a disease, disorder, and / or condition has a predisposition to develop such a disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition may not be diagnosed with such a disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition may exhibit symptoms of such a disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition may not exhibit symptoms of such a disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition will develop such a disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition will not develop such a disease, disorder, and / or condition.

[0164] Therapeutic Agent: As used herein, the term "therapeutic agent" generally refers to any agent that, when administered to a subject, causes the desired effect (e.g., the desired biological, clinical, or pharmaceutical effect). In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, an appropriate population is a group of subjects who have and / or are susceptible to a disease, disorder, or condition. In some embodiments, an appropriate population is a population of model organisms. In some embodiments, an appropriate population may be defined by one or more criteria, such as age group, sex, genetic background, or pre-existing clinical condition prior to receiving the therapy. In some embodiments, when administered to a subject in an effective amount, a therapeutic agent is a substance that reduces, improves, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition in the subject. In some embodiments, a "therapeutic agent" is an agent that has been or requires approval by a government agency before it can be marketed and administered to humans. In some embodiments, a "therapeutic agent" is an agent that requires a prescription for administration to humans. In some embodiments, a therapeutic agent is a compound provided, such as an oligonucleotide provided.

[0165] Therapeutic Effective Amount: As used herein, the term "therapeutic effective amount" means the amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject who has or is susceptible to such a disease, disorder, and / or condition. As will be understood by those skilled in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance to be delivered, and the target cells or tissues. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, improves, reduces, inhibits, prevents, delays, reduces, and / or reduces the incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, the therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver the therapeutically effective amount.

[0166] Treatment: As used herein, the terms “treat,” “treatment,” or “treating” refer to any method used to partially or completely relieve, improve, reduce, suppress, prevent, or delay the onset of a disease, symptom, and / or condition, reduce its severity, and / or decrease its incidence. Treatment may be administered to subjects who do not exhibit signs of a disease, symptom, and / or condition. In some embodiments, treatment may be administered to subjects who exhibit only early signs of a disease, symptom, and / or condition, for example, to reduce the risk of developing a pathology associated with the disease, symptom, and / or condition.

[0167] Unsaturated: As used herein, the term “unsaturated” means a portion having one or more unsaturated units.

[0168] Wild-type: As used herein, the term "wild-type" has its meaning as understood in the art, referring to an entity having the structure and / or activity found in nature in a "normal" state or background (as opposed to mutants, diseased, altered, etc.). Those skilled in the art will understand that wild-type genes and polypeptides typically exist in many different forms (e.g., alleles).

[0169] As those skilled in the art will understand, the methods and compositions described herein involving the provided compounds (e.g., oligonucleotides) are generally also applicable to pharmaceutically acceptable salts of such compounds.

[0170] Description of some embodiments

[0171] Oligonucleotides have applications in a variety of therapeutic, diagnostic, and research fields. The uses of naturally occurring nucleic acids are limited by factors such as their sensitivity to endonucleases and exonucleases. Therefore, various synthetic counterparts have been developed to circumvent these limitations and / or further improve various properties and activities. These synthetic counterparts include chemically modified oligonucleotides, such as base modifications, sugar modifications, and backbone modifications, which in particular make these molecules less susceptible to degradation and improve other properties and / or activities of the oligonucleotides.

[0172] From a structural perspective, modifications to the internucleotide linkages introduce chirality, and certain properties and activities may be affected by the configuration of the phosphorus atom in the oligonucleotide linkage. For example, the chirality of the phosphorus atom in the backbone linkage particularly affects binding affinity, sequence-specific binding to complementary RNA, stability, activity, delivery, and pharmacokinetics of nucleases.

[0173] In particular, this disclosure utilizes techniques for controlling various structural elements, such as sugar modifications and their patterns, nucleobase modifications and their patterns, modified nucleotide interlinking and their patterns, linking phosphorus stereochemistry and its patterns, additional chemical components (parts not typically in the oligonucleotide chain) and their patterns, etc. With the ability to fully control the structural elements of oligonucleotides, this disclosure provides oligonucleotides with improved and / or novel properties and / or activities for various applications, such as as therapeutic agents, probes, etc. For example, as demonstrated herein, the provided oligonucleotides and compositions thereof are particularly effective for editing target adenosine in target nucleic acids to correct G-to-A mutations by converting A to I in some embodiments.

[0174] In some embodiments, the oligonucleotide comprises the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 5 4, 55, 56, 57, 58, 59, 60, typically 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more sequences of consecutive identical or completely or substantially complementary bases. In some embodiments, the nucleic acid is a target nucleic acid containing one or more target adenosines. In some embodiments, the target nucleic acid contains one and no more than one target adenosine. In some embodiments, oligonucleotides may hybridize with the target nucleic acid. In some embodiments, such hybridization is favorable for the modification of A in the nucleic acid or its product by, for example, ADAR1, ADAR2, etc. (e.g., converting A to I).

[0175] In some embodiments, this disclosure provides oligonucleotides having a base sequence that is or comprises about 10-40, about 15-40, about 20-40, or at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, or at least 34 consecutive bases of oligonucleotides or nucleic acids disclosed herein (e.g., in the table), or sequences complementary to target RNA sequences, genes, transcripts, etc., disclosed herein, and wherein each T may optionally and independently be replaced by U and vice versa. In some embodiments, this disclosure provides oligonucleotides or oligonucleotide compositions as disclosed herein (e.g., in the table).

[0176] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide used for site-specific editing of nucleosides (e.g., target adenosine) in a target nucleic acid (e.g., RNA).

[0177] As described herein, oligonucleotides may contain one or more modified internucleotide links (non-natural phosphate links). In some embodiments, the modified internucleotide links are chiral internucleotide links, wherein the linking phosphorus is chiral. In some embodiments, the modified internucleotide links are phosphate thioester internucleotide links. In some embodiments, oligonucleotides contain one or more negatively charged internucleotide links (e.g., phosphate thioester internucleotide links, natural phosphate links, etc.). In some embodiments, oligonucleotides contain one or more unnegatively charged internucleotide links. In some embodiments, oligonucleotides contain one or more neutral internucleotide links.

[0178] In some embodiments, the oligonucleotide is chiral controlled. In some embodiments, the oligonucleotide is chiral pure (or "stereoisomer," "stereochemically pure"), wherein the oligonucleotide exists in a single stereoisomer form (in many cases a single diastereoisomer (or "diastereomeric") form, since multiple chiral centers can be present in the oligonucleotide, for example at the phosphate, sugar carbon, etc.). As those skilled in the art will understand, a chiral pure oligonucleotide is separated from its other stereoisomer forms (to the extent that some impurities may be present, as chemical and biological processes, selectivity and / or purification, etc., rarely (if any) achieve absolute completeness). In a chiral pure oligonucleotide, each chiral center is independently defined in terms of its configuration (for a chiral pure oligonucleotide, the internucleotide linkages are independently stereodefined or chiral controlled). In contrast to chiral and chiral-pure oligonucleotides containing a chiral phosphorus-linked phosphorus, oligonucleotides containing a chiral phosphorus-linked phosphorus that are “racemic” (or “stereochemically random”, “achirally controlled”) (e.g., from conventional phosphoramidine oligonucleotide synthesis, where there is no stereochemical control in the coupling step and it is combined with conventional sulfidation (forming a stereochemically random internucleotide linking of thiophosphate esters)) refer to various stereoisomers (typically a random mixture of diastereomers (or “diastereomers”, because there are multiple chiral centers in the oligonucleotide; e.g., from conventional oligonucleotide preparation using reagents that do not contain chiral elements other than those in the nucleoside and the phosphorus-linked phosphorus)). For example, for those in which It is the A of the internucleotide linkage of thiophosphate esters (which contains chiral linking phosphorus). A A, racemic oligonucleotide formulations include four diastereomers [2] 2 = 4, considering two chiral-bonded phosphorus molecules, each of which can exist in one of two configurations (Sp or Rp): A SA SA, A SA RA, A RA SA and A RA RA, where S represents the internucleotide linking of phosphothiophosphates. R represents Rp phosphate thioester internucleotide linkage. For chiral pure oligonucleotides, such as A... SA SA exists as a single stereoisomer and differs from other stereoisomers (e.g., diastereosome A). SA RA, A RA SA and A RA (RA) separate.

[0179] In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stereorandom nucleotides linked together (a mixture of Rp and Sp-linked phosphorus at the nucleotide link, for example from conventional achiral controlled oligonucleotide synthesis). In some embodiments, the oligonucleotide comprises one or more (e.g., 1-60, 1-50, 1-40, 1-30, 1-25, 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more) chiral-controlled internucleotide linkages (Rp or Sp-linked phosphorus at the internucleotide linkage, for example, from chiral-controlled oligonucleotide synthesis). In some embodiments, the internucleotide linkage is a phosphate thioester nucleotide linkage. In some embodiments, the internucleotide linkage is a stereorandom phosphate thioester nucleotide linkage. In some embodiments, the internucleotide linkage is a chiral controlled phosphate thioester nucleotide linkage.

[0180] In particular, this disclosure provides techniques for preparing chiral-controlled (in some embodiments, stereochemically pure) oligonucleotides. In some embodiments, the oligonucleotides are stereochemically pure. In some embodiments, the oligonucleotides disclosed herein are about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% stereochemically pure.

[0181] In some embodiments, this disclosure provides a variety of oligonucleotide compositions. In some embodiments, the oligonucleotide compositions are stereorandom or not chiral controlled. In some embodiments, chiral controlled internucleotide linkages are absent in the oligonucleotides of the provided compositions. In some embodiments, the internucleotide linkages of the oligonucleotides in the compositions comprise one or more chiral controlled internucleotide linkages (e.g., a chiral controlled oligonucleotide composition).

[0182] In some embodiments, the oligonucleotide composition comprises a plurality of oligonucleotides sharing a common base sequence, wherein one or more nucleotides in the oligonucleotides are chirally controlled and the internucleotide connections are stereoractic (chiral-controlled). In some embodiments, the oligonucleotide composition comprises a plurality of oligonucleotides sharing a common base sequence, wherein each nucleotide connection containing a chiral linking phosphorus is independently a chirally controlled internucleotide connection. In some embodiments, the plurality of oligonucleotides share the same base sequence, as well as the same base and sugar modifications. In some embodiments, the plurality of oligonucleotides share the same base sequence, as well as the same base, sugar, and nucleotide connection modifications. In some embodiments, the oligonucleotide composition comprises oligonucleotides having the same composition, wherein one or more nucleotides are chirally controlled and the internucleotide connections are stereoractic (chiral-controlled). In some embodiments, the oligonucleotide composition comprises oligonucleotides having the same composition, wherein each nucleotide connection containing a chiral linking phosphorus is independently a chirally controlled internucleotide connection. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides or all oligonucleotides having a common base sequence are the plurality of oligonucleotides.

[0183] In some embodiments, this disclosure provides techniques for preparing, evaluating, and / or utilizing the provided oligonucleotides and compositions thereof.

[0184] As used in this disclosure, in some embodiments, "one or more" is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In some embodiments, "one or more" means one. In some embodiments, "one or more" means two. In some embodiments, "one or more" means three. In some embodiments, "one or more" means four. In some embodiments, "one or more" means five. In some embodiments, "one or more" means six. In some embodiments, "one or more" means seven. In some embodiments, "one or more" means eight. In some embodiments, "one or more" means nine. In some embodiments, "one or more" means ten. In some embodiments, "one or more" means at least one. In some embodiments, "one or more" means at least two. In some embodiments, "one or more" means at least three. In some embodiments, "one or more" means at least four. In some embodiments, "one or more" means at least five. In some embodiments, "one or more" means at least six. In some embodiments, "one or more" means at least seven. In some embodiments, "one or more" means at least eight. In some embodiments, "one or more" means at least nine. In some embodiments, "one or more" means at least ten.

[0185] As used in this disclosure, in some embodiments, "at least one" means one or more.

[0186] As an example, variables such as R and R' are described. L Various embodiments of L, etc. Embodiments described for a variable (e.g., R) are generally applicable to all variables that can be such variables (e.g., R' ... L R L1 wait).

[0187] Oligonucleotides

[0188] In particular, this disclosure provides oligonucleotides with various designs that may include the various nucleobases and their patterns, sugars and their patterns, internucleotide bonds and their patterns, and / or additional chemical moieties and their patterns described in this disclosure. In some embodiments, the provided oligonucleotides can direct A to I editing in a target nucleic acid. In some embodiments, the oligonucleotides of this disclosure are single-stranded oligonucleotides capable of site-directed editing of adenosine (converting A to I) in a target RNA sequence.

[0189] In some embodiments, the oligonucleotide has a suitable length and sequence complementarity to specifically hybridize with the target nucleic acid. In some embodiments, the oligonucleotide is long enough and complementary enough to distinguish the target nucleic acid from other nucleic acids, thereby reducing off-target effects. In some embodiments, the oligonucleotide is short enough to facilitate delivery, reduce manufacturing complexity and / or cost, thereby maintaining the desired properties and activities (e.g., adenosine editing).

[0190] In some embodiments, the oligonucleotide has about 10-200 units (e.g., about 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-120, 10-150, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-120, 20-150, 20-200, 25-30, 25-40, 25-50, 25...). The length of the oligonucleotide sequence is approximately 10-60 nucleobases. In some embodiments, the length of the base sequence is approximately 15-50 nucleobases. In some embodiments, the length of the base sequence is approximately 15 to approximately 35 nucleobases. In some embodiments, the length of the base sequence is about 25 to about 34 nucleobases. In some embodiments, the length of the base sequence is about 26 to about 35 nucleobases. In some embodiments, the length of the base sequence is about 27 to about 32 nucleobases. In some embodiments, the length of the base sequence is about 29 to about 35 nucleobases. In some embodiments, the length of the base sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleobases. In some other embodiments, the base sequence is 35 or more nucleobases long. In some other embodiments, the base sequence is 34 or more nucleobases long. In some other embodiments, the base sequence is 33 or more nucleobases long. In some other embodiments, the base sequence is 32 or more nucleobases long. In some other embodiments, the base sequence is 31 or more nucleobases long. In some other embodiments, the base sequence is 30 or more nucleobases long. In some other embodiments, the base sequence is 29 or more nucleobases long. In some other embodiments, the base sequence is 28 or more nucleobases long.In some other embodiments, the base sequence is 27 or more nucleobases in length. In some other embodiments, the base sequence is 26 or more nucleobases in length. In some other embodiments, the base sequence of the complementary portion in the double strand is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 16, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more nucleobases in length. In some other embodiments, its length is at least 18 nucleobases. In some other embodiments, its length is at least 19 nucleobases. In some other embodiments, its length is at least 20 nucleobases. In some other embodiments, its length is at least 21 nucleobases. In some other embodiments, its length is at least 22 nucleobases. In some other embodiments, its length is at least 23 nucleobases. In some other embodiments, its length is at least 24 nucleobases. In some other embodiments, the length is at least 25 nucleobases. In particular, this disclosure provides oligonucleotides with comparable or better properties and / or comparable or higher activity but shorter length compared to previously reported adenosine-edited oligonucleotides.

[0191] In some embodiments, the oligonucleotide's base sequence is complementary to the target nucleic acid's base sequence (e.g., complementary to a portion of the target nucleic acid containing the target adenosine), and has 0-10 mismatches (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) that are not Watson-Crick base pairs (AT, AU, and CG). In some embodiments, there are no mismatches. In some embodiments, there is one mismatch. In some embodiments, there are two mismatches. In some embodiments, there are 3 mismatches. In some embodiments, there are 4 mismatches. In some embodiments, there are 5 mismatches. In some embodiments, there are 6 mismatches. In some embodiments, there are 7 mismatches. In some embodiments, there are 8 mismatches. In some embodiments, there are 9 mismatches. In some embodiments, there are 10 mismatches. In some embodiments, the oligonucleotide may contain portions not designed for complementarity (e.g., loops, protein-binding sequences, etc., used to recruit proteins, such as ADAR). As those skilled in the art will understand, such portions may be appropriately excluded when calculating mismatches and / or complementarity. In some embodiments, the complementarity between the oligonucleotide and the target nucleic acid is approximately 50%-100% (e.g., approximately 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%). (e.g., 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.). In some embodiments, complementarity is at least about 60%. In some embodiments, complementarity is at least about 65%. In some embodiments, complementarity is at least about 70%. In some embodiments, complementarity is at least about 75%. In some embodiments, complementarity is at least about 80%. In some embodiments, complementarity is at least about 85%. In some embodiments, complementarity is at least about 90%.In some embodiments, complementarity is at least about 95%. In some embodiments, complementarity is 100% along the length of the oligonucleotide. In some embodiments, complementarity is 100% along the length of the oligonucleotide, except at the nucleotide opposite to the target nucleotide (e.g., adenosine). Typically, complementarity is based on Watson-Crick base pairs AT, AU, and CG. Those skilled in the art will understand that when evaluating the complementarity of two sequences of different lengths (e.g., the provided oligonucleotide and target nucleic acid), complementarity may suitably be based on the length of the shorter sequence and / or the maximum complementarity between the two sequences. In many embodiments, the oligonucleotide and target nucleic acid have sufficient complementarity such that the modification is selectively targeted at the target adenosine site.

[0192] In some embodiments, one or more mismatches are independently oscillations. In some embodiments, each mismatch is an oscillation. In some embodiments, there are 0-10 oscillations (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.). In some embodiments, the number is 0. In some embodiments, the number is 1. In some embodiments, the number is 2. In some embodiments, the number is 3. In some embodiments, the number is 4. In some embodiments, the number is 5. In some embodiments, the wobble is GU, IA, GA, IU, IC, IT, AA, or reverse AT. In some embodiments, the wobble is GU, IA, GA, IU, or IC. In some embodiments, IC can be considered a match when I is the 3' adjacent nucleoside to the nucleoside opposite to the target nucleoside. In some embodiments, the base forming the wobble pair (e.g., U that can form a GU wobble) can replace the base forming the match pair (e.g., C that matches G) and can provide an oligonucleotide with editing activity.

[0193] In some embodiments, the duplex of the oligonucleotide and the target nucleic acid includes one or more protrusions, each protrusion independently containing one or more non-wobbly mismatches. In some embodiments, there are 0-10 protrusions (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.). In some embodiments, the number is 0. In some embodiments, the number is 1. In some embodiments, the number is 2. In some embodiments, the number is 3. In some embodiments, the number is 4. In some embodiments, the number is 5.

[0194] In some embodiments, the distance between two mismatches, between a mismatch and one or both ends of an oligonucleotide (or a portion thereof, such as a first domain, a second domain, a first subdomain, a second subdomain, or a third subdomain), and / or between a mismatch and a nucleotide opposite to the target adenosine, may independently be 0-50, 0-40, 0-30, 0-25, 0-20, 0-15, or 0-10 (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7). The nucleobases are 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 (excluding mismatches, terminal nucleosides, and nucleosides opposite to the target adenosine). In some embodiments, the number is 0-30. In some embodiments, the number is 0-20. In some embodiments, the number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the distance between two mismatches is 0-20. In some embodiments, the distance between two mismatches is 1-10. In some embodiments, the distance between a mismatch and the 5'-terminal nucleotide of the oligonucleotide is 0-20. In some embodiments, the distance between a mismatch and the 5'-terminal nucleotide of the oligonucleotide is 5-20. In some embodiments, the distance between a mismatch and the 3'-terminal nucleotide of the oligonucleotide is 0-40. In some embodiments, the distance between a mismatch and the 3'-terminal nucleotide of the oligonucleotide is 5-20. In some embodiments, the distance between a mismatch and the nucleotide opposite to the target adenosine is 0-20. In some embodiments, the distance between a mismatch and the nucleotide opposite to the target adenosine is 1-10. In some embodiments, the number of nucleobases is 0 for a given distance. In some embodiments, it is 1. In some embodiments, it is 2. In some embodiments, it is 3. In some embodiments, it is 4. In some embodiments, it is 5. In some embodiments, it is 6. In some embodiments, it is 7. In some embodiments, it is 8. In some embodiments, it is 9. In some embodiments, it is 10. In some embodiments, it is 11. In some embodiments, it is 12. In some embodiments, it is 13. In some embodiments, it is 14. In some embodiments, it is 15. In some embodiments, it is 16. In some embodiments, it is 17. In some embodiments, it is 18. In some embodiments, it is 19.In some embodiments, it is 20. In some embodiments, the mismatch is located at the end of the first domain, the second domain, the first subdomain, the second subdomain, or the third subdomain, for example, at the 5'-end or the 3'-end. In some embodiments, the mismatch is located at a nucleoside opposite to the target adenosine.

[0195] In some embodiments, the provided oligonucleotide can direct adenosine editing in a target nucleic acid (e.g., converting A to I) and has a base sequence consisting of the base sequence of the oligonucleotide disclosed herein, including or including a portion of the base sequence (e.g., a span of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more consecutive bases), wherein each T can be independently replaced by U and vice versa, and the oligonucleotide contains at least one non-naturally occurring modification of bases, sugars and / or internucleotide bonds.

[0196] In some embodiments, the provided oligonucleotide comprises one or more carbohydrate moieties. In some embodiments, the provided oligonucleotide comprises one or more GalNAc moieties. In some embodiments, the provided oligonucleotide comprises one or more targeting moieties. Non-limiting examples of such additional chemical moieties that can be conjugated to the oligonucleotide chain are described herein.

[0197] In some embodiments, the provided oligonucleotide can direct the correction of G-to-A mutations in a target sequence or its product. In some embodiments, the correction of G-to-A mutations is or includes converting A to I, which can be read as G in translation or other biological processes. In some embodiments, the provided oligonucleotide can direct the correction of G-to-A mutations in a target sequence or its product via ADAR-mediated deamination. In some embodiments, the provided oligonucleotide can direct the correction of G-to-A mutations in a target sequence or its product via ADAR-mediated deamination by recruiting endogenous ADAR (e.g., in target cells) and promoting ADAR-mediated deamination. However, in any case, this disclosure is not limited to any particular mechanism. In some embodiments, this disclosure provides oligonucleotides, compositions, methods, etc., capable of operating via double-stranded RNA interference, single-stranded RNA interference, RNase H-mediated knockdown, steric hindrance of translation, ADAR-mediated deamination, or a combination of two or more such mechanisms.

[0198] In some embodiments, the oligonucleotide comprises structural elements or portions thereof as described herein, for example, in the tables. In some embodiments, the oligonucleotide has a base sequence comprising the base sequence (or portions thereof) (where each T may be independently substituted with U), a chemical modification pattern (or portions thereof), and / or the form of an oligonucleotide disclosed herein (e.g., in tables or figures, or otherwise disclosed herein). In some embodiments, such an oligonucleotide may direct the correction of G to A mutations in a target sequence or its product.

[0199] In particular, the provided oligonucleotide can hybridize with its target nucleic acid (e.g., precursor mRNA, mature mRNA, etc.). In some embodiments, the oligonucleotide can hybridize with the target RNA sequence nucleic acid (including but not limited to precursor mRNA or mature mRNA) at any stage of RNA processing. In some embodiments, the oligonucleotide can hybridize with any element of the oligonucleotide nucleic acid or its complement, including but not limited to: promoter region, enhancer region, transcription termination region, translation initiation signal, translation termination signal, coding region, non-coding region, exon, intron, intron / exon or exon / intron junction, 5' UTR or 3' UTR.

[0200] In some embodiments, the oligonucleotide hybridizes with two or more variants of a transcript derived from the sense strand of a target site (e.g., a target sequence).

[0201] In some embodiments, the provided oligonucleotides contain one or more isotopes at increased levels. In some embodiments, the provided oligonucleotides are labeled, for example, with one or more isotopes of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). In some embodiments, the provided oligonucleotides in the provided composition (e.g., multiple oligonucleotides of the composition) contain base modifications, sugar modifications, and / or internucleotide linking modifications, wherein these oligonucleotides contain enriched levels of deuterium. In some embodiments, the provided oligonucleotides are labeled with deuterium at one or more positions (using - 2 H replacement- 1 H). In some embodiments, one or more of the oligonucleotide chain or any portion (e.g., a targeting portion, etc.) conjugated to the oligonucleotide chain. 1 H was 2 H substitution. Such oligonucleotides can be used in the compositions and methods described herein.

[0202] In some embodiments, the oligonucleotide comprises one or more modified nucleosides, one or more modified sugars, and / or one or more modified internucleotide bonds as described herein. In some embodiments, the oligonucleotide comprises a certain level of modified nucleosides, modified sugars, and / or modified internucleotide bonds, for example, approximately 5%-100%, approximately 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95 ... 5%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc.

[0203] In some embodiments, the oligonucleotide comprises one or more modified sugars. In some embodiments, the oligonucleotide comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.) modified sugars. In some embodiments, the oligonucleotide comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.) modified sugars having 2'-F modification.In some embodiments, the oligonucleotide comprises about 2 to 50 (e.g., about 2, 3, 4, 5, 6, 7, 8, 9 or 10, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.; 2-40, 2-30, 2-25, 2-20, 2-15, 2-10, 3-40, 3-30, 3-25, 3-20, 3-15, 3-10, 4-40, 4-30, 4-25, 4-20, 4-15 4-10, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 6-40, 6-30, 6-25, 6-20, 6-15, 6-10, 7-40, 7-30, 7-25, 7-20, 7-15, 7-10, 8-40, 8-30, 8-25, 8-20, 8-15, 8-10, 9- The oligonucleotide comprises 40, 9-30, 9-25, 9-20, 9-15, 9-10, 10-40, 10-30, 10-25, 10-20, 10-15, or approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive modified sugars having 2'-F modification. In some embodiments, the oligonucleotide comprises 2 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 3 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 4 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 5 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 6 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 7 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises eight consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises nine consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises ten consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises two or more 2'-F modified sugar blocks, wherein each sugar in the 2'-F modified sugar block is independently a 2'-F modified sugar. In some embodiments, each 2'-F modified sugar block independently comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive 2'-F modified sugars as described herein. In some embodiments, two consecutive 2'-F modified sugar blocks are independently separated by a separating block, the separating block comprising one or more sugars that are independently not 2'-F modified sugars.In some embodiments, the oligonucleotide comprises one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) 2'-F blocks and one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) separable blocks. In some embodiments, the first structural domain includes one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) 2'-F blocks and one or more (e.g., 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 3-15, 4-15, 5-15, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) separate blocks. In some embodiments, each first domain block bonded to the 2'-F block of the first domain is a segregated block. In some embodiments, each first domain block bonded to the segregated block of the first domain is a 2'-F block of the first domain. In some embodiments, each sugar in the segregated block is independently unmodified by 2'-F. In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) or all sugars in the segregated block are independently unmodified by 2'-F. In some embodiments, the segregated block comprises one or more bicyclic sugars (e.g., LNA sugars, cEt sugars, etc.) and / or one or more sugars modified by 2'-OR (where R is optionally a substituted C). 1-6 Aliphatic groups (e.g., 2'-OMe, 2'-MOE, etc.). In some embodiments, the separable block comprises one or more sugars modified with 2'-OR, wherein R is optionally a substituted C 1-6 Aliphatic groups (e.g., 2'-OMe, 2'-MOE, etc.). In some embodiments, two or more non--2'-F modified sugars are sequential. In some embodiments, two or more 2'-OR modified sugars (where R is optionally a substituted C) 1-6The aliphatic group (e.g., 2'-OMe, 2'-MOE, etc.) is continuous. In some embodiments, the segregated block comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OR modified sugars (where R is optionally a substituted C). 1-6 Aliphatic groups (e.g., 2'-OMe, 2'-MOE, etc.). In some embodiments, the separable block comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) consecutive 2'-OR modified sugars (where R is optionally a substituted C). 1-6 Aliphatic groups (e.g., 2'-OMe, 2'-MOE, etc.). In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe sugar. In some embodiments, each 2'-OR modified sugar is independently a 2'-MOE sugar. In some embodiments, the separating block contains one or more 2'-F modified sugars. In some embodiments, the 2'-F modified sugars in the separating block are not adjacent to each other. In some embodiments, the separating block does not contain 2'-F modified sugars. In some embodiments, each sugar in the separating block is independently a 2'-OR modified sugar (where R is optionally substituted C). 1-6 (Aliphatic group) or bicyclic sugar. In some embodiments, each sugar in each separate block is independently a 2'-OR modified sugar (where R is optionally a substituted C). 1-6 (Aliphatic group) or bicyclic sugar. In some embodiments, each sugar in the separated block is independently a 2'-OR modified sugar, wherein R is optionally a substituted C 1-6 Aliphatic group. In some embodiments, each sugar in each separate block is independently a 2'-OR modified sugar, wherein R is optionally a substituted C 1-6 Aliphatic group. In some embodiments, each sugar in the segregating block is independently a sugar modified with 2'-OMe or 2'-MOE. In some embodiments, each sugar in each segregating block is independently a sugar modified with 2'-OMe or 2'-MOE. In some embodiments, each sugar in the segregating block is independently a sugar modified with 2'-OMe. In some embodiments, each sugar in the segregating block is independently a sugar modified with 2'-MOE. In some embodiments, the segregating block comprises a 2'-OMe sugar and a 2'-MOE-modified sugar. In some embodiments, each 2'-F block and each segregating block independently contains 1, 2, 3, 4, or 5 nucleosides. In some embodiments, each 2'-F block and each segregating block independently contains 1, 2, or 3 nucleosides.

[0204] In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-90% of all sugars 5%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are modified sugars. In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85% All sugars of 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are modified sugars, which are independently selected from sugars modified by 2'-F and sugars modified by 2'-OR (where R is an optionally substituted C). 1-6Aliphatic groups) and bicyclic sugars (e.g., LNA sugars, cEt sugars, etc.). In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

[0205] In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-80%-95%-100%- ... %-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are modified sugars, which are independently selected from sugars modified by 2'-F and sugars modified by 2'-OR (where R is an optionally substituted C). 1-6(Aliphatic groups). In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75% -85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are modified sugars, which are independently selected from sugars modified with 2'-F, sugars modified with 2'-OMe, and sugars modified with 2'-MOE. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

[0206] In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80% of all sugars 0%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are modified sugars, which are independently selected from 2'-F modified sugars and 2'-OMe modified sugars. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

[0207] In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95% of all sugars The percentages of 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are 2'-F modified sugars. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%. In some embodiments, 10 or more sugars (e.g., about or at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more, 10-50, 10-40, 10-30, 10-25, 15-50, 15-40, 15-30, 15-25, 20-50, 20-40, 20-30, 20-25, etc.) are 2'-F modified sugars. In some embodiments, the oligonucleotide comprises two or more (e.g., 2-30, 2-25, 2-20, 2-15, 3-10, 3-30, 3-25, 3-20, 3-15, 3-10, 4-30, 4-25, 4-20, 4-15, 4-10, 5-30, 5-25, 5-20, 5-15, 5-10, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) consecutive 2'-F modified sugars.In some embodiments, the oligonucleotide comprises one or more 2'-F blocks, each independently comprising two or more (e.g., 2-30, 2-25, 2-20, 2-15, 3-10, 3-30, 3-25, 3-20, 3-15, 3-10, 4-30, 4-25, 4-20, 4-15, 4-10, 5-30, 5-25, 5-20, 5-15, 5-10, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises two or more 2'-F blocks as described herein, which are separated by one or more separating blocks as described herein. In some embodiments, the 2'-F block has 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F modified sugars. In some embodiments, the 2'-F block has no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 10 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 9 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 8 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 7 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 6 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 5 2'-F modified sugars. In some embodiments, each sugar in each 2'-F block is a 2'-F modified sugar, and each 2'-F block independently has no more than 4 2'-F modified sugars.In some embodiments, each block bonded to the 2'-F block is independently a block that does not contain a 2'-F modified sugar. In some embodiments, each block bonded to the 2'-F block is independently a sugar containing a natural DNA or RNA sugar, modified with 2'-OR (where R is optionally a substituted C). 1-6 Blocks of aliphatic groups or bicyclic sugars. In some embodiments, each block bonded to the 2'-F block is independently a block comprising a natural DNA or RNA sugar, a sugar modified with 2'-OMe, a sugar modified with 2'-MOE, or a bicyclic sugar. In some embodiments, each block bonded to the 2'-F block is independently a block comprising a natural DNA or RNA sugar, a sugar modified with 2'-OMe, or a sugar modified with 2'-MOE. In some embodiments, each nucleotide in the first domain bonded to the 2'-F block in the first domain is independently a sugar modified with 2'-OR (where R is optionally a substituted C). 1-6 (Aliphatic group) or bicyclic sugar. In some embodiments, each nucleotide in the first domain bonded to the 2'-F block in the first domain is independently a 2'-OR modified sugar, wherein R is optionally a substituted C 1-6 Aliphatic group. In some embodiments, each nucleotide in the first domain bonded to the 2'-F block in the first domain is independently a sugar modified with 2'-OMe or 2'-MOE. In some embodiments, each nucleotide in the second domain bonded to the 2'-F block in the second domain is independently a sugar modified with 2'-OR (where R is optionally a substituted C). 1-6 (Aliphatic group) or bicyclic sugar. In some embodiments, each nucleotide in the second domain bonded to the 2'-F block in the second domain is independently a 2'-OR modified sugar, wherein R is optionally a substituted C 1-6 Aliphatic group. In some embodiments, each nucleotide in the second domain bonded to the 2'-F block in the second domain is independently a sugar modified with 2'-OMe or 2'-MOE.

[0208] In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-1 00%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are sugars modified with 2'-OR, where R is an optionally substituted C. 1-6 Aliphatic groups. In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-95% -100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are sugars modified with 2'-OMe or 2'-MOE. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

[0209] In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95% of all sugars 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are 2'-OMe modified sugars. In some embodiments, the percentage is about or at least about 30%. In some embodiments, the percentage is about or at least about 40%. In some embodiments, the percentage is about or at least about 50%. In some embodiments, the percentage is about or at least about 60%. In some embodiments, the percentage is about or at least about 70%. In some embodiments, the percentage is about or at least about 80%. In some embodiments, the percentage is about or at least about 90%. In some embodiments, the percentage is about or at least about 95%.

[0210] In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-1 00%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are sugars modified with 2'-OR, where R is an optionally substituted C.1-6 Aliphatic groups. In some embodiments, approximately 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95% of all sugars. 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are sugars modified with 2'-MOE.

[0211] In some embodiments, one or more of the front (5'-end) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, etc.) and / or the rear The sugars of one or more (3'-terminal) nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, etc.) are independently modified sugars. In some embodiments, the first one or more sugars are independently modified sugars. In some embodiments, the second one or more sugars are independently modified sugars. In some embodiments, both the first and second one or more sugars are independently modified sugars. In some embodiments, the modified sugars are independently non--2'-F modified sugars, such as bicyclic sugars, sugars modified with 2'-OR (where R is as described herein and not -H (e.g., optionally substituted C)). 1-6 Aliphatic groups). In some embodiments, they are independently selected from bicyclic sugars and sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 (Aliphatic group). In some embodiments, it is independently a 2'-OR modified sugar, wherein R is an optionally substituted C.1-6 Aliphatic group. In some embodiments, it is independently a sugar modified with 2'-OMe and a sugar modified with 2'-MOE. In some embodiments, the first few sugars comprise one or more sugars modified with 2'-OR as described herein (where R is optionally a substituted C). 1-6 Aliphatic groups) or bicyclic sugars (e.g., LNA, cEt, etc.). In some embodiments, the first few sugars comprise one or more sugars modified with 2'-OR, wherein R is optionally a substituted C 1-6 Aliphatic group. In some embodiments, the first few sugars comprise one or more sugars modified with 2'-OMe. In some embodiments, the first few sugars comprise one or more sugars modified with 2'-MOE. In some embodiments, the first few sugars comprise one or more sugars modified with 2'-OMe and one or more sugars modified with 2'-MOE. In some embodiments, the later few sugars comprise one or more sugars modified with 2'-OR as described herein (where R is optionally a substituted C). 1-6 Aliphatic groups) or bicyclic sugars (e.g., LNA, cEt, etc.). In some embodiments, the latter few sugars comprise one or more sugars modified with 2'-OR, wherein R is optionally a substituted C 1-6 Aliphatic group. In some embodiments, the latter few sugars comprise one or more sugars modified with 2'-OMe. In some embodiments, the latter few sugars comprise one or more sugars modified with 2'-MOE. In some embodiments, the latter few sugars comprise one or more sugars modified with 2'-OMe and one or more sugars modified with 2'-MOE. In some embodiments, the latter few sugars are independently sugars modified with 2'-OMe. In some embodiments, the former few sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive bicyclic sugars or sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 (Aliphatic group). In some embodiments, the first few sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-OR modified sugars, wherein R is optionally a substituted C 1-6Aliphatic group. In some embodiments, the first few sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive modified sugars, wherein each modified sugar is independently a 2'-OMe-modified sugar or a 2'-MOE-modified sugar. In some embodiments, the first few sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-OMe-modified sugars. In some embodiments, the first few sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-MOE-modified sugars. In some embodiments, the latter few sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-OR-modified sugars, wherein R is optionally a substituted C. 1-6 Aliphatic group. In some embodiments, the latter few sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive modified sugars, wherein each modified sugar is independently a 2'-OMe-modified sugar or a 2'-MOE-modified sugar. In some embodiments, the latter few sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-OMe-modified sugars. In some embodiments, the latter few sugars comprise three or more consecutive 2'-OMe-modified sugars. In some embodiments, the latter few sugars comprise four or more consecutive 2'-OMe-modified sugars. In some embodiments, the latter few sugars comprise five or more consecutive 2'-OMe-modified sugars. In some embodiments, the latter few sugars comprise six or more consecutive 2'-OMe-modified sugars. In some embodiments, the latter few sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) consecutive 2'-MOE-modified sugars.

[0212] In some embodiments, one or more of the first few (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are modified sugars. In some embodiments, one or more of the first few (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 Aliphatic groups) and bicyclic sugars (e.g., sugars containing 2'-O-CH2-4', wherein -CH2- is optionally substituted (e.g., LNA sugars, cET sugars (e.g., (S)-cEt))). In some embodiments, two or more of the first few sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally substituted C).1-6 Aliphatic groups) and bicyclic sugars. In some embodiments, three or more of the first few sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 Aliphatic groups) and bicyclic sugars. In some embodiments, four or more of the first few sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 (Aliphatic group) and bicyclic sugar. In some embodiments, one or more sugars are sequential. In some embodiments, the first one, two, three, or four sugars are modified sugars. In some embodiments, the first two sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 Aliphatic groups) and bicyclic sugars. In some embodiments, the first three sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 Aliphatic groups) and bicyclic sugars. In some embodiments, the first four sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 (Aliphatic group) and bicyclic sugar. In some embodiments, each 2'-OR modified sugar is independently a sugar modified with 2'-OMe or 2'-MOE. In some embodiments, each bicyclic sugar is independently an LNA sugar or a cEt sugar. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) of the first few sugars or the first few (e.g., 1, 2, 3, 4, or 5) sugars are each independently a 2'-OR modified sugar, wherein R is optionally a substituted C 1-6Aliphatic group. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) of the first few sugars are each independently modified with 2'-OMe or 2'-MOE. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) of the first few sugars are each independently modified with 2'-OMe. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) of the first few sugars are each independently modified with 2'-MOE. In some embodiments, the first one, two, three, four, or more sugars are independently modified with 2'-OMe. In some embodiments, the first sugar is modified with 2'-OMe. In some embodiments, the first two sugars are independently modified with 2'-OMe. In some embodiments, the first three sugars are independently 2'-OMe modified sugars. In some embodiments, the first four sugars are independently 2'-OMe modified sugars. In some embodiments, the first one, two, three, four or more sugars are independently 2'-MOE modified sugars. In some embodiments, the first sugar is a 2'-MOE modified sugar. In some embodiments, the first two sugars are independently 2'-MOE modified sugars. In some embodiments, the first three sugars are independently 2'-MOE modified sugars. In some embodiments, the first four sugars are independently 2'-MOE modified sugars. In some embodiments, each of these modified sugars is independently a sugar whose nucleobase is a nucleoside of a nucleoside of an optionally substituted or protected A, T, C, G or U or an optionally substituted or protected tautomer of A, T, C, G or U. In some embodiments, one or more of these sugars are independently linked to an uncharged nucleotide. In some embodiments, one or more such sugars are independently linked to a neutral nucleotide (e.g., n001). In some embodiments, the uncharged or neutral nucleotide link (e.g., n001) is chiral. In some embodiments, it is Rp. In some embodiments, one or more such sugars are independently linked to a phosphate thioester nucleotide link. In some embodiments, the phosphate thioester nucleotide link is chiral. In some embodiments, it is Sp. In some embodiments, as described herein, the nucleotide link between the first and second nucleosides is an uncharged nucleotide link. In some embodiments, it is a neutral nucleotide link. In some embodiments, it is a phosphorylguanidine nucleotide link. In some embodiments, it is n001. In some embodiments, it is chiral. In some embodiments, it is Rp.In some embodiments, apart from the nucleotide-to-nucleotide linkage between the first and second nucleosides, each nucleotide-to-nucleotide linkage bonded to a nucleoside comprising one or more of the first few modified sugars is independently a phosphate thionucleotide linkage. In some embodiments, each is chiral controlled. In some embodiments, each is Sp. In some embodiments, the first nucleoside is optionally linked to another portion, such as Mod001, via a linker (e.g., L001) through its 5'-terminal carbon (in some embodiments, through a phosphate group).

[0213] In some embodiments, one or more of the latter few (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are modified sugars. In some embodiments, one or more of the latter few (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 Aliphatic groups) and bicyclic sugars (e.g., sugars containing 2'-O-CH2-4', wherein -CH2- is optionally substituted (e.g., LNA sugars, cET sugars (e.g., (S)-cEt))). In some embodiments, two or more of the latter sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally substituted C). 1-6 Aliphatic groups) and bicyclic sugars. In some embodiments, three or more of the latter sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 Aliphatic groups) and bicyclic sugars. In some embodiments, four or more of the latter sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 (Aliphatic group) and bicyclic sugar. In some embodiments, one or more sugars are sequential. In some embodiments, the last one, two, three, or four sugars are modified sugars. In some embodiments, the last two sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 Aliphatic groups) and bicyclic sugars. In some embodiments, the latter three sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6 Aliphatic groups) and bicyclic sugars. In some embodiments, the latter four sugars are modified sugars, each independently selected from sugars modified with 2'-OR (where R is optionally a substituted C). 1-6(Aliphatic group) and bicyclic sugar. In some embodiments, each 2'-OR modified sugar is independently a sugar modified with 2'-OMe or 2'-MOE. In some embodiments, each bicyclic sugar is independently an LNA sugar or a cEt sugar. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) of the latter sugars or the latter few (e.g., 1, 2, 3, 4, or 5) sugars are each independently a 2'-OR modified sugar, wherein R is optionally a substituted C 1-6Aliphatic group. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) of the last few sugars are each independently modified with 2'-OMe or 2'-MOE. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) of the last few sugars are each independently modified with 2'-OMe. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) of the last few sugars are each independently modified with 2'-MOE. In some embodiments, the last one, two, three, four, or more sugars are independently modified with 2'-OMe. In some embodiments, the last sugar is modified with 2'-OMe. In some embodiments, the last two sugars are independently modified with 2'-OMe. In some embodiments, the last three sugars are independently 2'-OMe modified sugars. In some embodiments, the last four sugars are independently 2'-OMe modified sugars. In some embodiments, the last one, two, three, four or more sugars are independently 2'-MOE modified sugars. In some embodiments, the last sugar is a 2'-MOE modified sugar. In some embodiments, the last two sugars are independently 2'-MOE modified sugars. In some embodiments, the last three sugars are independently 2'-MOE modified sugars. In some embodiments, the last four sugars are independently 2'-MOE modified sugars. In some embodiments, each of these modified sugars is independently a sugar whose nucleobase is a nucleotide of a nucleoside that is optionally substituted or protected with an A, T, C, G or U, or an alternatively substituted or protected tautomer of A, T, C, G or U. In some embodiments, one or more of these sugars are independently linked to an uncharged nucleotide. In some embodiments, one or more such sugars are independently linked to a neutral nucleotide (e.g., n001). In some embodiments, the uncharged or neutral nucleotide link (e.g., n001) is chiral. In some embodiments, it is Rp. In some embodiments, one or more such sugars are independently linked to a phosphate thioester nucleotide link. In some embodiments, the phosphate thioester nucleotide link is chiral. In some embodiments, it is Sp. In some embodiments, as described herein, the nucleotide link between the last and penultimate nucleotides is an uncharged nucleotide link. In some embodiments, it is a neutral nucleotide link. In some embodiments, it is a phosphorylguanidine nucleotide link. In some embodiments, it is n001. In some embodiments, it is chiral. In some embodiments, it is Rp.In some embodiments, apart from the nucleotide link between the last and penultimate nucleosides, each nucleotide link bonded to a nucleoside comprising one or more of the latter modified sugars is independently a phosphate thionucleotide link. In some embodiments, each is chiral controlled. In some embodiments, each is sp.

[0214] In some embodiments, the sugar at position +1 is a 2'-F modified sugar. In some embodiments, the sugar at position +1 is a native DNA sugar. In some embodiments, the sugar at position 0 is a native DNA sugar (the nucleotide at position 0 is opposite to the target adenosine upon alignment). In some embodiments, the sugar at position -1 is a DNA sugar. In some embodiments, the sugar at position -2 is a 2'-OR modified sugar (where R is optionally a substituted C). 1-6 Aliphatic groups) or bicyclic sugars (e.g., sugars containing 2'-O-CH2-4', where -CH2- is optionally substituted (e.g., LNA sugars, cET sugars (e.g., (S)-cEt))). In some embodiments, it is a 2'-OR modified sugar, where R is optionally substituted C 1-6 Aliphatic group. In some embodiments, it is a sugar modified with 2'-OMe. In some embodiments, it is a sugar modified with 2'-MOE. In some embodiments, it is a bicyclic sugar. In some embodiments, it is an LNA sugar. In some embodiments, it is a cEt sugar. In some embodiments, the sugar at position -3 is a sugar modified with 2'-F. In some embodiments, each sugar after position -3 (e.g., positions -4, -5, -6, etc.) is independently a sugar modified with 2'-OR (where R is optionally a substituted C). 1-6 Aliphatic groups) or bicyclic sugars (e.g., sugars containing 2'-O-CH2-4', wherein -CH2- is optionally substituted (e.g., LNA sugars, cET sugars (e.g., (S)-cEt))). In some embodiments, each is independently a 2'-OR modified sugar (where R is optionally substituted C). 1-6(Aliphatic group) or bicyclic sugar. In some embodiments, each is independently a sugar modified with 2'-OMe or 2'-MOE. In some embodiments, each is a sugar modified with 2'-OMe. In some embodiments, each is a sugar modified with 2'-MOE. In some embodiments, one or more are independently 2'-OMe modified sugars, and one or more are independently 2'-MOE modified sugars. In some embodiments, as described herein, the internucleotide link between the nucleosides at positions -1 and -2 is an uncharged internucleotide link. In some embodiments, it is a neutral internucleotide link. In some embodiments, it is a phosphorylguanidine internucleotide link. In some embodiments, it is n001. In some embodiments, it is chiral controlled. In some embodiments, it is Sp. In some embodiments, it is Rp. In some embodiments, the internucleotide link between the nucleosides at positions -2 and -3 is a native phosphate ester link. In some embodiments, as described herein, the internucleotide link between the last and penultimate nucleosides is an uncharged internucleotide link. In some embodiments, it is a neutral internucleotide link. In some embodiments, it is a phosphorylguanidine nucleotide link. In some embodiments, it is n001. In some embodiments, it is chiral controlled. In some embodiments, it is Rp. In some embodiments, each nucleotide link between the 3' side of the nucleoside and the nucleoside opposite to the target adenosine (except for those nucleotide links between nucleosides at positions -1 and -2, between nucleosides at positions -2 and -3, and between the last and penultimate nucleosides) is independently a phosphate thioester nucleotide link. In some embodiments, each phosphate thioester nucleotide link is chiral controlled. In some embodiments, each is Sp.

[0215] In some embodiments, the first and / or the latter one or more sugars are modified sugars (e.g., bicyclic sugars and / or sugars modified with 2'-OR) (where R is optionally a substituted C). 1-6 Aliphatic groups (e.g., sugars modified with 2'-OMe, sugars modified with 2'-MOE, etc.). In some embodiments, such sugars can increase the stability, affinity, and / or activity of the oligonucleotide. In some embodiments, when conjugated with one or more additional chemical moieties, the sugar at the 5'- and / or 3'-terminus of the oligonucleotide is not a bicyclic sugar or a 2'-OR modified sugar (where R is optionally a substituted C). 1-6 Aliphatic group). In some embodiments, the 5'-terminal sugar is a bicyclic sugar or a sugar modified with 2'-OR (where R is optionally a substituted C). 1-6(Aliphatic group). In some embodiments, such a 5'-terminal sugar is not linked to an additional chemical moiety. In some embodiments, the 5'-terminal sugar is a 2'-F modified sugar. In some embodiments, the 5'-terminal sugar is a 2'-F modified sugar conjugated to an additional chemical moiety. In some embodiments, the 3'-terminal sugar is a bicyclic sugar or a 2'-OR modified sugar (where R is optionally a substituted C). 1-6 Aliphatic group). In some embodiments, such 3'-terminal sugars are not attached to additional chemical moieties. In some embodiments, the 3'-terminal sugar is a 2'-F modified sugar. In some embodiments, the 3'-terminal sugar is a 2'-F modified sugar conjugated to additional chemical moieties. In some embodiments, the latter few sugars are 3'-side sugars relative to the nucleotide opposite to the target adenosine (e.g., sugars of 3'-side nucleotides, such as N). -1 N -2 (etc.). In some embodiments, the last few sugars or 3'-side sugars comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-F modified sugars. In some embodiments, the last few sugars or 3'-side sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) consecutive 2'-F modified sugars. In some embodiments, the last few sugars or 3'-side sugars comprise one or more, or two or more consecutive 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a bicyclic sugar or a 2'-OR modified sugar (where R is optionally a substituted C). 1-6 (Aliphatic group). In some embodiments, as described herein, the 2'-OR modified sugar is a 2'-OMe modified sugar or a 2'-MOE modified sugar; in some embodiments, it is a 2'-OMe modified sugar; in some embodiments, it is a 2'-MOE modified sugar. In some embodiments, the later sugars or 3'-side sugars comprise one or more, or two or more consecutive 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-OR modified sugar, wherein R is optionally a substituted C. 1-6Aliphatic group. In some embodiments, the last few sugars or 3'-side sugars comprise one or more, or two or more consecutive 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-OMe modified sugar or a 2'-MOE modified sugar. In some embodiments, the last few sugars or 3'-side sugars comprise one or more, or two or more consecutive 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-OMe modified sugar. In some embodiments, the last few sugars or 3'-side sugars comprise one or more, or two or more consecutive 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a 2'-MOE modified sugar. In some embodiments, two, but no more than two, nucleosides on the 3'-side of the nucleoside opposite to adenosine independently have 2'-F modified sugars. In some embodiments, this is at positions -4 and -5. In some embodiments, this is the penultimate and penultimate nucleosides of the oligonucleotide. In some embodiments, one and more than one nucleotide on the 3'-side of a nucleotide opposite to adenosine has a sugar modified by 2'-F. In some embodiments, it is at position -3. In some embodiments, it is the fourth-to-last nucleotide of an oligonucleotide.

[0216] In some embodiments, the bicyclic sugar or the 2'-OR modified sugar (where R is optionally a substituted C) 1-6 Aliphatic groups are present in regions comprising one or more (e.g., 1-30, 1-25, 1-20, 1-15, 1-10, 2-30, 2-25, 2-20, 2-25, 2-10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) 2'-F modified sugars. In some embodiments, most sugars in such regions, as described herein, are 2'-F modified sugars. In some embodiments, two or more 2'-F modified sugars are consecutive. In some embodiments, the region is a first structural domain. In some embodiments, bicyclic sugars are present in such regions. In some embodiments, 2'-OR modified sugars (where R is optionally a substituted C) are present. 1-6 Aliphatic groups are present in such regions. In some embodiments, sugars modified with 2'-OMe are present in such regions. In some embodiments, sugars modified with 2'-MOE are present in such regions.

[0217] In some embodiments, positions -5, -4, -3, +1, +2, +4, +5, +6, +7, and +8 (position 0 is the position of the nucleoside opposite to the target adenosine; "+" indicates the direction from the nucleoside opposite to the target adenosine toward the 5' end of the oligonucleotide, and "-" indicates the direction from the nucleoside opposite to the target adenosine toward the 3' end of the oligonucleotide; for example, at 5'-N1N0N)-1 In -3', if N0 is a nucleoside opposite to the target adenosine, then it is located at position 0, and N1 is located at position +1, and N -1 One or more sugars located at position -1) are independently 2'-F modified sugars. In some embodiments, the sugar at position +1, and one or more sugars at positions -5, -4, -3, +2, +4, +5, +6, +7, and +8 are independently 2'-F modified sugars. In some embodiments, the sugar at position +1, and one sugar at positions -5, -4, -3, +2, +4, +5, +6, +7, and +8 are independently 2'-F modified sugars.

[0218] In some embodiments, the oligonucleotide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 2-10, 3-10, 2-5, 2-4, 2-3, 3-5, 3-4, etc.) native DNA sugars. In some embodiments, one or more native DNA sugars are located in the edit region, e.g., at positions +1, 0, and / or -1. In some embodiments, the native DNA sugars are within the first few nucleotides of the oligonucleotide (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). In some embodiments, the first, second, and / or third nucleotide of the oligonucleotide independently contains native DNA sugars. In some embodiments, the native DNA sugars are bonded to modified nucleotide inter-links (such as uncharged nucleotide inter-links, neutral nucleotide inter-links, phosphorylguanidine nucleotide inter-links, n001, or phosphate thioester nucleotide inter-links (Sp in various embodiments)).

[0219] Oligonucleotides can contain various types of internucleotide linkages. In some embodiments, an oligonucleotide contains one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphate thioester internucleotide linkage. In some embodiments, the modified internucleotide linkage is a negatively charged internucleotide linkage. In some embodiments, the modified internucleotide linkage is a neutral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorylguanidine internucleotide linkage. In some embodiments, the modified internucleotide linkage is n001. In some embodiments, an oligonucleotide contains one or more native phosphate linkages. In some embodiments, the native phosphate linkage is coupled to a nucleoside containing a modified sugar that improves stability (e.g., resistance to nucleases). In some embodiments, the native phosphate linkage is coupled to a bicyclic sugar. In some embodiments, the native phosphate linkage is coupled to a 2'-modified sugar. In some embodiments, the native phosphate linkage is coupled to a 2'-OR modified sugar (where R is optionally a substituted C). 1-6(Aliphatic group) bonding. In some embodiments, the native phosphate ester link is bonded to a sugar modified with 2'-OMe. In some embodiments, the native phosphate ester link is bonded to a sugar modified with 2'-MOE. In some embodiments, the oligonucleotide comprises a phosphate thioester nucleotide link, a neutral nucleotide link, and a native phosphate ester link. In some embodiments, the oligonucleotide comprises a phosphate thioester nucleotide link, a neutral nucleotide link, and a native phosphate ester link. In some embodiments, the oligonucleotide comprises a phosphate thioester nucleotide link, a phosphoryl guanidine nucleotide link, and a native phosphate ester link. In some embodiments, the oligonucleotide comprises a phosphate thioester nucleotide link, n001, and a native phosphate ester link. In some embodiments, each chiral nucleotide link is independently chiral controlled. In some embodiments, one or more chiral nucleotide links are not chiral controlled. In some embodiments, each phosphate thioester nucleotide link is independently chiral controlled. In some embodiments, each chiral nucleotide link is independently chiral controlled. In some embodiments, most or every phosphate thioester nucleotide link is Sp as described herein. In some embodiments, most or every uncharged nucleotide link, such as n001, is Rp. In some embodiments, most or every uncharged nucleotide link, such as n001, is Sp.

[0220] In some embodiments, the oligonucleotide comprises phosphate-thioester nucleotide links and uncharged nucleotide links. In some embodiments, the oligonucleotide comprises phosphate-thioester nucleotide links and neutral nucleotide links. In some embodiments, the oligonucleotide comprises phosphate-thioester nucleotide links and phosphorylguanidine nucleotide links. In some embodiments, the oligonucleotide comprises phosphate-thioester nucleotide links and n001. In some embodiments, each chiral nucleotide link is independently chiral controlled. In some embodiments, one or more chiral nucleotide links are not chiral controlled. In some embodiments, each phosphate-thioester nucleotide link is independently chiral controlled. In some embodiments, each chiral nucleotide link is independently chiral controlled. In some embodiments, most or each phosphate-thioester nucleotide link is Sp as described herein. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) phosphate-thioester nucleotide links are Rp. In some embodiments, most or each uncharged nucleotide link, such as n001, is Rp. In some embodiments, most or every uncharged nucleotide link, such as n001, is sp. In some embodiments, the oligonucleotide does not contain native phosphate ester links. In some embodiments, each nucleotide link is independently a phosphate thioester or an uncharged nucleotide link. In some embodiments, each nucleotide link is independently a phosphate thioester or a neutral nucleotide link. In some embodiments, each nucleotide link is independently a phosphate thioester or a phosphorylguanidine nucleotide link. In some embodiments, each nucleotide link is independently a phosphate thioester or an n001 nucleotide link. In some embodiments, the last nucleotide link of the oligonucleotide is an uncharged nucleotide link, a neutral nucleotide link, a phosphorylguanidine nucleotide link, or n001.

[0221] In some embodiments, the oligonucleotides of this disclosure comprise one or more modified nucleobases. In some embodiments, the oligonucleotides of this disclosure comprise one or more modified sugars. In some embodiments, the oligonucleotides of this disclosure comprise one or more modified internucleotide bonds. According to this disclosure, various modifications may be introduced into the sugars, nucleobases, and / or internucleotide bonds. For example, in some embodiments, the modifications are those described in US 9006198. In some embodiments, the modification is found in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399, US20180216108, US 20180216107, US 9598458, WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 022473, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO The modifications described in WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 032612, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, WO 2019 / 032612, WO 2020 / 191252, WO 2021 / 071858, WO 2022 / 099159 and / or WO 2023 / 201095, the respective sugar, base and nucleotide linkages of which are independently incorporated herein by reference. In some embodiments, the modifications are those described in WO 2023 / 152371, WO 2024 / 110565, WO 2024 / 115635, WO 2024 / 121373, WO 2024 / 175550, or WO 2024 / 114908. In some embodiments, combinations or patterns of several modifications from these publications may be utilized according to this disclosure.

[0222] In some embodiments, the nucleobases in the nucleoside are or comprise cyclic BA having the following structures: BA-I, BA-Ia, BA-Ib, BA-Ic, BA-Id, BA-II, BA-II-a, BA-II-b, BA-II-c, BA-II-d, BA-III, BA-III-a, BA-III-b, BA-III-c, BA-III-d, BA-III-e, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-Va, BA-Vb, or BA-VI, or tautomers of cyclic BA, wherein the nucleobases are optionally substituted or protected. Many useful nucleobases are described herein. In particular, examples of the present invention demonstrate that various nucleobases can be used to provide targeted adenosine editing.

[0223] In some embodiments, the sugar is a modified sugar containing a 2'-modification (e.g., 2'-F, 2'-OR (where R is an optionally substituted aliphatic group)), or a bicyclic sugar (e.g., LNA sugar), or an acyclic sugar (e.g., UNA sugar). In some embodiments, the modified sugar contains a ring that is 6-membered or larger than that of the natural DNA or RNA sugar (e.g., 6-membered to 9-membered). In particular, examples of the present invention demonstrate that various sugars can be used to provide targeted adenosine editing.

[0224] In some embodiments, as described herein, the provided oligonucleotides comprise one or more domains, each independently having certain lengths, modifications, phosphorus-linked stereochemistry, etc., as described herein. In some embodiments, this disclosure provides oligonucleotides comprising one or more modified sugars and / or one or more modified nucleotides linked together, wherein the oligonucleotides comprise a first domain and a second domain, each independently comprising one or more nucleobases. In some embodiments, this disclosure provides oligonucleotides comprising one or more domains and / or subdomains as described herein. In some embodiments, this disclosure provides oligonucleotides comprising a first domain as described herein. In some embodiments, this disclosure provides oligonucleotides comprising a second domain as described herein. In some embodiments, this disclosure provides oligonucleotides comprising a first subdomain as described herein. In some embodiments, this disclosure provides oligonucleotides comprising a second subdomain as described herein. In some embodiments, this disclosure provides oligonucleotides comprising a third subdomain as described herein. In some embodiments, this disclosure provides oligonucleotides comprising one or more regions, each region independently selected from a first domain, a second domain, a first subdomain, a second subdomain, and a third subdomain, each region independently as described herein. In some embodiments, this disclosure provides oligonucleotides comprising:

[0225] The first structural domain; and

[0226] Second structural domain,

[0227] in:

[0228] The first structural domain contains one or more 2'-F modifications;

[0229] The second domain contains one or more sugars that do not have 2'-F modification.

[0230] In some embodiments, the oligonucleotide or portions thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) contain a level of modified sugar. In some embodiments, the modified sugar contains a 2'-modification. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar is an acyclic sugar (e.g., by breaking the C2-C3 bond of the corresponding cyclic sugar). In some embodiments, the modified sugar contains a 5'-modification. Typically, unless otherwise stated, such as by context, the oligonucleotides of this disclosure have a free 5'-OH at their 5'-end and a free 3'-OH at their 3'-end. In some embodiments, the 5'-terminal sugar of the oligonucleotide may contain a modified 5'-OH.

[0231] In some embodiments, the levels are approximately, for example, about 5%-100%, about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-80%, etc., representing approximately 5%-100%, 10%-100%, 2 ... 5%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%.

[0232] In some embodiments, the majority are at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the majority are about 50%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%. %, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the majority is about or at least about 50%. In some embodiments, the majority is about or at least about 55%. In some embodiments, the majority is about or at least about 60%. In some embodiments, the majority is about or at least about 65%. In some embodiments, the majority is about or at least about 70%. In some embodiments, the majority is about or at least about 75%. In some embodiments, the majority is about or at least about 80%. In some embodiments, the majority is about or at least about 85%. In some embodiments, the majority is about or at least about 90%. In some embodiments, the majority is about or at least about 95%.

[0233] In some embodiments, the oligonucleotide or portions thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a level of modified internucleotide linkages. In some embodiments, the oligonucleotide or portions thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a level of chiral internucleotide linkages. In some embodiments, the levels are approximately, for example, about 5%-100%, about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-80%, 70%-80%, 70%-90%, 65%-100%, 70%-80%, 70%-90%, 60%-100%, 70%-80%, 70%-90%, 60%-100%, 70%-80%, 70%-90%, 60%-100%, 70%-80%, 70%-90%, 60%-100%, 70%-80%, 70%-90%, 60%-100%, 70%-90%, 70%-10 ... %-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%.

[0234] In some embodiments, the oligonucleotide or portions thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a certain level of chiral-controlled internucleotide bonding. In some embodiments, the oligonucleotide or portions thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a certain level of sp nucleotide bonding. In some embodiments, the levels are approximately, for example, about 5%-100%, about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-80%, 70%-80%, 70%-90%, 65%-100%, 70%-80%, 70%-90%, 60%-100%, 70%-80%, 70%-90%, 60%-100%, 70%-80%, 70%-90%, 60%-100%, 70%-80%, 70%-90%, 60%-100%, 70%-80%, 70%-90%, 60%-100%, 70%-90%, 70%-10 ... %-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc. In some embodiments, the levels are approximately, for example, about 5%-100%, about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-90%, 60%-95%, 60%-100%, 70%-8 ... 0%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%.In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%.

[0235] In some embodiments, the oligonucleotide or a portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a certain level of sp nucleotide inter-linking. In some embodiments, the level is approximately, for example, about 5%-100%, about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-80%, 70%-95%, 60%-10 ...95%, 60%-100%, 70%-95%, 60%-100%, 70%-95%, 60%-100%, 70%-95%, 60%-100%, 70%-95 %-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc. In some embodiments, the levels are approximately, for example, about 5%-100%, about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-90%, 60%-95%, 60%-100%, 70%-8 ... 0%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc.In some embodiments, the levels are approximately, for example, about 5%-100%, about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, and 70%-80% of the chiral-controlled internucleotide bonds in the oligonucleotide or its fraction. 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, about 1-50, 1-40, 1-30, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides are independently Sp chiral nucleotides. In many embodiments, it has been observed that a high percentage of Sp nucleotides in the oligonucleotide or certain portions thereof (e.g., relative to Rp nucleotides and / or native phosphate esters) can provide improved properties and / or activity, such as high stability and / or high adenosine editing activity.

[0236] In some embodiments, the oligonucleotide or a portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a certain level of Rp nucleotide cohesion. In some embodiments, the level is approximately, for example, about 5%-100%, about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-80%, 70%-95%, 60%-10 ...95%, 60%-100%, 70%-95%, 60%-100%, 70%-95%, 60%-100%, 70%-95%, 60%-100%, 70%-95%, 60%-100%, 70%-95%, 60%-10 %-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc. In some embodiments, the levels are approximately, for example, about 5%-100%, about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-80%, 70%-90%, 60%-95%, 60%-100%, 70%-8 ... 0%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc.In some embodiments, the levels are approximately, for example, about 5%-100%, about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, and 70%-80% of the chiral-controlled internucleotide bonds in the oligonucleotide or its fraction. 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 55%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 65%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is about 100%. In some embodiments, the percentage is about or no more than about 5%. In some embodiments, the percentage is about or no more than about 10%. In some embodiments, the percentage is about or no more than about 15%. In some embodiments, the percentage is about or no more than about 20%. In some embodiments, the percentage is about or no more than about 25%. In some embodiments, the percentage is about or no more than about 30%. In some embodiments, the percentage is about or no more than about 35%. In some embodiments, the percentage is about or no more than about 40%. In some embodiments, the percentage is about or no more than about 45%. In some embodiments, the percentage is about or no more than about 50%. In some embodiments, about 1-50, 1-40, 1-30, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides are independently Rp chiral nucleotides. In some embodiments, the number is about or no more than about 1. In some embodiments, the number is about or no more than about 2. In some embodiments, the number is about or no more than about 3.In some embodiments, the number is about or no more than about 4. In some embodiments, the number is about or no more than about 5. In some embodiments, the number is about or no more than about 6. In some embodiments, the number is about or no more than about 7. In some embodiments, the number is about or no more than about 8. In some embodiments, the number is about or no more than about 9. In some embodiments, the number is about or no more than about 10.

[0237] While not wishing to be bound by theory, it is noted that in some cases, the Rp and Sp conformations of internucleotide linkages can influence structural changes in the helical conformation of double-stranded complexes formed by oligonucleotides and target nucleic acids such as RNA, and that ADAR proteins can recognize and interact with a variety of targets (e.g., double-stranded complexes formed by oligonucleotides and target nucleic acids such as RNA) through multiple domains. In some embodiments, the provided oligonucleotides and compositions thereof promote and / or enhance the interaction profiles of oligonucleotides, target nucleic acids, and / or ADAR proteins to provide efficient adenosine modifications to ADAR proteins through the incorporation of various modifications and / or control over stereochemistry.

[0238] In some embodiments, oligonucleotides may have or contain a base sequence; internucleotide linkages, base modifications, sugar modifications, additional chemical motifs or patterns thereof; and / or any other structural elements described herein, such as those in the table.

[0239] In some embodiments, the provided oligonucleotide or composition is characterized in that, when it is contacted in a system (e.g., an ADAR-mediated deamination system) with a target nucleic acid containing a target adenosine, the modification of the target adenosine (e.g., deamination of target A) is improved relative to the modification of the target adenosine observed under reference conditions (e.g., selected from the group consisting of the absence of the composition, the presence of the reference oligonucleotide or composition, and combinations thereof). In some embodiments, modifications, such as ADAR-mediated deamination (e.g., endogenous ADAR-mediated deamination), are increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 times or more.

[0240] In some embodiments, the oligonucleotide is provided in the form of a salt. In some embodiments, the oligonucleotide is provided in the form of a salt comprising a negatively charged internucleotide link (e.g., a thiophosphate nucleotide link, a native phosphate link, etc.) present as a salt. In some embodiments, the oligonucleotide is provided in the form of a pharmaceutically acceptable salt. In some embodiments, the oligonucleotide is provided in the form of a metal salt. In some embodiments, the oligonucleotide is provided in the form of a sodium salt. In some embodiments, the oligonucleotide is provided in the form of an ammonium salt. In some embodiments, the oligonucleotide is provided in the form of a metal salt, such as a sodium salt, wherein each negatively charged internucleotide link is independently in the form of a salt (e.g., for a sodium salt, for a thiophosphate nucleotide link it is -OP(O)(SNa)-O-, for a native phosphate link it is -OP(O)(ONa)-O-, etc.).

[0241] In some embodiments, the oligonucleotide is chiral controlled and comprises one or more chiral controlled internucleotide bonds. In some embodiments, the provided oligonucleotide is stereochemically pure. In some embodiments, the provided oligonucleotide or composition thereof is substantially free of other stereoisomers. In some embodiments, this disclosure provides chiral controlled oligonucleotide compositions.

[0242] As described herein, the oligonucleotides of this disclosure can be provided with high purity (e.g., 50%-100%). In some embodiments, the oligonucleotides of this disclosure have high stereochemical purity (e.g., 50%-100%). In some embodiments, the oligonucleotides in the provided compositions have high stereochemical purity (e.g., a high percentage (e.g., 50%-100%) of one stereoisomer compared to other stereoisomers of the same oligonucleotide). In some embodiments, the percentage is at least or about 50%. In some embodiments, the percentage is at least or about 60%. In some embodiments, the percentage is at least or about 70%. In some embodiments, the percentage is at least or about 75%. In some embodiments, the percentage is at least or about 80%. In some embodiments, the percentage is at least or about 85%. In some embodiments, the percentage is at least or about 90%. In some embodiments, the percentage is at least or about 95%.

[0243] In some embodiments, the oligonucleotides of this disclosure are at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% stereochemically pure at the linking phosphorus sites between the chiral nucleotides. In some embodiments, the oligonucleotides of this disclosure are stereoselectively prepared and substantially free of stereoisomers. In some embodiments, in a provided composition comprising a plurality of oligonucleotides sharing the same base sequence of a chiral linking phosphorus stereochemistry (e.g., comprising one or more of Rp and / or Sp, wherein each chiral linking phosphorus is independently Rp or Sp), at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition sharing the same base sequence with the plurality of oligonucleotides share the same chiral linking phosphorus stereochemistry pattern or the plurality of oligonucleotides. In some embodiments, in the provided composition comprising a plurality of oligonucleotides sharing the same base sequence with the same chiral phosphorus-linked stereochemical pattern, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides sharing the same composition with the plurality of oligonucleotides share the same chiral phosphorus-linked stereochemical pattern or the plurality of oligonucleotides. In some embodiments, the diastereomeric excess of each chiral phosphorus is independently about or at least about 90%. In some embodiments, the diastereomeric excess of each chiral phosphorus is independently about or at least about 95%. In some embodiments, the diastereomeric excess of each chiral phosphorus is independently about or at least about 97%. In some embodiments, the diastereomeric excess of each chiral phosphorus is independently about or at least about 98%. In some embodiments, the diastereomeric purity is about or at least about (DS). nc Where DS is about 90%-100%, and nc is the number of chiral linked phosphorus. In some embodiments, DS is about 90% or greater. In some embodiments, DS is about 95% or greater. In some embodiments, DS is about 96% or greater. In some embodiments, DS is about 97% or greater. In some embodiments, DS is about 98% or greater. In some embodiments, DS is about 99% or greater. In some embodiments, diastereomeric purity is expressed as the product of the diastereomeric purity of each chiral linked phosphorus. Various oligonucleotide designs and features, such as sugars, nucleobases, internucleotide linkages and their patterns, first domains, second domains, first subdomains, second subdomains, third subdomains, etc., are described in WO 2023 / 201095 and used in accordance with this disclosure; they are incorporated herein by reference.

[0244] First structural domain

[0245] As described herein, in some embodiments, the oligonucleotide comprises a first domain and a second domain. In some embodiments, the oligonucleotide consists of a first domain and a second domain. Some embodiments are described below by way of example.

[0246] In some embodiments, the first domain has a length of about 2 to 50 nucleotides (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc.). In some embodiments, the first domain has a length of about 5 to 30 nucleotides. In some embodiments, the first domain has a length of about 10 to 30 nucleotides. In some embodiments, the first domain has a length of about 10 to 25 nucleotides. In some embodiments, the first domain has a length of about 10 nucleotides. In some embodiments, the first domain has a length of 15 nucleotides. In some embodiments, the first domain has a length of 20 nucleotides. In some embodiments, the first domain has a length of about 20-25 nucleotides.

[0247] In some embodiments, the first domain is about or at least about 5%-95%, 10%-90%, 20%-80%, 30%-70%, 40%-70%, 40%-60%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. In some embodiments, the percentage is about 30%-80%. In some embodiments, the percentage is about 30%-70%. In some embodiments, the percentage is about 40%-60%. In some embodiments, the percentage is about 20%. In some embodiments, the percentage is about 25%. In some embodiments, the percentage is about 30%. In some embodiments, the percentage is about 35%. In some embodiments, the percentage is about 40%. In some embodiments, the percentage is about 45%. In some embodiments, the percentage is about 50% or more. In some embodiments, the percentage is about 50%. In some embodiments, the percentage is about 55%. In some embodiments, the percentage is about 60%. In some embodiments, the percentage is about 60% or more. In some embodiments, the percentage is about 55%. In some embodiments, the percentage is about 60%. In some embodiments, the percentage is about 65%. In some embodiments, the percentage is about 70%. In some embodiments, the percentage is about 75%. In some embodiments, the percentage is about 80%. In some embodiments, the percentage is about 85%. In some embodiments, the percentage is about 90%.

[0248] In some embodiments, when the oligonucleotide is complementary to the target nucleic acid, there are one or more mismatches (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) in the first domain. In some embodiments, when the oligonucleotide is complementary to the target nucleic acid, there are one or more wiggles (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) in the first domain.

[0249] In some embodiments, the double strands of the oligonucleotide and the target nucleic acid in the first domain region include one or more protrusions, each protrusion independently containing one or more non-wobbly mismatches. In some embodiments, there are 0-10 protrusions (e.g., 0-1, 0-2, 0-3, 0-4, 0-5, 0-6, 0-7, 0-8, 0-9, 0-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.).

[0250] In some embodiments, the first domain is completely complementary to the target nucleic acid.

[0251] In some embodiments, the first domain comprises one or more modified nucleobases.

[0252] In some embodiments, the first domain comprises one or more sugars containing two 2'-H atoms (e.g., natural DNA sugars). In some embodiments, the first domain comprises one or more sugars containing 2'-OH atoms (e.g., natural RNA sugars). In some embodiments, the first domain comprises one or more modified sugars. In some embodiments, the modified sugars contain 2'-modification. In some embodiments, the modified sugars are bicyclic sugars, such as LNA sugars. In some embodiments, the modified sugars are acyclic sugars (e.g., by cleaving the C2-C3 bonds of the corresponding cyclic sugar).

[0253] In some embodiments, the first domain comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.) modified sugars. In some embodiments, the first domain comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.) modified sugars having 2'-F modification. In some embodiments, the first structural domain comprises about 2 to 50 (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 1...). 4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.; 2-40, 2-30, 2-25, 2-20, 2-15, 2-10, 3-40, 3-30, 3-25, 3-20, 3-15, 3-10, 4-40, 4-30, 4-25, 4-20, 4-1 5, 4-10, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 6-40, 6-30, 6-25, 6-20, 6-15, 6-10, 7-40, 7-30, 7-25, 7-20, 7-15, 7-10, 8-40, 8-30, 8-25, 8-20, 8-15, 8-10, 9- 40, 9-30, 9-25, 9-20, 9-15, 9-10, 10-40, 10-30, 10-25, 10-20, 10-15, approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc.) consecutive modified sugars with 2'-F modification.In some embodiments, the first domain comprises two consecutive 2'-F modified sugars. In some embodiments, the first domain comprises three consecutive 2'-F modified sugars. In some embodiments, the first domain comprises four consecutive 2'-F modified sugars. In some embodiments, the first domain comprises five consecutive 2'-F modified sugars. In some embodiments, the first domain comprises six consecutive 2'-F modified sugars. In some embodiments, the first domain comprises seven consecutive 2'-F modified sugars. In some embodiments, the first domain comprises eight consecutive 2'-F modified sugars. In some embodiments, the first domain comprises nine consecutive 2'-F modified sugars. In some embodiments, the first domain comprises ten consecutive 2'-F modified sugars. In some embodiments, the first domain comprises two or more 2'-F modified sugar blocks, wherein each sugar in the 2'-F modified sugar block is independently a 2'-F modified sugar. In some embodiments, each 2'-F modified sugar block independently comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive 2'-F modified sugars as described herein. In some embodiments, two consecutive 2'-F modified sugar blocks are independently separated by a separating block comprising one or more sugars that are independently not 2'-F modified. In some embodiments, each sugar in the separating block is independently not 2'-F modified. In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) or all sugars in the separating block are independently not 2'-F modified. In some embodiments, the separating block comprises one or more bicyclic suga...

Claims

1. A method for modifying a target adenosine in a target nucleic acid in a system, the method comprising administering or delivering an oligonucleotide or a combination thereof to the system, wherein: The oligonucleotide contains 5'-N1N0N -1 -3', where N1, N0 and N -1 Each is an independent nucleoside and they are linked by internucleotide bonds; The oligonucleotide is capable of binding to the target nucleic acid, wherein NO is opposite to the target adenosine; and N0 sugar is , , , , ... , , , , , , , , , , , Or, the nucleobase of N0 is .

2. A compound containing 5'-N1N0N -1 -3' oligonucleotides, wherein: N1, N0 and N -1 Each is an independent nucleoside and they are linked by internucleotide bonds; The oligonucleotide is capable of binding to the target nucleic acid, wherein NO is opposite to the target adenosine; and N0 sugar is , , , , ... , , , , , , , , , , , Or, the nucleobase of N0 is .

3. A compound containing 5'-N1N0N -1 -3' oligonucleotides, wherein N1, N0 and N -1 Each is an independent nucleoside and is linked by internucleotide bonds, wherein the oligonucleotide is capable of binding to the target nucleic acid, and N0 is opposite to the target adenosine.

4. An oligonucleotide comprising one or more modified sugars and / or one or more modified nucleotides linked together, wherein the oligonucleotide comprises a first domain and a second domain, each independently comprising one or more nucleobases.

5. The oligonucleotide according to any one of claims 1 to 4, wherein when the oligonucleotide is contacted with a target nucleic acid containing a target adenosine in a system containing ADAR1 or ADAR2, the target adenosine in the target nucleic acid is modified.

6. The oligonucleotide according to any one of the preceding claims, wherein the oligonucleotide has about 10-200 (e.g., about 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-120, 10-150, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-120, 20-150, 20-200, 25-30, 25-40) The length of nucleobases (e.g., 25-50, 25-60, 25-70, 25-80, 25-90, 25-100, 25-120, 25-150, 25-200, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-120, 30-150, 30-200, 10, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, etc.).

7. The oligonucleotide according to any one of the preceding claims, wherein the first domain has a length of about 15 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases.

8. The oligonucleotide according to any one of the preceding claims, wherein the first domain comprises about 1 to 50 sugars (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.) having 2'-F modification.

9. The oligonucleotide according to any one of the preceding claims, wherein the first domain comprises about 2 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.) consecutive sugars having a 2'-F modification.

10. The oligonucleotide according to any one of the preceding claims, wherein the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars comprising 2'-OR modification, wherein R is optionally a substituted C 1-6 Aliphatic groups.

11. The oligonucleotide according to any one of the preceding claims, wherein the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars containing 2'-MOE modification.

12. The oligonucleotide according to any one of the preceding claims, wherein the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars containing 2'-OMe modification.

13. The oligonucleotide according to any one of the preceding claims, wherein the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) natural DNA sugars.

14. The oligonucleotide according to any one of the preceding claims, wherein the first about 1 to 5 sugars, for example 1, 2, 3, 4 or 5 sugars from the 5' end of the first domain are independently 2'-OR modified sugars, wherein R is independently an optionally substituted C 1-6 Aliphatic groups.

15. The oligonucleotide according to any one of the preceding claims, wherein the first domain comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.) modified nucleotides linked together.

16. The oligonucleotide according to any one of the preceding claims, wherein the first domain comprises one or more natural phosphate ester bonds.

17. The oligonucleotide according to any one of the preceding claims, wherein the nucleotide link between the first nucleoside and the second nucleoside of the first domain is a modified nucleotide link.

18. The oligonucleotide according to any one of the preceding claims, wherein the internucleotide linking between the first nucleoside and the second nucleoside of the first domain is a phosphorylguanidine internucleotide linking.

19. The oligonucleotide according to any one of the preceding claims, wherein the internucleotide link between the first nucleoside and the second nucleoside of the first domain is an n001 internucleotide link.

20. The oligonucleotide according to any one of claims 1 to 17, wherein the nucleotide linking between the first nucleoside and the second nucleoside in the first domain is a phosphate thioester nucleotide linking.

21. The oligonucleotide according to any one of the preceding claims, wherein the second domain has a length of about 2 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases.

22. The oligonucleotide according to any one of the preceding claims, wherein the second domain comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.) modified sugars having 2'-F modification.

23. The oligonucleotide according to any one of the preceding claims, wherein the second domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars comprising 2'-OR modification, wherein R is optionally a substituted C 1-6 Aliphatic groups.

24. The oligonucleotide according to any one of the preceding claims, wherein the second domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars containing 2'-OMe modification.

25. The oligonucleotide according to any one of the preceding claims, wherein the second domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars containing 2'-MOE modification.

26. The oligonucleotide according to any one of the preceding claims, wherein the second domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) LNA sugars.

27. The oligonucleotide according to any one of the preceding claims, wherein the second domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) natural DNA sugars.

28. The oligonucleotide according to any one of the preceding claims, wherein the second domain comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.) modified nucleotides linked together.

29. The oligonucleotide according to any one of the preceding claims, wherein the internucleotide link between the last nucleoside and the penultimate nucleoside of the second domain is a modified internucleotide link.

30. The oligonucleotide according to any one of the preceding claims, wherein the internucleotide link between the last nucleoside and the penultimate nucleoside of the second domain is a phosphorylguanidine internucleotide link.

31. The oligonucleotide according to any one of the preceding claims, wherein the internucleotide link between the last nucleoside and the penultimate nucleoside of the second domain is an n001 internucleotide link.

32. The oligonucleotide according to any one of claims 1 to 29, wherein the internucleotide link between the last nucleoside and the penultimate nucleoside of the second domain is a phosphate thioester internucleotide link.

33. The oligonucleotide according to any one of the preceding claims, wherein the second domain comprises one or more natural phosphate ester bonds.

34. The oligonucleotide according to any one of the preceding claims, wherein the second domain from 5' to 3' comprises or consists of a first subdomain, a second subdomain, and a third subdomain.

35. The oligonucleotide according to any one of the preceding claims, wherein the second domain comprises 5'-N1N0N -1 -3', where N0 is opposite to the target adenosine, or where the second subdomain contains 5'-N1N0N -1 -3', where N0 is the counterpart to target adenosine.

36. The oligonucleotide according to any one of the preceding claims, wherein the third subdomain has a length of about 1 to 50 (e.g., about 5, 6, 7, 8, 9 or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40 or 50, etc.) nucleobases.

37. The oligonucleotide according to any one of the preceding claims, wherein the oligonucleotide comprises a targeting portion.

38. The oligonucleotide according to any one of claims 1 to 37, wherein the portion is or comprises a ligand of a desialyl glycoprotein receptor.

39. The oligonucleotide according to any one of the preceding claims, wherein the nucleobase of N0 is hypoxanthine, T, A, G, U, C, b001U, b002U, b003U, b004U, b005U, b006U, b007U, b008U, b009U, b011U, b012U, b013U, b014U, b015U, b001A, b002A, b003A, b004A, b005A, b006A, b007A, b001G, b002G, b0 01C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b009C, b002I, b003I, b004I, b014I, [3ne5U], [3nT], [3nfl5U], [3npry5U], [3ncn5U], [napth6o8A], [ipr6o8A], [c7In], [c39z48In], [z2c3In], [z5C], zndp, or selected from Table BA-1.

40. The oligonucleotide according to any one of claims 1 to 38, wherein the nucleobase of NO is .

41. The oligonucleotide according to any one of claims 1 to 38, wherein the nucleobase of NO is .

42. The oligonucleotide according to any one of claims 1 to 38, wherein the nucleobase of NO is .

43. The oligonucleotide according to any one of the preceding claims, wherein the sugar of NO is a 2'-F modified sugar or a natural DNA sugar.

44. The oligonucleotide according to any one of claims 1 to 42, wherein the sugar of NO is a 2'-OR modified sugar, wherein R is optionally a substituted C 1-6 Aliphatic groups.

45. The oligonucleotide according to any one of claims 1 to 42, wherein the sugar of NO is a sugar modified with 2'-OMe.

46. ​​The oligonucleotide according to any one of claims 1 to 42, wherein the sugar of N0 comprises , or ,in: X s -O-, -S-, or optionally substituted -CH2-; The sugar is bonded to a nucleobase at s1; and X s2 For optional substituted divalent C 1-6 Aliphatic chains.

47. The oligonucleotide according to any one of claims 1 to 42, wherein the sugar of N0 is , , ,in: X s -O-, -S-, or optionally substituted -CH2-; The sugar is bonded to a nucleobase at s1; and X s2 For optional substituted divalent C 1-6 Aliphatic chains.

48. The oligonucleotide according to any one of claims 46 to 47, wherein X s It is -O-.

49. The oligonucleotide according to any one of claims 46 to 47, wherein X s For -S-.

50. The oligonucleotide according to any one of claims 46 to 47, wherein X s It is -CH2-.

51. The oligonucleotide according to any one of claims 46 to 50, wherein X s2 For optional substituted divalent C 2-3 Aliphatic chains.

52. The oligonucleotide according to any one of claims 46 to 50, wherein X s2 It is -CH2-CH2-.

53. The oligonucleotide according to any one of claims 46 to 50, wherein X s2 For -CH=CH-.

54. The oligonucleotide according to any one of claims 46 to 50, wherein X s2 It is -CH2-CH2-CH2-.

55. The oligonucleotide according to any one of claims 46 to 50, wherein X s2 It is -CH=CH-CH2-.

56. The oligonucleotide of claim 55, wherein the -CH2- is bonded to the carbon at position s1.

57. The oligonucleotide according to any one of claims 1 to 42, wherein the sugar of N0 is [thpyr], [S6thpyr], [B5thpyr], [R3thpyr], [h2367oxi], [h23pyr], [oxa], [m1d], [25d3r], [Ld], [ALd], [23tfu], [Bcm2fl2r], [c4d], [s4d], [As4d], [Lh23pyr], [S6Lh23pyr] or [Lthpyr].

58. The oligonucleotide according to any one of the preceding claims, wherein the sugar of N1 is a natural DNA sugar or a modified sugar.

59. The oligonucleotide according to any one of the preceding claims, wherein N -1 The sugar in it is a natural DNA sugar.

60. The oligonucleotide according to any one of the preceding claims, comprising 5'-N1N0N -1 N -2 -3', where N -2 To link N via internucleotide bonds -1 Linked nucleosides.

61. The oligonucleotide according to any one of the preceding claims, wherein N -2 The sugars are modified sugars.

62. The oligonucleotide according to any one of the preceding claims, wherein N -2 The sugar is a 2'-OR modified sugar, where R is an optionally substituted C. 1-6 Aliphatic groups or bicyclic sugars.

63. The oligonucleotide according to any one of the preceding claims, wherein N -2 The sugar is either 2'-OMe modified or 2'-MOE modified.

64. The oligonucleotide according to any one of the preceding claims, wherein N -1 With N -2 The nucleotide bonds between them are uncharged nucleotide bonds.

65. The oligonucleotide according to any one of the preceding claims, wherein N -1 With N -2 The nucleotide linkages between them are phosphorylguanidine nucleotide linkages.

66. The oligonucleotide according to any one of the preceding claims, wherein N -1 With N -2 The nucleotide bonds between them are n001.

67. The oligonucleotide according to any one of claims 1 to 63, wherein N -1 With N -2 The nucleotide bonds between them are methylphosphonate bonds.

68. The oligonucleotide according to any one of claims 1 to 63, wherein N -1 With N -2 The nucleotide bonds between them are phosphate bonds of thiophosphate.

69. The oligonucleotide according to any one of claims 1 to 63, wherein N -1 With N -2 The nucleotide bonds between them are natural phosphate ester bonds.

70. The oligonucleotide according to any one of the preceding claims, comprising 5'-N1N0N -1 N -2 N -3 -3', where N -3 To link N via internucleotide bonds -2 Linked nucleosides.

71. The oligonucleotide according to any one of the preceding claims, wherein N -3 The sugars are modified sugars.

72. The oligonucleotide according to any one of the preceding claims, wherein N -3 The sugar is a sugar modified by 2'-F.

73. The oligonucleotide according to any one of the preceding claims, wherein N -2 With N -3 The nucleotide bonds between them are natural phosphate ester bonds.

74. The oligonucleotide according to any one of claims 1 to 72, wherein N -2 With N -3 The nucleotide linkages between them are modified nucleotide linkages.

75. The oligonucleotide according to any one of the preceding claims, comprising 5'-N1N0N -1 N -2 N -3 N -4 -3', where N -4 To link N via internucleotide bonds -3 Linked nucleosides.

76. The oligonucleotide according to any one of the preceding claims, wherein N -4 The sugars are modified sugars.

77. The oligonucleotide according to any one of claims 1 to 75, wherein N -4 The sugar is a 2'-OR modified sugar, where R is an optionally substituted C. 1-6 Aliphatic groups or bicyclic sugars.

78. The oligonucleotide according to any one of claims 1 to 75, wherein N -4 The sugar is a sugar modified with 2'-OMe.

79. The oligonucleotide according to any one of claims 1 to 75, wherein N -4 The sugar is a sugar modified with 2'-MOE.

80. The oligonucleotide according to any one of claims 1 to 75, wherein N -4 The sugar is a sugar modified by 2'-F.

81. The oligonucleotide according to any one of claims 1 to 80, wherein N -3 With N -4 The nucleotide linkages between them are modified nucleotide linkages, such as phosphate thioester nucleotide linkages.

82. The oligonucleotide according to any one of claims 1 to 80, wherein N -3 With N -4 The nucleotide bonds between them are natural phosphate ester bonds.

83. The oligonucleotide according to any one of the preceding claims, comprising 5'-N1N0N -1 N -2 N -3 N -4 N -5 -3', where N -5 To link N via internucleotide bonds -4 Linked nucleosides.

84. The oligonucleotide according to any one of the preceding claims, wherein N -5 The sugars are modified sugars.

85. The oligonucleotide according to any one of claims 1 to 83, wherein N -5 The sugar is a 2'-OR modified sugar, where R is an optionally substituted C. 1-6 Aliphatic groups or bicyclic sugars.

86. The oligonucleotide according to any one of claims 1 to 83, wherein N -5 The sugar is a sugar modified with 2'-OMe.

87. The oligonucleotide according to any one of claims 1 to 83, wherein N -5 The sugar is a sugar modified with 2'-MOE.

88. The oligonucleotide according to any one of claims 1 to 87, wherein N -4 With N -5 The nucleotide linkages between them are phosphate thionucleotide linkages.

89. The oligonucleotide according to any one of claims 1 to 87, wherein N -4 With N -5 The nucleotide linkages between them are n001 nucleotide linkages.

90. The oligonucleotide according to any one of the preceding claims, comprising 5'-N1N0N -1 N -2 N -3 N -4 N -5 N -6 -3', where N -6 To link N via internucleotide bonds -5 Linked nucleosides.

91. The oligonucleotide according to any one of the preceding claims, wherein N -6 The sugars are modified sugars.

92. The oligonucleotide according to any one of claims 1 to 90, wherein N -6 The sugar is a 2'-OR modified sugar, where R is an optionally substituted C. 1-6 Aliphatic groups or bicyclic sugars.

93. The oligonucleotide according to any one of claims 1 to 90, wherein N -6 The sugar is a sugar modified with 2'-OMe.

94. The oligonucleotide according to any one of claims 1 to 90, wherein N -6 The sugar is a sugar modified with 2'-MOE.

95. The oligonucleotide according to any one of claims 1 to 94, wherein N -5 With N -6 The nucleotide linkages between them are called PN, such as phosphoryl guanidine (e.g., n001) nucleotide linkages.

96. The oligonucleotide according to any one of claims 1 to 94, wherein N -5 With N -6 The nucleotide linkages between them are phosphate thionucleotide linkages.

97. The oligonucleotide according to any one of the preceding claims, wherein N -6 It is the last nucleoside starting from the 5' end.

98. An oligonucleotide comprising a double-stranded region and a targeting region, wherein the targeting region is or comprises a second region according to any one of the preceding claims; or an oligonucleotide comprising a double-stranded region and a targeting region, wherein the targeting region is or comprises 5'-N1N0N according to any one of the preceding claims. -1 -3'.

99. The oligonucleotide according to any one of the preceding claims, wherein the oligonucleotide is in salt form.

100. The oligonucleotide according to any one of the preceding claims, wherein one or more chiral phosphate linkages are independently chiral controlled.

101. The oligonucleotide according to any one of the preceding claims, wherein the diastereomeric purity of said oligonucleotide is about or at least about (DS). nc Where DS is about 85%-100% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more), and nc is the number of chiral phosphorus-linked centers, or one or more of them (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of chiral phosphorus-linked centers in diastereomeric excess independently of about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or wherein the oligonucleotide has about 10%-100% (e.g., about 10%-95%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%). 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95% Purity of 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or approximately or at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.

102. A pharmaceutical composition comprising or delivering an effective amount of an oligonucleotide according to any one of the preceding claims or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

103. An oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides share: 1) Common base sequence, and 2) The same binding phosphorus stereochemistry independently at one or more (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more) chiral nucleotide interlinkings ("chiral-controlled nucleotide interlinkings"); Each of the plurality of oligonucleotides is independently an oligonucleotide according to any one of the preceding claims, or in its acid, base or salt form.

104. The composition of claim 103, wherein the plurality of oligonucleotides share a common structure, and the level of the plurality of oligonucleotides sharing the common structure in the oligonucleotides of the composition is about or at least about (DS). nc , where DS is about 85%-100% (e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more), and nc is the number of chiral-bonded phosphorus.

105. An oligonucleotide composition comprising an oligonucleotide according to any one of claims 1 to 99, wherein the composition is a stereorandom composition.

106. The composition according to any one of the preceding claims, wherein the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

107. An oligonucleotide, wherein a -O group is present at the site of internucleotide linkage, except that the oligonucleotides are modified. 5 -P L (R CA )-O 3 Apart from the structural bonding, the oligonucleotide is otherwise identical to the oligonucleotide according to any one of the preceding claims, wherein: P L For P or P(=W); W represents O, S, or W. N ; R CA It may contain, or optionally substituted or capped, chiral adjuvant portions. O 5 For the oxygen bonded to the 5'-carbon of the sugar, and O 3 The oxygen is bonded to the 3'-carbon of the sugar.

108. A phosphoramid, wherein the nucleobase of the phosphoramid is the nucleobase or tautomer of any one of claims 1 to 101, wherein the nucleobase or tautomer of the phosphoramid is optionally substituted or protected; or a phosphoramid, wherein the sugar of the phosphoramid is the sugar of any one of claims 1 to 101, wherein the sugar is optionally protected.

109. A method for preparing oligonucleotides or compositions, the method comprising coupling an -OH group of an oligonucleotide or nucleoside to a phosphoramidite according to claim 108.

110. A method comprising: Evaluating a drug agent or a combination thereof in cells, tissues, or animals, wherein the cells, tissues, or animals are or contain cells, tissues, or organs associated with or belonging to a condition, disorder, or disease, and / or contain nucleotide sequences associated with a condition, disorder, or disease; and Administering or delivering an effective amount of a medicine or composition for the prevention or treatment of a condition, disorder, or disease to a subject who is susceptible to or suffers from such condition, disorder, or disease; or A method comprising: Administering or delivering an effective amount of a drug or composition for the prevention or treatment of a condition, disorder, or disease to a subject susceptible to or suffering from such condition, disorder, or disease, wherein the drug or composition is evaluated in cells, tissues, or animals, wherein said cells, tissues, or animals are or contain cells, tissues, or organs associated with or belonging to the condition, disorder, or disease, and / or contain nucleotide sequences associated with the condition, disorder, or disease; or A method for characterizing oligonucleotides or compositions, the method comprising: The oligonucleotide or composition is administered to cells or populations of cells containing or expressing the ADAR1 polypeptide or its characteristic moiety, or encoding a polynucleotide of the ADAR1 polypeptide or its characteristic moiety; or A method for characterizing oligonucleotides or compositions, the method comprising: Administer the oligonucleotide or composition to a non-human animal or population thereof that contains or expresses the ADAR1 polypeptide or its characteristic moiety, or a polynucleotide encoding the ADAR1 polypeptide or its characteristic moiety; or A method for modifying a target adenosine in a target nucleic acid, the method comprising contacting the target nucleic acid with an oligonucleotide or composition according to any one of the preceding claims; or A method for deamination of a target adenosine in a target nucleic acid, the method comprising contacting the target nucleic acid with an oligonucleotide or composition according to any one of the preceding claims; or A method for producing a product of a specific nucleic acid or restoring or increasing the level of a product of a specific nucleic acid, the method comprising contacting a target nucleic acid with an oligonucleotide or composition according to any one of the preceding claims, wherein the target nucleic acid comprises a target adenosine, and the specific nucleic acid differs from the target nucleic acid in that the specific nucleic acid has I or G instead of the target adenosine; or A method for reducing the level of a product of a target nucleic acid, the method comprising contacting the target nucleic acid with an oligonucleotide or composition according to any one of the preceding claims, wherein the target nucleic acid comprises a target adenosine; or A method comprising: The oligonucleotide or composition according to any one of the preceding claims is contacted with a sample containing the target nucleic acid and adenosine deaminase, wherein: The base sequence of one or more oligonucleotides in the oligonucleotide composition is substantially complementary to the base sequence of the target nucleic acid; and The target nucleic acid contains target adenosine; The target adenosine is modified; or A method, which includes 1) Obtaining a first modification level of target adenosine in the target nucleic acid, said level being observed when a first oligonucleotide composition is contacted with a sample containing said target nucleic acid and adenosine deaminase, wherein said first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence substantially complementary to the base sequence of said target nucleic acid; and 2) Obtain a reference modification level of the target adenosine in the target nucleic acid, said level being observed when a reference oligonucleotide composition is contacted with a sample containing the target nucleic acid and adenosine deaminase, wherein said reference oligonucleotide composition comprises a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to the base sequence of the target nucleic acid; in: The first plurality of oligonucleotides contains more sugars with 2'-F modification, more sugars with 2'-OR modification, wherein R is not -H, and / or more chiral nucleotides linked together, than the reference plurality of oligonucleotides; and Compared to the oligonucleotides of the reference oligonucleotide composition, the first oligonucleotide composition provides a higher level of modification; or A method, which includes A first modification level of target adenosine in the target nucleic acid is obtained, said level being observed when a first oligonucleotide composition is contacted with a sample containing the target nucleic acid and adenosine deaminase, wherein said first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence substantially complementary to the base sequence of the target nucleic acid; and The first modification level of the target adenosine is higher than the reference modification level of the target adenosine, wherein the reference level is observed when the reference oligonucleotide composition is contacted with a sample containing the target nucleic acid and adenosine deaminase, wherein the reference oligonucleotide composition comprises a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to the base sequence of the target nucleic acid. in: The first plurality of oligonucleotides contains more sugars with 2'-F modification, more sugars with 2'-OR modification, where R is not -H, and / or more chiral nucleotides linked together than the reference plurality of oligonucleotides; or A method, which includes 1) Obtaining a first modification level of target adenosine in the target nucleic acid, said level being observed when a first oligonucleotide composition is contacted with a sample containing said target nucleic acid and adenosine deaminase, wherein said first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence substantially complementary to the base sequence of said target nucleic acid; and 2) Obtain a reference modification level of the target adenosine in the target nucleic acid, said level being observed when a reference oligonucleotide composition is contacted with a sample containing the target nucleic acid and adenosine deaminase, wherein said reference oligonucleotide composition comprises a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to the base sequence of the target nucleic acid; in: The first plurality of oligonucleotides contains more sugars with 2'-F modification, more sugars with 2'-OR modification, wherein R is not -H, and / or more chiral-controlled chiral nucleotide linkages than the reference plurality of oligonucleotides; and Compared to the oligonucleotides of the reference oligonucleotide composition, the first oligonucleotide composition provides a higher level of modification; or A method, which includes A first modification level of target adenosine in the target nucleic acid is obtained, said level being observed when a first oligonucleotide composition is contacted with a sample containing the target nucleic acid and adenosine deaminase, wherein said first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence substantially complementary to the base sequence of the target nucleic acid; and The first modification level of the target adenosine is higher than the reference modification level of the target adenosine, wherein the reference level is observed when the reference oligonucleotide composition is contacted with a sample containing the target nucleic acid and adenosine deaminase, wherein the reference oligonucleotide composition comprises a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to the base sequence of the target nucleic acid. in: The first plurality of oligonucleotides contains more sugars with 2'-F modification, more sugars with 2'-OR modification, where R is not -H, and / or more chiral-controlled chiral nucleotide linkages than the reference plurality of oligonucleotides; or A method, which includes 1) Obtaining a first modification level of target adenosine in the target nucleic acid, said level being observed when a first oligonucleotide composition is contacted with a sample containing said target nucleic acid and adenosine deaminase, wherein said first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence substantially complementary to the base sequence of said target nucleic acid; and 2) Obtain a reference modification level of the target adenosine in the target nucleic acid, said level being observed when a reference oligonucleotide composition is contacted with a sample containing the target nucleic acid and adenosine deaminase, wherein said reference oligonucleotide composition comprises a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to the base sequence of the target nucleic acid; in: The first plurality of oligonucleotides comprises one or more chiral-controlled chiral nucleotide linkages; and The reference oligonucleotides do not contain chiral-controlled inter-chiral nucleotide bonds (the reference oligonucleotide composition is a "stereo-random composition"); and Compared to the oligonucleotides of the reference oligonucleotide composition, the first oligonucleotide composition provides a higher level of modification; or A method, which includes A first modification level of target adenosine in the target nucleic acid is obtained, said level being observed when a first oligonucleotide composition is contacted with a sample containing the target nucleic acid and adenosine deaminase, wherein said first oligonucleotide composition comprises a first plurality of oligonucleotides sharing the same base sequence substantially complementary to the base sequence of the target nucleic acid; and The first modification level of the target adenosine is higher than the reference modification level of the target adenosine, wherein the reference level is observed when the reference oligonucleotide composition is contacted with a sample containing the target nucleic acid and adenosine deaminase, wherein the reference oligonucleotide composition comprises a plurality of reference oligonucleotides sharing the same base sequence substantially complementary to the base sequence of the target nucleic acid. in: The first plurality of oligonucleotides comprises one or more chiral-controlled chiral nucleotide linkages; and The reference oligonucleotides do not contain chiral-controlled inter-chiral nucleotide bonds (the reference oligonucleotide composition is a "stereo-random composition").

111. A method for preventing or treating a condition, disorder, or disease, the method comprising administering or delivering an effective amount of an oligonucleotide or composition according to any one of the preceding claims to a subject susceptible to or suffering from said condition, disorder, or disease; or a method for preventing or treating a condition, disorder, or disease associated with a G to A mutation, the method comprising administering or delivering an effective amount of an oligonucleotide or composition according to any one of the preceding claims to a subject susceptible to or suffering from said condition, disorder, or disease.

112. The method of claim 111, wherein the condition, disorder, or disease is responsive to A to G or A to I modifications.

113. A method for regulating protein-protein interactions in a system, wherein a protein is translated from its encoding RNA, the method comprising contacting the encoding RNA with an oligonucleotide or composition according to any one of the preceding claims, wherein adenosine in the encoding RNA is edited, wherein the protein is translated from the edited mRNA ("edited protein"), wherein the edited protein differs from the unedited protein at amino acid residues involved in the protein-protein interaction; or A method for regulating protein-drug interactions in a system, wherein the protein is translated from its encoding RNA, the method comprising contacting the encoding RNA with an oligonucleotide or composition according to any one of the preceding claims, wherein adenosine in the encoding RNA is edited, wherein the protein is translated from the edited mRNA ("edited protein"), wherein the edited protein differs from the unedited protein at amino acid residues involved in the protein-drug interaction; or A method for regulating protein-drug interactions in a system, wherein the protein is translated from its encoding RNA, the method comprising administering or delivering to the system an oligonucleotide or composition according to any one of the preceding claims, wherein the adenosine in the encoding RNA is edited, wherein the protein is translated from the edited mRNA ("edited protein"), wherein the edited protein differs from the unedited protein at amino acid residues involved in the protein-drug interaction; or A method for modulating the interaction between a protein and its partner protein in a system, the method comprising administering or delivering to the system an oligonucleotide or composition according to any one of the preceding claims, wherein the oligonucleotide or composition is capable of editing adenosine in a nucleic acid encoding the protein or its partner protein, and the edited nucleic acid encodes a protein that is different from a protein encoded by an unedited nucleic acid at at least one amino acid residue relating to the interaction between the protein and its partner protein. or A method for regulating the level of nucleic acids in a system, the method comprising contacting the nucleic acids with an oligonucleotide or composition according to any one of the preceding claims, wherein adenosine in the nucleic acids is edited; A method for regulating the level of nucleic acids in a system, the method comprising administering or delivering to the system an oligonucleotide or composition according to any one of the preceding claims, wherein adenosine in the nucleic acid is edited; or A method for modifying a target adenosine in a target nucleic acid in a system, the method comprising administering or delivering to the system an oligonucleotide or composition according to any one of the preceding claims; or A method for deamination of target adenosine in a target nucleic acid in a system, the method comprising administering or delivering to the system an oligonucleotide or composition according to any one of the preceding claims; or A method for generating a product of a specific nucleic acid in a system, or restoring or increasing the level of a product of a specific nucleic acid, the method comprising administering or delivering to the system an oligonucleotide or composition according to any one of the preceding claims, wherein the target nucleic acid comprises a target adenosine, and the specific nucleic acid differs from the target nucleic acid in that the specific nucleic acid has I or G instead of the target adenosine; or A method for reducing the level of a target nucleic acid product in a system, the method comprising administering or delivering to the system an oligonucleotide or composition according to any one of the preceding claims, wherein the target nucleic acid comprises a target adenosine; or A method for regulating the level, structure, and / or activity of nucleic acids and / or products encoded therein in a system, the method comprising contacting the nucleic acid with an oligonucleotide or composition according to any one of the preceding claims, wherein adenosine in the nucleic acid is edited; or A method for regulating the level, structure, and / or activity of nucleic acids and / or products encoded therein in a system, the method comprising administering or delivering to the system an oligonucleotide or composition according to any one of the preceding claims, wherein adenosine in the nucleic acid is edited.

114. An oligonucleotide or oligonucleotide composition according to any one of the preceding claims, used in a method according to any one of the preceding claims, or used to manufacture a medicament for use in a method according to any one of the preceding claims.

115. The use of the oligonucleotide or oligonucleotide composition according to any one of the preceding claims for the method according to any one of the preceding claims, or the use of the oligonucleotide or oligonucleotide composition according to any one of the preceding claims for the manufacture of a medicament for the method according to any one of the preceding claims.

116. The oligonucleotide, composition, compound, method, or use according to any one of Examples 1 to 1076.

Images