Oligonucleotide compositions and methods related thereto

Abstract

The disclosure features useful oligonucleotide compositions and methods related thereto. The disclosure encompasses the knowledge that structural elements of oligonucleotides, such as base sequences, chemical modifications (e.g., modifications of sugars, bases, and / or inter-nucleotide linkages), or patterns thereof, may have significant effects on oligonucleotide characteristics and activity. The disclosure also provides methods of treating a disorder for which deamination of adenosine in mRNA produces a therapeutic outcome, such as in a subject in need thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 331,756, filed April 15, 2022, U.S. Provisional Application No. 63 / 397,320, filed August 11, 2022, U.S. Provisional Application No. 63 / 411,062, filed September 28, 2022, and U.S. Provisional Application No. 63 / 454,299, filed March 23, 2023, the entire contents of each of which are incorporated herein by reference. Background Art

[0002] Oligonucleotides can be used in a variety of applications, such as therapeutic, diagnostic and / or research applications. For example, oligonucleotides targeting a variety of genes can be used to treat conditions, disorders or diseases associated with such target genes. Summary of the Invention

[0003] In particular, the present disclosure provides designed oligonucleotides and compositions thereof comprising modifications as described herein (e.g., modifications to nucleobase sugars and / or internucleotide linkages, and patterns thereof). In some embodiments, the technology (compounds (e.g., oligonucleotides), compositions, methods, etc.) disclosed herein (e.g., oligonucleotides, oligonucleotide compositions, methods, etc.) are particularly useful for editing nucleic acids, such as site-directed editing in nucleic acids (e.g., editing of target adenosine). In some embodiments, as demonstrated herein, the technology provided 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, the present disclosure provides technology for editing in RNA (e.g., for modifying A residues, such as converting A to I). In some embodiments, the present disclosure provides technology for editing in transcripts (e.g., mRNA) (e.g., for modifying A residues, such as converting A to I). In particular, the technology provided provides the benefit of utilizing 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 a G to A mutation). Those skilled in the art will recognize that such utilization of endogenous proteins can avoid many challenges and / or provide various benefits compared to those technologies that require the delivery of exogenous components (e.g., proteins (e.g., those engineered to bind to oligonucleotides (and / or their duplexes with target nucleic acids) to provide the desired activity), nucleic acids encoding proteins, viruses, etc.).

[0004] In particular, in some embodiments, the oligonucleotides of the provided technology contain 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 linkage modifications and / or stereochemistry and / or patterns thereof [e.g., the type, modification and / or configuration of chiral phosphorus linkages (Rp or Sp), etc.], etc., which, when combined with one or more other structural elements described herein (e.g., additional chemical moieties), can provide high activity and / or a variety of desirable 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 to oligonucleotides having a high percentage of natural RNA sugars for adenosine editing. In some embodiments, the provided oligonucleotides provide high activity, such as adenosine editing activity. In some embodiments, provided oligonucleotides provide high selectivity, for example, in some embodiments, provided oligonucleotides provide selective modification of a target adenosine in a target nucleic acid relative to other adenosines in the same target nucleic acid (e.g., 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 greater than modification at another adenosine or all other adenosines in the target nucleic acid).

[0005] In particular, the present disclosure provides designed oligonucleotides and compositions having improved properties and / or activity compared to reference oligonucleotides and compositions (e.g., those described herein or reported in the art). For example, in some embodiments, as demonstrated herein, provided oligonucleotides and compositions can provide improved stability, pharmacokinetic properties, pharmacodynamic properties, and / or improved activity (e.g., for A to I editing). Various designed oligonucleotides and compositions are described herein. For example, in some embodiments, the present disclosure provides oligonucleotides and compositions thereof, including chirality-controlled oligonucleotide compositions thereof, wherein these oligonucleotides comprise several (e.g., 1, 2, 3, 4, or 5 or more; in some embodiments, 3 or more) nucleosides that independently comprise a sugar modification (e.g., a 2'-OR modification, wherein R is an optionally substituted C 1-6In some embodiments, the first nucleoside is a nucleotide sequence that is selected from the group consisting of alkyl (e.g., 2'-OMe, 2'-MOE, etc.), bicyclic sugar (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 comprise sugar modifications. In some embodiments, the first 3 or more and the last 3 or more nucleosides independently comprise sugar modifications. In some embodiments, the one or more internucleotide linkages bonded to such nucleosides are non-negatively charged internucleotide linkages, such as phosphorylguanidine internucleotide linkages, like n001. In some embodiments, the first and last internucleotide linkages are independently non-negatively charged internucleotide linkages. In some embodiments, the first and last internucleotide linkages are independently phosphorylguanidine internucleotide linkages. In some embodiments, the first and last internucleotide linkages are independently n001. In some embodiments, they are all chirally controlled and are Rp. In some embodiments, the oligonucleotide comprises a nucleoside N0 comprising a natural DNA sugar (two 2'-H), a natural RNA sugar, or a 2'-F modified sugar. In some embodiments, when the oligonucleotide is used for adenosine editing, N0 is the nucleoside opposite the target adenosine. In some embodiments, the sugar of N0 is a natural DNA sugar. In some embodiments, the sugar of N1 (a "+" or nothing before the number indicates counting toward the 5'-direction (5'...N1N0N) is the nucleoside opposite the target adenosine. -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 Sugar ("-" indicates counting toward 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 of is a DNA sugar. In some embodiments, N -3 In some embodiments, the oligonucleotide comprises multiple 2'-F modified sugars and multiple 2'-modified sugars (e.g., 2'-OR modified sugars, wherein R is an optionally substituted C 1-6In some embodiments, the oligonucleotide 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) 2'-F blocks from N2 to its 5'-end (e.g., when the first subdomain of the second domain terminates with and includes N2, the first domain and the first subdomain are combined). In some embodiments, the present invention provides a 2'-F block or a 2'-F block, wherein each nucleoside in the 2'-F block independently comprises a 2'-F modification, each nucleoside in the 2'-F block independently does not comprise a 2'-F modification, and each block independently 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) nucleoside. In some embodiments, there are two or more such 2'-F blocks and two or more such 2'-F blocks. In some embodiments, one or more or all such 2'-F blocks are independently bonded to two 2'-F blocks. In some embodiments, each nucleoside in one or more or all of the separating blocks independently comprises a 2'-OR modification, wherein R is an optionally substituted C 1-6 Alkyl or bicyclic sugars such as LNA sugars, cEt sugars, etc. In some embodiments, each nucleoside in one or more or all isolated blocks independently comprises a 2'-OR modification, wherein R is an optionally substituted C 1-6 In some embodiments, each nucleoside in one or more or all separating blocks independently comprises a 2'-OMe or 2'-MOE modification. In some embodiments, each of such 2'-F and separating blocks independently comprises 1, 2, 3, 4, or 5 nucleosides. In some embodiments, nucleosides close to NO, such as N2, N1, NO, N -1 、N -2 etc., without large 2'-modifications such as 2'-MOE. In some embodiments, N2, N1, N0, N -1 and N -2 The sugars of N1, N0, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, N16, N17, N18, N19, N20, N21, N22, N33, N34, N35, N46, N57, N68, N79, N80, N91, N10, N -1 The sugars of are each natural DNA sugars. In some embodiments, each chiral internucleotide linkage is independently chirality controlled.

[0006] In some embodiments, the present disclosure provides oligonucleotides 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 oligonucleotides comprise one or more chirally modified internucleotide linkages. In some embodiments, the present disclosure provides oligonucleotides comprising: (a) a first domain; and (b) the second domain, wherein 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 comprising a 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 comprising a 2'-OR modification, wherein R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-OL B -4', where L B is optionally substituted -CH2-, etc.); and 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 sugars, each independently comprising a 2'-OR modification, wherein R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-OL B -4', where L B is optionally substituted -CH2-, etc.).

[0007] In some embodiments, the present disclosure provides an oligonucleotide comprising: (a) a first domain; and (b) the second domain, wherein about 20%-80% (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%) of all carbohydrates in the first domain comprise 2'-F The modification comprises a 2'-OR modification, wherein R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-OL, 2'-OMe, 2'-MOE, 2'-OL, 2'-OMe, 2'-MOE, 2'-OL, 2'-OMe, 2'-MOE, 2'-OL, 2'-OMe, 2'-MOE, 2'-OL, 2'-OMe, 2'-MOE, 2'-OL, 2'-OMe, 2'-MOE, 2'-OL, 2'-OMe, 2'-MOE, 2'-OL, 2'-OMe, 2'-MOE, 2'-OL, 2'-OMe, 2'-MOE, 2'-OL, 2'-OMe, 2'-MOE, 2'-OL B -4', where L B is optionally substituted -CH2-, etc.); and 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 do not have a 2'-F modification, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars in the second domain do not have a 2'-F modification.

[0008] In some embodiments, the second domain comprises a first subdomain, a second subdomain, and a third subdomain as described herein, or consists of these domains. 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 comprising a 2'-OR modification, wherein R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-O-LB-4', wherein L B is optionally substituted -CH2-, etc.). In some embodiments, there are more such sugars in the first subdomain than 2'-F modified sugars. In some embodiments, none of the sugars in the second subdomain contain any 2'-OR modifications, where R is optionally substituted C 1-6 Aliphatic group, or 2'-OL BIn certain embodiments, each sugar of the second subdomain is independently a natural DNA sugar, a natural RNA sugar, or a 2'-F modified sugar. In certain embodiments, each sugar of the second subdomain is independently a natural DNA sugar or a natural RNA sugar. In certain embodiments, each sugar of the second subdomain is independently a natural DNA sugar or a 2'-F modified sugar. In certain embodiments, each sugar of the second subdomain is independently a natural DNA sugar or a 2'-F modified sugar. In certain embodiments, each sugar of the second subdomain is independently a natural DNA sugar. In certain embodiments, there are three nucleosides in the second subdomain. In certain embodiments, when combined with the target, the second nucleoside in the three nucleosides is opposite to the target adenosine. In certain embodiments, the sugar of the second nucleoside does not contain any 2'-OR modifications (e.g., 2'-OMe, 2'-MOE, etc.) as described herein. In certain embodiments, such sugar is a natural DNA sugar. In certain embodiments, it is a natural RNA sugar. In certain 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, each independently comprising a 2'-OR modification, wherein R is not -H (e.g., 2'-OMe, 2'-MOE, 2'-O-LB-4', wherein L B is optionally substituted -CH2-, etc.). In some embodiments, there are more such sugars in the third subdomain than 2'-F modified sugars.

[0009] 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 comprise a 2'-OR modification, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars in the second domain comprise a 2'-OR modification, wherein R is an optionally substituted C 1-6 In some embodiments, R is a methyl group. In some embodiments, R is -CH2CH2OCH3. As described herein, other sugar modifications can also be utilized according to the present disclosure, optionally with base modifications and / or internucleotide linkage modifications as described herein.

[0010] In certain embodiments, the oligonucleotide comprises or has a 5'-first domain-second domain-3' structure. In certain embodiments, the second domain comprises or has a 5'-first subdomain-second subdomain-third subdomain-3' structure. In certain embodiments, the oligonucleotide comprises or has a 5'-first domain-first subdomain-second subdomain-third subdomain-3' structure. In certain embodiments, the oligonucleotide is conjugated to another part, such as various other chemical parts as described herein. In certain embodiments, the oligonucleotide comprises another part, such as another part as described herein. In certain embodiments, another chemical part is or comprises a small molecule part, a carbohydrate part (e.g., a GalNAc part), a nucleic acid part (e.g., an oligonucleotide part, a nucleic acid part (e.g., a part of RNAse H dependent oligonucleotides, RNAi oligonucleotides, aptamers, gRNA, etc.)) and / or a peptide part.

[0011] In certain embodiments, the base sequence of the oligonucleotide provided is substantially complementary to the base sequence of the target nucleic acid comprising the target adenosine. In certain embodiments, when compared with the target nucleic acid, the oligonucleotide provided includes one or more mismatches (non-Watson-Crick base pairs). In certain embodiments, when compared with the target nucleic acid, the oligonucleotide provided includes one or more wobbles (wobble) (e.g., GU, IA, GA, IU, I-C, etc.). In certain embodiments, mismatches and / or wobbles can help one or more proteins (e.g., ADAR1, ADAR2, etc.) identify the duplex formed by the oligonucleotide provided and the target nucleic acid. In certain embodiments, the oligonucleotide provided forms a duplex with the target nucleic acid. In certain embodiments, ADAR proteins recognize and bind to such duplexes. In certain embodiments, the nucleoside opposite to the target adenosine is located in the middle of the oligonucleotide provided, for example, there are 5-50 nucleosides on the 5' side, and 1-50 nucleosides on its 3' side. In certain embodiments, the 5' side has more nucleosides than the 3' side. In certain embodiments, the 5' side has fewer nucleosides than the 3' side. In certain embodiments, the 5' side has the same number of nucleosides as the 3' side. In some embodiments, the oligonucleotides provided comprise 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 oligonucleotides provided is or comprises the base sequence of the oligonucleotides described in the table.

[0012] In some embodiments, utilizing various structural elements (e.g., various modifications, stereochemistries, and patterns thereof), the present disclosure can achieve desired properties and high activity with short oligonucleotides (e.g., those of about 20-40, 25-40, 25-35, 26-32, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length).

[0013] In certain embodiments, the oligonucleotides provided include modified nucleobases. In certain embodiments, modified nucleobases promote the modification of target adenine. In certain embodiments, compared with the presence of U, the nucleobase opposite to the target adenine maintains interaction with an enzyme such as ADAR, and the interaction with the target adenine is not as strong as with U (e.g., forming less hydrogen bonds). In certain embodiments, opposite nucleobases and / or their associated sugars provide certain flexibility (e.g., when compared with U) to promote modification of the target adenosine by an enzyme (e.g., ADAR1, ADAR2, etc.). In certain embodiments, nucleobases 5' or 3' next to the opposite nucleobase (target adenine), such as I and its derivatives, enhance the modification of the target adenine. In particular, the present disclosure recognizes that when the duplex of the oligonucleotides provided and their target nucleic acid interacts with a modifying enzyme (e.g., ADAR1 or ADAR2), such nucleobases can result in a steric hindrance smaller than G. In some embodiments, the base sequence of the oligonucleotide is selected (e.g., when several adenosine residues are suitable targets) and / or designed (e.g., by utilizing a variety of nucleobases described herein) such that steric hindrance is reduced or eliminated (e.g., no G is adjacent to the opposite nucleoside of the target A).

[0014] According to the present disclosure, a variety of internucleotide linkages can be used in oligonucleotides. In some embodiments, the oligonucleotide comprises one or more types of internucleotide linkages. In some embodiments, the oligonucleotide comprises two or more types of internucleotide linkages. In some embodiments, the oligonucleotide comprises at least three types of internucleotide linkages. In some embodiments, the linkage contains a linkage phosphorus atom bonded to an oxygen atom, which is not bonded to a backbone sugar or is not a part of a backbone sugar ("PO linkage", such as a natural phosphate linkage). In some embodiments, the linkage contains a linkage phosphorus atom bonded to a sulfur atom, which is not bonded to a backbone sugar or is not a part of a backbone sugar ("PS linkage", such as a thiophosphate internucleotide linkage). In some embodiments, the linkage contains a linkage phosphorus atom bonded to a nitrogen atom, which is not bonded to a backbone sugar or is not a part of a backbone sugar ("PN linkage", such as n001). In some embodiments, the oligonucleotide comprises one or more PS linkages. In some embodiments, the oligonucleotide comprises one or more PO linkages. In some embodiments, the oligonucleotide comprises one or more PN linkages. In some embodiments, the oligonucleotide comprises one or more PS linkages and one or more PO linkages. In some embodiments, the oligonucleotide comprises one or more PS linkages and one or more PN linkages. In some embodiments, the oligonucleotide comprises one or more PS linkages, one or more PN linkages, and one or more PO linkages. In some embodiments, the PS linkage is a phosphorothioate linkage. In some embodiments, each PS linkage is independently a phosphorothioate linkage. In some embodiments, the PO linkage is a natural phosphate linkage. In some embodiments, each PO linkage is independently a natural phosphate linkage. In some embodiments, the PN linkage is a phosphoguanidine linkage. In some embodiments, each PN linkage is independently a phosphoguanidine linkage.

[0015] In some embodiments, the first domain comprises one or more PO linkages, one or more PS linkages, and one or more PN linkages. In some embodiments, the first subdomain comprises one or more PO linkages, one or more PS linkages, and / or one or more PN linkages. In some embodiments, the first subdomain comprises one or more PO linkages. In some embodiments, the first subdomain comprises one or more natural phosphate linkages. In some embodiments, the second subdomain comprises one or more modified internucleotide linkages. In some embodiments, each internucleotide linkage to a nucleoside of the second subdomain is independently a modified internucleotide linkage. In some embodiments, each internucleotide linkage to a nucleoside of the second subdomain is independently a PS or PN linkage. In some embodiments, the third subdomain comprises one or more PO linkages, one or more PS linkages, and / or one or more PN linkages. In some embodiments, the third subdomain comprises one or more PO linkages. In some embodiments, the third subdomain comprises one or more natural phosphate linkages. In some embodiments, the third subdomain comprises one or more PS linkages. In some embodiments, the third subdomain comprises one or more PN linkages. In some embodiments, the third subdomain comprises one or more PO linkages, one or more PS linkages, and one or more PN linkages. In some embodiments, the first internucleotide linkage of the first domain or oligonucleotide is a PN linkage. In some embodiments, the last internucleotide linkage of the third subdomain or oligonucleotide is a PN linkage. In some embodiments, a natural DNA sugar is bonded to a modified internucleotide linkage. In some embodiments, a natural DNA sugar is bonded to a PN or PS internucleotide linkage. In some embodiments, each natural DNA sugar in an oligonucleotide or portion thereof (e.g., the first domain, the first subdomain, the second subdomain, the third subdomain, etc.) is independently bonded to a modified internucleotide linkage. In some embodiments, each natural DNA sugar is independently bonded to a PN or PS internucleotide linkage. In some embodiments, a natural RNA sugar is bonded to a modified internucleotide linkage. In some embodiments, a natural RNA sugar is bonded to a PN or PS internucleotide linkage. In some embodiments, each natural RNA sugar in an oligonucleotide or portion thereof (e.g., the first domain, the first subdomain, the second subdomain, the third subdomain, etc.) is independently bonded to a modified internucleotide linkage. In some embodiments, each natural RNA sugar is independently bonded to a PN or PS internucleotide linkage.

[0016] In some embodiments, the 2'-F modified sugar is bonded to a modified internucleotide linkage. In some embodiments, the 2'-F modified sugar is bonded to a PN or PS internucleotide linkage. In some embodiments, each 2'-F modified sugar in an oligonucleotide or portion thereof (e.g., the first domain, the first subdomain, the second subdomain, the third subdomain, etc.) is independently bonded to a modified internucleotide linkage. In some embodiments, each 2'-F modified sugar is independently bonded to a PN or PS internucleotide linkage. In some embodiments, each PO linkage is independently a natural phosphate linkage. In some embodiments, each PS linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, one or more PN linkages are independently non-negatively charged internucleotide linkages. In some embodiments, one or more PN linkages are independently neutral internucleotide linkages. In some embodiments, one or more PN linkages are independently phosphorylguanidine linkages. In some embodiments, each PN linkage is independently phosphorylguanidine linkages. In some embodiments, one or more PN linkages are independently n001. In some embodiments, each PN linkage is independently n001.

[0017] In some embodiments, the oligonucleotides disclosed herein provide modified internucleotide linkages (i.e., internucleotide linkages that are not natural phosphate linkages). In some embodiments, the linkage phosphorus of the modified internucleotide linkage (e.g., chiral internucleotide linkage) is chiral and can exist in different configurations (Rp and Sp). In particular, the disclosure demonstrates that the incorporation of modified internucleotide linkages, particularly with control of the stereochemistry of the linkage phosphorus center (such that a configuration is enriched at this control center compared to a stereorandom oligonucleotide preparation), can significantly improve properties (e.g., stability) and / or activity (e.g., adenosine modification activity (e.g., adenosine is converted to inosine)). In some embodiments, the oligonucleotides provided have a stereochemical purity significantly higher than that of a stereorandom preparation. In some embodiments, the oligonucleotides provided are chirally controlled.

[0018] In some embodiments, the oligonucleotides of the present disclosure comprise one or more chiral internucleotide linkages whose linkage phosphorus is chiral (e.g., phosphorothioate internucleotide 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 internucleotide linkages in the oligonucleotide, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 50%-100%, 60%-100%, 70%-100%) of all internucleotide linkages in the oligonucleotide. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, the chiral internucleotide linkages are chiral internucleotide linkages. In some embodiments, one or more chiral internucleotide linkages are independently non-negatively charged internucleotide linkages or neutral internucleotide linkages. In some embodiments, one or more chiral internucleotide linkages are independently phosphorylguanidine internucleotide linkages. In some embodiments, one or more chiral internucleotide linkages are independently chirality-controlled. In some embodiments, each chiral internucleotide linkage is independently chirality-controlled. In some embodiments, one or more chiral internucleotide linkages are not chirality-controlled. In some embodiments, each phosphorothioate internucleotide linkage is independently chirality-controlled. In some embodiments, each modified internucleotide linkage is independently phosphorothioate or non-negatively charged internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently phosphorothioate or neutral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently phosphorothioate or neutral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently phosphorothioate or phosphorylguanidine internucleotide linkage. In some embodiments, the phosphorylguanidine internucleotide linkage is n001. In some embodiments, each phosphorylguanidine internucleotide linkage is n001. In some embodiments, each non-negatively charged internucleotide linkage is n001. In some embodiments, each neutral internucleotide linkage is n001. In some embodiments, the modified internucleotide linkage 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 linkage phosphorus can be Rp or Sp. In some embodiments, at least one linkage phosphorus is Rp. In some embodiments, at least one linkage 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 internucleotide linkages in an oligonucleotide, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 50%-100%, 60%-100%, 85%-90%, 95%-100%) of all chiral internucleotide linkages in an oligonucleotide are chiral. or 99% etc.), or all chiral internucleotide linkages 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 phosphorothioate internucleotide linkages, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 50%-100%, 60%-100%) of all phosphorothioate internucleotide linkages in an oligonucleotide. In some embodiments, at least 50% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 60% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 70% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 75% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 80% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 85% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 90% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 95% of all phosphorothioate internucleotide linkages are Sp.In some embodiments, at least 96% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 97% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, at least 98% of all phosphorothioate internucleotide linkages are Sp. In some embodiments, all phosphorothioate internucleotide linkages are Sp. In some embodiments, no more than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than 3 consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than 4 consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than 5 consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than 6 consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than 7 consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than 8 consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than 9 consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, no more than 10 consecutive phosphorothioate internucleotide linkages are Rp. In some embodiments, consecutive Rp phosphorothioate internucleotide linkages are not used in moieties where the majority (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) or all of the sugars are native DNA and / or RNA and / or 2'-F modified sugars. In some embodiments, when consecutive Rp phosphorothioate internucleotide linkages are used, one or more or the majority (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) or all of the such internucleotide linkages are independently bonded to sugars, which can improve stability. In some embodiments, when continuous Rp phosphorothioate internucleotide linkages are used, one or more or a majority (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) or all such internucleotide linkages are independently to a bicyclic sugar or a 2'-OR modified sugar (wherein R is an optionally substituted C. 1-6 In some embodiments, when consecutive Rp phosphorothioate internucleotide linkages are used, one or more or a majority (e.g., greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more) or all such internucleotide linkages are independently linked to a 2'-OR modified sugar (wherein R is an optionally substituted C 1-6In 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.

[0019] In some embodiments, the stereochemistry of one or more chiral linkage phosphorus of a provided oligonucleotide is controlled in the composition. In some embodiments, the disclosure provides a composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides share a common base sequence and independently have one or more chiral internucleotide linkages ("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 In some embodiments, the present invention provides a plurality of oligonucleotides having at least one chiral oligonucleotide and at least one chiral oligonucleotide. In some embodiments, the present invention provides a plurality of oligonucleotides having at least one chiral oligonucleotide and at least one chiral oligonucleotide. In some embodiments, the present invention provides a plurality of oligonucleotides having at least one chiral oligonucleotide and at least one chiral oligonucleotide. In some embodiments, the present invention provides a plurality of oligonucleotides having at least one chiral oligonucleotide and at least one chiral oligonucleotide. In some embodiments, the present invention provides a plurality of oligonucleotides having at least one chiral oligonucleotide and at least one chiral oligonucleotide. 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 backbone 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.

[0020] In some embodiments, the present disclosure provides oligonucleotide compositions of controlled chirality of oligonucleotides, 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, independently have one or more chiral internucleotide linkages (e.g., about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-2 ... In some embodiments, the chiral oligonucleotides are selected from the group consisting of: -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 internucleotide linkages, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all chiral internucleotide linkages (e.g., all Rp or all Sp for the chiral linkage phosphorus). In some embodiments, the present disclosure provides oligonucleotide compositions of controlled chirality of oligonucleotides, 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 in one or more forms of oligonucleotides (e.g., acid form, salt form (e.g., a pharmaceutically acceptable salt form; as will be understood by one skilled in the art, where the oligonucleotide is a salt, other salt forms of the corresponding acid or base form of the oligonucleotide), etc.).

[0021] In some embodiments, as demonstrated herein, chirality-controlled oligonucleotide compositions provide numerous advantages over corresponding stereorandom oligonucleotide compositions, such as increased stability, activity, etc. In some embodiments, it was observed that chirality-controlled oligonucleotide compositions provide high levels of adenosine modification (e.g., converting A to I) activity with various isoforms of ADAR proteins (e.g., p150 and p110 forms of ADAR1), whereas corresponding stereorandom compositions provide high levels of adenosine modification (e.g., converting A to I) activity only with certain isoforms of ADAR proteins (e.g., the p150 isoform of ADAR1).

[0022] In certain embodiments, the oligonucleotides provided include other moieties, such as targeting moieties, carbohydrate moieties, etc. In certain embodiments, other moieties are or include the ligand of asialoglycoprotein receptors. In certain embodiments, other moieties are or include GalNAc or derivatives thereof. In particular, other moieties can be conducive to the delivery of certain target locations, such as cells, tissues, organs, etc. (such as, comprising the position of the receptor interacting with other moieties). In certain embodiments, other moieties are conducive to the delivery of the liver.

[0023] In certain embodiments, present disclosure provides the technology for preparing oligonucleotide and composition thereof, particularly the oligonucleotide composition of chirality control.In certain embodiments, provided oligonucleotide and composition thereof are high purity.In certain embodiments, the oligonucleotide of present disclosure is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% stereochemically pure at the linkage phosphorus of chiral internucleotide linkage.In certain embodiments, the oligonucleotide of present disclosure is stereoselectively prepared and is substantially free of stereoisomers.In certain embodiments, in the composition of the multiple oligonucleotides of the same base sequence of the provided comprising sharing the same chiral linkage phosphorus stereochemistry (for example, comprising one or more of Rp and / or Sp, wherein each chiral linkage phosphorus is independently Rp or Sp) pattern, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of all oligonucleotides sharing the same base sequence with these multiple oligonucleotides in the composition share the same chiral linkage phosphorus stereochemistry pattern or are these multiple oligonucleotides. In some embodiments, in provided compositions comprising a plurality of oligonucleotides having the same base sequence that share the same chiral phosphorus-bonded stereochemistry pattern, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition that share the same constitution as the plurality of oligonucleotides share the same chiral phosphorus-bonded stereochemistry pattern or are the plurality of oligonucleotides.

[0024] In some embodiments, the present disclosure describes useful techniques for evaluating oligonucleotides and compositions thereof. For example, the various techniques disclosed herein can be used to evaluate adenosine modifications. As will be appreciated by those skilled in the art, in some embodiments, the modification / editing of adenosine can be assessed by sequencing, mass spectrometry, assessment of the product (e.g., RNA, protein, etc.) of a modified nucleic acid (e.g., wherein the adenosine of the target nucleic acid is converted into inosine) (e.g., level, activity, etc.), optionally taking into account other components (e.g., ADAR proteins) present in the modification system (e.g., in vitro system, in vitro system, cell, tissue, organ, organism, subject, etc.). It will be understood by those skilled in the art that the oligonucleotides providing the adenosine modification of the target nucleic acid can also provide modified nucleic acids (e.g., wherein the target adenosine is converted into I) and one or more products thereof (e.g., mRNA, protein, etc.). Certain useful techniques are described in the examples.

[0025] As described herein, the oligonucleotides and compositions of the present disclosure can be provided / utilized in a variety of forms. In some embodiments, the present disclosure provides compositions comprising oligonucleotides in one or more forms, such as acid forms (e.g., wherein the natural phosphate linkage is present as -O(P(O)(OH)-O- and the phosphorothioate internucleotide linkage is present as -O(P(O)(SH)-O-), base forms, salt forms (e.g., wherein the natural phosphate linkage is present as a salt form (e.g., a sodium salt (-O(P(O)(O-)))), or a salt form (e.g., wherein the natural phosphate linkage is present as a salt form (e.g., a sodium salt (-O(P(O)(O-)))). - Na - )-O-)) exists, the phosphorothioate linkage exists as a salt form (e.g., sodium salt (-O(P(O)(S - Na + )-O-)) exists) etc. As will be appreciated by those skilled in the art, oligonucleotides can exist in various salt forms, including pharmaceutically acceptable salts, and in solutions (e.g., various aqueous buffer systems), cations can dissociate from anions. In certain embodiments, present disclosure provides pharmaceutical compositions comprising provided oligonucleotides and / or one or more pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is a chiral controlled oligonucleotide composition.

[0026] The provided technology can be used for a variety of purposes. For example, those skilled in the art will appreciate that the provided technology can be used for many purposes involving adenosine modification, for example, correcting G to A mutations, regulating the levels of certain nucleic acids and / or products encoded therefrom (e.g., by introducing A to G / I modifications to reduce the level of a protein), regulating splicing, regulating translation (e.g., by introducing A to G / I modifications to regulate translation start and / or stop sites), etc.

[0027] In some embodiments, the present disclosure provides techniques for preventing or treating disorders, disorders or diseases suitable for adenosine modification (e.g., converting A to I or G). As will be appreciated by those skilled in the art, I can perform one or more functions of G, such as base pairing, translation, etc. In some embodiments, the G to A mutation can be corrected by the conversion of A to I, so that one or more products of G-form nucleic acid, such as proteins, can be produced. In some embodiments, the present disclosure provides techniques for preventing or treating disorders, disorders or diseases associated with mutations, including administering to a subject susceptible to or suffering from the disorder, disorder or disease an oligonucleotide or composition thereof, which oligonucleotide or composition can edit mutations. In some embodiments, the present disclosure provides techniques for preventing or treating disorders, disorders or diseases associated with G to A mutations, including administering to a subject susceptible to or suffering from the disorder, disorder or disease an oligonucleotide or composition thereof, which oligonucleotide or composition can modify A. In some embodiments, the technology provided modifies A in transcripts (e.g., RNA transcripts). In some embodiments, A is converted to I. In some embodiments, during translation, the protein synthesis machinery reads I as G. In some embodiments, the A form encodes one or more proteins that have one or more higher desired activities and / or one or more better desired properties than the proteins encoded by its corresponding G form. In some embodiments, the A form provides higher levels of one or more proteins with one or more higher desired activities and / or one or more better desired properties than its corresponding G form. 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.

[0028] As will be appreciated by those skilled in the art, many conditions, disorders, or diseases are associated with mutations that can be modified by the provided technologies and can be prevented and / or treated using the provided technologies. For example, it has been reported that more than 20,000 conditions, disorders, or diseases are associated with G to A mutations and can benefit from A to I editing.

[0029] This application incorporates by reference the following applications: U.S. Provisional Application No. 63 / 111,079 filed on November 8, 2020, U.S. Provisional Application No. 63 / 175036 filed on April 14, 2021, U.S. Provisional Application No. 63 / 188,415 filed on May 13, 2021, U.S. Provisional Application No. 63 / 196,178 filed on June 2, 2021, U.S. Provisional Application No. 63 / 248,520 filed on September 26, 2021, and WO 2021 / 071858 and WO 2022 / 099159. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 .The technology provided comprising oligonucleotides of various lengths can provide editing. For various oligonucleotide compositions, editing of the SERPINA1-Z allele in primary mouse hepatocytes transgenic for human ADARp110 and SERPINA1-Z alleles was confirmed (N=2 biological replicates). Primary hepatocytes were treated for 48 hours with a specified oligonucleotide composition targeting the SERPINA1-Z allele. Compositions with oligonucleotides of different lengths (29-26 polymers) can provide different levels of editing. RNA editing was quantified by Sanger sequencing.

[0031] Figure 2 The provided technology including various modifications can provide editing. For various oligonucleotide compositions, editing of the SERPINA1-Z allele in primary mouse hepatocytes transgenic for human ADARp110 and SERPINA1-Z allele was confirmed (N=2 biological replicates). Primary hepatocytes were treated with designated oligonucleotides targeting the SERPINA1-Z allele for 48 hours. Various modifications (e.g., LNA sugars) were included at or around the editing region (e.g., at N0, N1, etc.) and at the junction between N0 and N1. -1 Compositions of oligonucleotides containing phosphorothioate or phosphorylguanidine internucleotide linkages between Rp or Sp can provide editing. RNA editing was quantified by Sanger sequencing.

[0032] Figure 3 .The technology provided, including a variety of modifications, can provide editing. For various oligonucleotide compositions, editing of the SERPINA1-Z allele in primary mouse hepatocytes transgenic for human ADARp110 and SERPINA1-Z alleles was confirmed (N=2 biological replicates). Primary hepatocytes were treated for 48 hours with a specified oligonucleotide composition targeting the SERPINA1-Z allele. Compositions of oligonucleotides comprising various sugar and / or base modifications (including at N0) can provide editing. RNA editing was quantified by Sanger sequencing.

[0033] Figure 4 .The technology provided, including a variety of modifications, can provide editing. For various oligonucleotide compositions, editing of the SERPINA1-Z allele in primary mouse hepatocytes transgenic for human ADARp110 and SERPINA1-Z alleles was confirmed (N=2 biological replicates). Primary hepatocytes were treated for 48 hours with a specified oligonucleotide composition targeting the SERPINA1-Z allele. Compositions of oligonucleotides comprising various sugar and / or base modifications (including at N0 and / or N1) can provide editing. RNA editing was quantified by Sanger sequencing.

[0034] Figure 5 .The technology provided, including a variety of modifications, can provide editing. For various oligonucleotide compositions, editing of the SERPINA1-Z allele in primary mouse hepatocytes transgenic for human ADARp110 and SERPINA1-Z alleles was confirmed (N=2 biological replicates). Primary hepatocytes were treated for 48 hours with a specified oligonucleotide composition targeting the SERPINA1-Z allele. Compositions of oligonucleotides comprising various sugar and / or base modifications (including at N0) can provide editing. NA editing was quantified by Sanger sequencing.

[0035] Figure 6 .The technology provided, including a variety of modifications, can provide editing. For various oligonucleotide compositions, editing of the SERPINA1-Z allele in primary mouse hepatocytes transgenic for human ADARp110 and SERPINA1-Z alleles was confirmed (N=2 biological replicates). Primary hepatocytes were treated for 48 hours with a specified oligonucleotide composition targeting the SERPINA1-Z allele. Oligonucleotides comprising various sugar and / or base modifications (including at N0) can provide editing. In some embodiments, 6-membered ring sugars are utilized. RNA editing is quantified by Sanger sequencing.

[0036] Figure 7The technology provided can provide editing in vivo. Editing of UGP2 transcripts in mice was confirmed in various tissue types (e.g., heart, kidney, lung, spleen, white adipose tissue (WAT), brown adipose tissue (BAT), liver) and various cell types (e.g., liver-related cells, such as CD3+, EpCAM+, CD146+ (LSEC), F4 / 80+ (M0) cells). Mice (N=5 / group) were subcutaneously administered 100 mg / kg of WV-50168 or PBS (control). After 7 days, the animals were euthanized and the designated tissues were collected. In addition, the liver was digested into a single cell suspension, and the designated cells were isolated using a commercially available kit. RNA editing was quantified by Sanger sequencing. Error bars represent standard deviations.

[0037] Figure 8 .The technology provided can provide editing in vivo. On days 0, 2, and 4, mice were administered 10 mg / kg of a specified oligonucleotide composition targeting NRF2 or UGP2. All oligonucleotides were GalNAc conjugated. On day 7, the animals were sacrificed and specified tissues (e.g., liver, kidney) were collected. In some embodiments, the technology provided can provide editing of target transcripts, which can provide, for example, regulation of protein-protein interactions, which can provide, for example, regulation of gene expression of downstream genes. RNA editing was quantified by Sanger sequencing. N=5 / group; error bars represent SEM.

[0038] Figure 9 .The provided technology can provide changes in gene activation in vivo. On days 0, 2, and 4, mice were administered 10 mg / kg of a specified oligonucleotide composition targeting NRF2 or UGP2. All oligonucleotides were GalNAc conjugated. On day 7, the animals were sacrificed and the livers were collected. In some embodiments, the provided technology can provide editing of target transcripts, which can provide, for example, regulation of protein-protein interactions, which can provide, for example, regulation of gene expression of downstream genes. (a) The provided technology can provide regulation of protein-protein interactions and / or regulation of gene expression. The expression of Nrf2-regulated genes NQO1 and a control (HPRT1) was analyzed via qPCR. (b) The provided technology can provide regulation of protein-protein interactions and / or regulation of gene expression. The expression of Nrf2-regulated genes GSTM1 and a control (HPRT1) was analyzed via qPCR. N=5 / group; error bars represent SEM.

[0039] Figure 10.The provided technology can provide modulation of protein-protein interactions. Primary mouse hepatocytes were administered a specified oligonucleotide composition targeting NRF2 at a specified concentration (e.g., 5, 1, 0.2, 0.04, 0.008, 0.0016 uM) via naked uptake for 48 hours. All oligonucleotides were GalNAc conjugated. In some embodiments, the provided technology can provide editing of target transcripts, which can provide, for example, modulation of protein-protein interactions, which can provide, for example, modulation of gene expression of downstream genes. N=2 / group; error bars represent SEM. (a) The provided oligonucleotide composition can provide editing of target NRF2 transcripts. RNA editing was quantified by Sanger sequencing. (b) The provided technology can provide modulation of protein-protein interactions and / or modulation of gene expression. The expression fold change of the Nrf2-regulated gene NQO1 was measured by qPCR. (c) The provided technology can provide modulation of protein-protein interactions and / or modulation of gene expression. The expression fold change of the Nrf2-regulated gene SLC17a was measured by qPCR.

[0040] Figure 11 The provided technology can provide upregulation of RNA expression. In some embodiments, editing of target transcripts provides editing of RNA sequence motifs (e.g., ARE motifs), which can provide disruption of interactions between RNA and RNA-binding proteins, alterations in RNA half-life, and / or upregulation of RNA expression. Human primary T cells were treated with 10 uM of the specified oligonucleotide composition for 48 hours via naked uptake. RNA expression was quantified by qPCR. N = 4 / group; error bars represent SEM.

[0041] Figure 12 The provided technology can provide upregulation of RNA expression. In some embodiments, editing of target transcripts provides editing of RNA sequence motifs (e.g., ARE motifs), which can provide disruption of interactions between RNA and RNA-binding proteins, alterations in RNA half-life, and / or upregulation of RNA expression. Human primary hepatocytes were treated with 10 uM of the specified oligonucleotide composition for 48 hours via naked uptake. RNA expression was quantified by qPCR. N = 4 / group; error bars represent SEM.

[0042] Figure 13The provided technology can provide upregulation of RNA expression. In some embodiments, editing of target transcripts provides editing of RNA sequence motifs (e.g., ARE motifs), which can provide disruption of interactions between RNA and RNA-binding proteins, alterations in RNA half-life, and / or upregulation of RNA expression. Human primary hepatocytes were treated with 10 uM of the specified oligonucleotide composition for 48 hours via naked uptake. RNA expression was quantified by qPCR. N = 4 / group; error bars represent SEM.

[0043] Figure 14 The provided technology can provide upregulation of RNA expression. In some embodiments, editing of target transcripts provides editing of RNA sequence motifs (e.g., ARE motifs), which can provide disruption of interactions between RNA and RNA-binding proteins, alterations in RNA half-life, and / or upregulation of RNA expression. Human primary hepatocytes were treated with 10 uM of the specified oligonucleotide composition for 48 hours via naked uptake. RNA expression was quantified by qPCR. N = 4 / group; error bars represent SEM.

[0044] Figure 15 .The technology provided can provide editing of target transcripts. Editing of the SERPINA1-Z allele was confirmed (N=2 biological replicates). Human patient iPSC-derived hepatocytes with a ZZ genotype were plated on day 0 and treated with specified oligonucleotides (e.g., WV-46312, WV-49090, WV-49092) at different concentrations (5, 1.25, 0.31, and 0.08 uM per oligonucleotide from left to right) on day 2. The culture medium was updated every 2 days (e.g., on days 4, 6, and 8). RNA was collected on day 10 and RNA editing was quantified by Sanger sequencing. Error bars represent standard error of the mean (SEM).

[0045] Figure 16 .The technology provided can provide editing of target transcripts. Editing of the SERPINA1-Z allele was confirmed (N=2 biological replicates). Human patient iPSC-derived hepatocytes with a ZZ genotype were plated on day 0 and treated with different concentrations (e.g., 5, 1.25, 0.31, and 0.08 uM) of specified oligonucleotides (WV-46312 on the left, WV-44515 on the right) on day 2. The culture medium was replaced every 2 days (e.g., on days 4, 6, and 8), and the specified oligonucleotides were added back every 2 days (e.g., on days 4, 6, and 8). RNA was collected on day 10 and RNA editing was quantified by Sanger sequencing. Error bars represent standard error of the mean (SEM).

[0046] Figure 17.The technology provided can provide editing in vivo. Editing of transcripts from the SERPINA1PiZ allele was confirmed. Seven-week-old NSG-PiZ mice (JAX catalog number 028842; N=5 per treatment group) were subcutaneously administered with a specified oligonucleotide composition (e.g., WV-49090) at a dose of 10 mg / kg per dose. One group of mice received a loading dose at week 1 (days 0, 2, and 4), while the other group received a single dose (no loading dose) at week 1 (day 0). These groups subsequently received an additional dose every 2 weeks (e.g., during weeks 2, 4, 6, 8, 10, and 12). The control group of mice received PBS. Liver biopsies were collected from mice at week 13 after treatment. RNA was collected from liver biopsies and RNA editing was quantified by Sanger sequencing. Error bars represent the standard error of the mean (SEM). The difference in editing % between the loading dose and the no-loading dose was tested using a one-way ANOVA adjusted for multiple comparisons (Tukey) (ns: not significant).

[0047] Figure 18 .The technology provided can increase SERPINA1 mRNA levels in vivo. Seven-week-old NSG-PiZ mice (JAX catalog number 028842; N=5 per treatment group) were subcutaneously administered a specified oligonucleotide composition (e.g., WV-49090) at a dose of 10 mg / kg per dose. One group of mice received a loading dose on week 1 (days 0, 2, and 4), while the other group received a single dose (no loading dose) on week 1 (day 0). These groups subsequently received an additional dose every 2 weeks (e.g., during weeks 2, 4, 6, 8, 10, and 12). Liver biopsies were collected from mice at week 13 after treatment. The control group of mice received PBS. RNA was collected from liver biopsies, and relative SERPINA1 mRNA levels (SERPINA1 / HPRT) were quantified using qPCR. Baseline measurements of relative SERPINA1 mRNA levels were determined based on liver biopsies from mice collected before dosing (week 0). Error bars represent standard error of the mean (SEM). Differences in relative SERPINA1 mRNA levels were tested using one-way analysis of variance (Dunnett) adjusted for repeated measures and multiple comparisons (***: P value < 0.001; ns: not significant).

[0048] Figure 19.The technology provided can reduce the level of mutant Z-AAT protein in serum and increase the level of wild-type (M) AAT protein. Seven-week-old NSG-PiZ mice (JAX catalog number 028842; N=5 per treatment group) were subcutaneously administered with a specified oligonucleotide composition (e.g., WV-49090) at a dose of 10 mg / kg per dose. One group of mice received a loading dose on week 1 (days 0, 2, and 4), while the other group received a single dose (no loading dose) on week 1 (day 0). These groups then received an additional dose every 2 weeks (e.g., during weeks 2, 4, 6, 8, 10, and 12). The control group of mice received PBS. Serum was collected from the mice at week 13 after treatment. The relative abundance of Z (mutant) and M (wild-type) AAT isoforms was determined by liquid chromatography-mass spectrometry (LC-MS). The error bars represent the standard error of the mean (SEM).

[0049] Figure 20 . Functional wild-type AAT protein can be produced by editing with various provided oligonucleotide compositions. Seven-week-old NSG-PiZ mice (JAX catalog number 028842; N=5 per treatment group) were subcutaneously administered the specified oligonucleotide composition at a dose of 10 mg / kg per dose. One group of mice received a loading dose at week 1 (days 0, 2, and 4), while the other groups received a single dose at week 1 (day 0). These groups subsequently received an additional dose every 2 weeks (e.g., during weeks 2, 4, 6, 8, 10, and 12). The control group of mice received PBS. Serum was collected from mice before administration and at week 13 after treatment. The relative elastase inhibitory activity in serum was determined in an in vitro reaction using a commercially available kit. The treatment groups at each time point were, from left to right: PBS control, WV-49090 (with loading doses on days 0, 2, and 4), and WV-49090 (no loading dose). The error bars represent the standard error of the mean (SEM). Two-way ANOVA adjusted for multiple comparisons (Bonferroni) was used to test differences in elastase inhibitory activity in serum between treatment groups receiving the indicated oligonucleotide compositions and the PBS control group (***: P value < 0.001; ns: not significant).

[0050] Figure 21 The provided technology can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphorylguanidine linkages, such as n001), etc.) and stereochemistry and their patterns were designed and evaluated. Hep3B cells were plated in 96-well plates and treated with specified concentrations of UGP2-targeting oligonucleotides (AIMers) via naked uptake for 48 hours. RNA was isolated and RNA editing was quantified using Sanger sequencing. Error bars represent standard deviations. N=3.

[0051] Figure 22 The provided technology can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphorylguanidine linkages, such as n001), etc.) and stereochemistry and their patterns were designed and evaluated. Hep3B cells were plated in 96-well plates and treated with designated oligonucleotides (AIMers) targeting ACTB at designated concentrations via naked uptake for 48 hours. RNA was isolated and RNA editing was quantified using Sanger sequencing. Error bars represent standard deviations. N=3.

[0052] Figure 23 The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphorylguanidine linkages, such as n001), etc.) and stereochemistry and their patterns were designed and evaluated. Hep3B cells were plated in 96-well plates and treated with designated oligonucleotides (AIMers) targeting SRSF1 at designated concentrations via naked uptake for 48 hours. RNA was isolated and RNA editing was quantified using Sanger sequencing. Error bars represent standard deviations. N=3.

[0053] Figure 24 The provided technology can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphorylguanidine linkages, such as n001), etc.) and stereochemistry and their patterns were designed and evaluated. Hep3B cells were plated in 96-well plates. On the second day, the cells were transfected with designated oligonucleotides (AIMers) targeting UGP2 at designated concentrations for 48 hours. RNA was isolated and RNA editing was quantified using Sanger sequencing. Error bars represent standard deviations. N=3.

[0054] Figure 25 The provided technology can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphorylguanidine linkages, such as n001), etc.) and stereochemistry and their patterns were designed and evaluated. Hep3B cells were plated in 96-well plates. On the second day, the cells were transfected with designated oligonucleotides (AIMers) targeting ACTB at designated concentrations for 48 hours. RNA was isolated and RNA editing was quantified using Sanger sequencing. Error bars represent standard deviations. N=3.

[0055] Figure 26.The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphorylguanidine linkages, such as n001), etc.) and stereochemistry and their patterns were designed and evaluated. Hep3B cells were plated in 96-well plates. On the second day, the cells were transfected with the specified concentrations of the specified oligonucleotides (AIMers) targeting SRSF1 for 48 hours. After treatment, RNA was isolated and RNA editing was quantified using Sanger sequencing. Error bars represent standard deviations. N=3.

[0056] Figure 27 .The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns are designed and evaluated. HEK293T cells are transfected with a plasmid encoding ADARp110 and incubated for 48 hours. The cells are dissociated from the flask and precipitated, then resuspended in an equal volume of RNA editing buffer. The resulting lysate is sonicated and clarified by centrifugation. The lysate is plated into a 96-well thermal cycler plate, and a specified oligonucleotide (AIMer) targeting UGP2 is added at a specified concentration. The reaction mixture is incubated at 30°C for 1 hour, after which RNA is extracted and edited quantitatively using Sanger sequencing. Error bars represent standard deviations. N=3.

[0057] Figure 28 .The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns are designed and evaluated. HEK293T cells are transfected with a plasmid encoding ADARp110 and incubated for 48 hours. The cells are dissociated from the flask and precipitated, then resuspended in an equal volume of RNA editing buffer. The resulting lysate is sonicated and clarified by centrifugation. The lysate is plated into a 96-well thermal cycler plate, and the specified oligonucleotides (AIMers) targeting ACTB are added at the specified concentrations. The reaction mixture is incubated at 30°C for 1 hour, after which RNA is extracted and edited quantitatively using Sanger sequencing. Error bars represent standard deviations. N=3.

[0058] Figure 29.The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns are designed and evaluated. HEK293T cells were transfected with a plasmid encoding ADARp110 and incubated for 48 hours. The cells were dissociated from the flask and pelleted, then resuspended in an equal volume of RNA editing buffer. The resulting lysate was sonicated and clarified by centrifugation. The lysate was plated into a 96-well thermal cycler plate, and the specified oligonucleotides (AIMers) targeting SRSF1 were added at the specified concentrations. The reaction mixture was incubated at 30°C for 1 hour, after which RNA was extracted and editing was quantified using Sanger sequencing. Error bars represent standard deviations. N=3.

[0059] Figure 30 .The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns were designed and evaluated. Primary mouse hepatocytes derived from hADAR mice were plated into 96-well plates. On the second day, the cells were treated with 3 μM of the specified oligonucleotides targeting UGP2 for 6 hours. The cells were then washed thoroughly with PBS and then refreshed with maintenance medium. Cells were collected and RNA was extracted at the specified times after the addition of the oligonucleotides. RNA editing was quantified by Sanger sequencing. A mixed effects model was used to test statistical significance. **: p < 0.01, ***: p < 0.0001, ns: not significant. Error bars represent standard deviations. N = 2.

[0060] Figure 31 .The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns were designed and evaluated. Primary mouse hepatocytes derived from hADAR mice were plated into 96-well plates. On the second day, the cells were treated with 3 μM of the specified oligonucleotides targeting SRSF1 for 6 hours. The cells were then washed thoroughly with PBS and then refreshed with maintenance medium. Cells were collected and RNA was extracted at the specified times after the addition of the oligonucleotides. RNA editing was quantified by Sanger sequencing. A mixed effects model was used to test statistical significance. *: p < 0.05, **: p < 0.01, ***: p < 0.0001, ns: not significant. Error bars represent standard deviations. N = 2.

[0061] Figure 32.The technology provided can provide efficient oligonucleotide delivery. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns are designed and evaluated. Primary mouse hepatocytes derived from hADAR mice were plated into 96-well plates. On the second day, the cells were treated with 3 μM of the specified oligonucleotides targeting UGP2 for 6 hours. The cells were then washed thoroughly with PBS and then lysed in RIPA buffer. The concentration of oligonucleotides (AIMer) in the resulting lysate was quantified using hybridization ELISA. One-way analysis of variance was used to test significance. ***: p < 0.001, ***: p < 0.0001. Error bars represent standard deviations. N = 4.

[0062] Figure 33 .The technology provided can provide efficient oligonucleotide delivery. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and patterns thereof were designed and evaluated. Primary mouse hepatocytes derived from hADAR mice were plated into 96-well plates. On the second day, the cells were treated with 3 μM of the specified oligonucleotides targeting SRSF1 for 6 hours. The cells were then washed thoroughly with PBS and then lysed in RIPA buffer. The concentration of oligonucleotides (AIMer) in the resulting lysate was quantified using hybridization ELISA. Significance was tested using one-way analysis of variance. ***p<0.0001. Error bars represent standard deviations. N=4.

[0063] Figure 34 .The technology provided can provide efficient oligonucleotide delivery. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns were designed and evaluated. Primary mouse hepatocytes derived from hADAR mice were plated into 96-well plates. On the second day, the cells were treated with 3 μM of the specified oligonucleotide targeting ACTB for 6 hours. The cells were then washed thoroughly with PBS and then lysed in RIPA buffer. The concentration of oligonucleotides (AIMer) in the resulting lysate was quantified using hybridization ELISA. Significance was tested using one-way analysis of variance. ***p<0.0001. Error bars represent standard deviations. N=4.

[0064] Figure 35.The technology provided can provide efficient oligonucleotide delivery. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns are designed and evaluated. Primary mouse hepatocytes derived from hADAR mice are plated into 96-well plates. On the second day, the cells are treated with 3 μM of the specified oligonucleotide targeting UGP2 for 6 hours. The cells are then washed thoroughly with PBS and then refreshed with maintenance medium. 96 hours after treatment, the cells are lysed in RIPA buffer. The concentration of oligonucleotides (AIMer) in the resulting lysate is quantified using hybridization ELISA. Compared with cells collected directly after treatment (e.g., Figure 32 The proportion of oligonucleotides remaining compared to the control (described in [ 0.05 ]) is shown on the Y-axis. Significance was tested using one-way ANOVA. **: p < 0.01, ***: p < 0.0001. Error bars represent standard deviation. N = 4.

[0065] Figure 36 .The technology provided can provide efficient oligonucleotide delivery. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns are designed and evaluated. Primary mouse hepatocytes derived from hADAR mice are plated into 96-well plates. On the second day, the cells are treated with 3 μM of the specified oligonucleotides targeting SRSF1 for 6 hours. The cells are then washed thoroughly with PBS and then refreshed with maintenance medium. 96 hours after treatment, the cells are lysed in RIPA buffer. The concentration of oligonucleotides (AIMer) in the resulting lysate is quantified using hybridization ELISA. Compared with cells collected directly after treatment (e.g., as Figure 33 The proportion of oligonucleotides remaining compared to the control (described in [ 0.05 ]) is shown on the Y-axis. Significance was tested using one-way ANOVA. ***: p < 0.001, ns: not significant. Error bars represent standard deviation. N = 4.

[0066] Figure 37 .The technology provided can provide efficient oligonucleotide delivery. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns are designed and evaluated. Primary mouse hepatocytes derived from hADAR mice are plated into 96-well plates. On the second day, the cells are treated with 3 μM of the specified oligonucleotide targeting ACTB for 6 hours. The cells are then washed thoroughly with PBS and then refreshed with maintenance medium. 96 hours after treatment, the cells are lysed in RIPA buffer. The concentration of oligonucleotide (AIMer) in the resulting lysate is quantified using hybridization ELISA. Compared with cells collected directly after treatment (e.g., as Figure 34The proportion of oligonucleotides remaining compared to the control (described in [ 0.05] ) is shown on the Y-axis. Significance was tested using one-way ANOVA. *: p < 0.05, ns: not significant. Error bars represent standard deviation. N = 4.

[0067] Figure 38 .The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.) and stereochemistry and their patterns are designed and evaluated. For naked editing % (bottom bar for each oligonucleotide): primary mouse hepatocytes derived from hADAR mice are plated into 96-well plates. The cells are treated with 6 μM of the specified oligonucleotides targeting UGP2 for 48 hours. RNA is collected and editing is quantified using Sanger sequencing. For cell-free editing % (top bar for each oligonucleotide): HEK293T cells are transfected with a plasmid encoding ADARp110 and incubated for 48 hours. The cells are dissociated from the flask and precipitated, then resuspended in an equal volume of RNA editing buffer. The resulting lysate is sonicated and clarified by centrifugation. The lysate is plated into a 96-well thermal cycler plate, and the specified oligonucleotides targeting UGP2 (AIMer) are added at specified concentrations. The reaction mixture was incubated at 30°C for 1 hour, after which RNA was extracted and quantified by Sanger sequencing. Error bars represent standard deviation. N = 2.

[0068] Figure 39 The provided technology can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphorylguanidine linkages such as n001), sugar modifications (e.g., 2'-F, 2'-OMe, etc.) and stereochemistry and patterns were designed and evaluated. Hep3B cells were plated in 96-well plates and treated with the specified concentrations of the specified oligonucleotides targeting UGP2 for 48 hours. RNA was isolated and RNA editing was quantified using Sanger sequencing. Error bars represent standard deviation. N=3.

[0069] Figure 40 The provided technology can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphorylguanidine linkages such as n001), sugar modifications (e.g., 2'-F, 2'-OMe, etc.) and stereochemistry and patterns were designed and evaluated. Hep3B cells were plated in 96-well plates. On the second day, the cells were transfected with the specified concentrations of the specified oligonucleotides targeting UGP2 for 48 hours. RNA was isolated and RNA editing was quantified using Sanger sequencing. Error bars represent standard deviation. N=3.

[0070] Figure 41.The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, etc.) and stereochemistry and their patterns are designed and evaluated. HEK293T cells were transfected with a plasmid encoding ADARp110 and incubated for 48 hours. The cells were dissociated from the flask and precipitated, then resuspended in an equal volume of RNA editing buffer. The resulting lysate was sonicated and clarified by centrifugation. The lysate was plated into a 96-well thermal cycler plate, and the specified oligonucleotides (AIMer) targeting UGP2 were added at the specified concentrations. The reaction mixture was incubated at 30°C for 1 hour, after which RNA was extracted and edited quantified using Sanger sequencing. Error bars represent standard deviations. N=3.

[0071] Figure 42 .The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, 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 derived from hADAR mice were plated into 96-well plates. On the second day, the cells were treated with 3 μM of the specified oligonucleotides targeting UGP2 for 6 hours. The cells were then washed thoroughly with PBS and then refreshed with maintenance medium. Cells were collected and RNA was extracted at the specified times after the addition of the oligonucleotides. RNA editing was quantified by Sanger sequencing. A mixed effects model was used to test statistical significance. *: p < 0.05, ***: p < 0.0001, ns: not significant. Error bars represent standard deviations. N = 2.

[0072] Figure 43 .The technology provided can provide efficient oligonucleotide delivery. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, etc.) and stereochemistry and patterns thereof are designed and evaluated. Primary mouse hepatocytes derived from hADAR mice are plated into 96-well plates. On the second day, the cells are treated with 3 μM of the designated oligonucleotides targeting UGP2 for 6 hours. The cells are then washed thoroughly with PBS and then lysed in RIPA buffer. The concentration of oligonucleotides (AIMer) in the resulting lysate is quantified using hybridization ELISA. One-way analysis of variance is used to test significance. ***: p < 0.0001. Error bars represent standard deviations. N = 4.

[0073] Figure 44.The technology provided can provide efficient oligonucleotide delivery. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, etc.) and stereochemistry and patterns thereof are designed and evaluated. Primary mouse hepatocytes derived from hADAR mice are plated into 96-well plates. On the second day, the cells are treated with 3 μM of AIMer targeting UGP2 for 6 hours. The cells are then washed thoroughly with PBS and then refreshed with maintenance medium. 96 hours after treatment, the cells are lysed in RIPA buffer. The oligonucleotide (AIMer) concentration is quantified using hybridization ELISA. Compared to cells collected directly after treatment (e.g., Figure 43 The proportion of oligonucleotides remaining compared to the control (described in [ 0.05 ]) is shown on the Y-axis. Significance was tested using one-way ANOVA. ***: p < 0.0001, ns: not significant. Error bars represent standard deviation. N = 4.

[0074] Figure 45 The provided technology can provide upregulation of RNA expression in vivo. In some embodiments, editing of target transcripts provides editing of RNA sequence motifs (e.g., ARE motifs), which can provide disruption of interactions between RNA and RNA-binding proteins, alteration of RNA half-life, upregulation of RNA expression, and / or upregulation of protein levels. Transgenic hADAR mice were subcutaneously administered 10 mg / kg of the indicated oligonucleotide compositions targeting the ARE site in the 3" UTR of the FGF21 transcript on days 0, 2, and 4 (N=5 per treatment group). Mice in the reference group received PBS. Serum was collected on day 0 (before dosing) and day 7 (after dosing). Liver biopsies were collected from mice on day 7. FGF21 mRNA editing in liver biopsies was quantified by Sanger sequencing (a). FGF21 mRNA levels in liver biopsies were quantified by qPCR. Quantified FGF21 mRNA levels were normalized to the corresponding HPRT mRNA levels (as also quantified by qPCR), and the fold change of the normalized FGF21 mRNA levels relative to the mean normalized FGF21 mRNA levels of mice administered PBS was determined (b). FGF21 protein levels in serum were quantified by ELISA (c). Error bars represent the standard deviation of the mean.

[0075] Figure 46.The technology provided can provide regulation of protein-protein interactions. Under bare conditions, primary human hepatocytes were administered increasing concentrations (e.g., 0.0016, 0.008, 0.04, 0.2, 1, 5 uM) of designated oligonucleotides (AIMers) targeting NRF2 transcripts (N=2 per group). RNA was collected after 48 h. (a) The oligonucleotide compositions provided can provide editing of target NRF2 transcripts. RNA editing was quantified by Sanger sequencing. The solid line represents the nonlinear regression fit of the plotted data. The dotted line represents the 95% confidence interval of the nonlinear regression fit of the plotted data. (b) The technology provided can provide regulation of protein-protein interactions and / or regulation of gene expression. The expression fold change of the Nrf2-regulated gene SLC17A11 was measured by qPCR. For each oligonucleotide, the horizontal bars represent, from top to bottom, the fold change of SLC7A11 mRNA expression measured after treatment at concentrations of 5, 1, 0.2, 0.04, 0.008, and 0.0016 uM. Error bars represent standard error of the mean (SEM).

[0076] Figure 47 The provided technology can provide editing. In some embodiments, compared to others, N -1 Certain nucleobases at N provide higher levels of editing than others. -1 Certain sugars at N provide higher levels of editing. In some embodiments, N -1 Certain nucleosides at the nucleotide position provided higher levels of editing. These sequences containing various nucleobases (e.g., natural nucleobases, b003U, b004U, b005U, b008U, b009U, b010U, b011U, b012U, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b001A, b003A, b004A, b006 Oligonucleotides with different oligonucleotides (e.g., A, b007A, b002I, b003I, b004I, b001G, b002G, etc.), sugars (e.g., native DNA sugars, 2'-F modified sugars, 2'-OMe modified sugars, sm11, sm12, sm15, sm17, etc.), linkages (e.g., PS, PN (e.g., phosphoguanidine linkages such as n001), etc.), and stereochemistry and patterns thereof. HEK293T cells were transfected with the ACTB 5'-CAG-3' plasmid and the ADAR-p110 plasmid. After 24 h, the cells were transfected with the indicated oligonucleotides. After 48 h, the cells were harvested and RNA was collected and transcribed into cDNA. Quantification was performed by Sanger sequencing. Error bars represent standard deviation (SD). N=2.

[0077] Figure 48The provided technology can provide editing. In some embodiments, compared to others, N -1 Certain nucleobases at N provide higher levels of editing than others. -1 Certain sugars at N provide higher levels of editing. In some embodiments, N -1 Certain nucleosides at the nucleotide position provided higher levels of editing. These sequences containing various nucleobases (e.g., natural nucleobases, b003U, b004U, b005U, b008U, b009U, b010U, b011U, b012U, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b001A, b003A, b004A, b006 Oligonucleotides with different oligonucleotides (e.g., A, b007A, b002I, b003I, b004I, b001G, b002G, etc.), sugars (e.g., native DNA sugars, 2'-F modified sugars, 2'-OMe modified sugars, sm11, sm12, sm15, sm17, etc.), linkages (e.g., PS, PN (e.g., phosphoguanidine linkages such as n001), etc.), and stereochemistry and patterns thereof. HEK293T cells were transfected with the ACTB 5'-GAG-3' plasmid and the ADAR-p110 plasmid. After 24 h, the cells were transfected with the indicated oligonucleotides. After 48 h, the cells were harvested and RNA was collected and transcribed into cDNA. Quantification was performed by Sanger sequencing. Error bars represent standard deviation (SD). N=2.

[0078] Figure 49 The provided technology can provide editing. In some embodiments, compared to others, N -1 Certain nucleobases at N provide higher levels of editing than others. -1 Certain sugars at N provide higher levels of editing. In some embodiments, N -1Certain nucleosides at the nucleotide position provided higher levels of editing. These sequences containing various nucleobases (e.g., natural nucleobases, b003U, b004U, b005U, b008U, b009U, b010U, b011U, b012U, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b001A, b003A, b004A, b006 Oligonucleotides with different oligonucleotides (e.g., A, b007A, b002I, b003I, b004I, b001G, b002G, etc.), sugars (e.g., native DNA sugars, 2'-F modified sugars, 2'-OMe modified sugars, sm11, sm12, sm15, sm17, etc.), linkages (e.g., PS, PN (e.g., phosphoguanidine linkages such as n001), etc.), and stereochemistry and patterns thereof. HEK293T cells were transfected with the ACTB 5'-TAG-3' plasmid and the ADAR-p110 plasmid. After 24 h, the cells were transfected with the indicated oligonucleotides. Cells were harvested after 48 h, and RNA was collected and transcribed into cDNA. Quantification was performed by Sanger sequencing. Error bars represent standard deviation (SD). N=2.

[0079] Figure 50 The provided technology can provide editing. In some embodiments, certain nucleobases at N0 provide higher levels of editing and / or selectivity compared to others. In some embodiments, certain sugars at N0 provide higher levels of editing and / or selectivity compared to others. In some embodiments, certain nucleosides at N0 provide higher levels of editing and / or selectivity compared to others. In some embodiments, certain nucleosides at N0 provide higher levels of editing and / or selectivity compared to others. -1 Certain nucleobases at N provide higher levels of editing and / or selectivity than others. -1 Certain sugars at N provide higher levels of editing and / or selectivity. In some embodiments, N -1Certain nucleosides at these sites provide higher levels of editing and / or selectivity. These sequences containing various nucleobases (e.g., natural nucleobases, b003U, b004U, b005U, b008U, b009U, b010U, b011U, b012U, b001C, b002C, b003C, b004C, b005C, b006C, b007C, b008C, b001A, b003A, b004A, b006 Oligonucleotides with different oligonucleotides (e.g., A, b007A, b002I, b003I, b004I, b001G, b002G, etc.), sugars (e.g., native DNA sugars, 2'-F modified sugars, 2'-OMe modified sugars, sm11, sm12, sm15, sm17, etc.), linkages (e.g., PS, PN (e.g., phosphoguanidine linkages such as n001), etc.), and stereochemistry and patterns thereof. HEK293T cells were transfected with the ACTB 5'-AAG-3' plasmid and the ADAR-p110 plasmid. After 24 h, the cells were transfected with the indicated oligonucleotides. After 48 h, the cells were harvested and RNA was collected and transcribed into cDNA. Quantification was performed by Sanger sequencing. Error bars represent standard deviation (SD). N=2.

[0080] Figure 51 .The technology provided can provide editing. Oligonucleotides containing various modifications such as base modifications (e.g., 3nU), linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), etc.), sugar modifications (e.g., 2'-F, 2'-OMe, etc.) and stereochemistry and their patterns are designed and evaluated. 3uM (a) or 0.3uM (b) of the specified oligonucleotides were administered to primary mouse hepatocytes in a naked manner. Cells were harvested after 72h, and RNA was collected and transcribed into cDNA. Editing was quantified by Sanger sequencing. Error bars represent standard error of the mean (SEM). N=2. *Indicates that data do not exist.

[0081] Figure 52 .The technology provided can provide editing. Oligonucleotides containing various modifications such as linkage modifications (e.g., PS, PN (e.g., phosphoguanidine linkages, such as n001), sugar modifications (e.g., 2'-F, 2'-OMe, etc.) and stereochemistry and their patterns were designed and evaluated. The designated oligonucleotides targeting KEAP1 or NRF2 transcripts were transfected into iPSC-derived astrocytes at 25nM (a) or into SF8628 human glioma cells at 25nM (b) or introduced into primary human hepatocytes at 10uM naked (c). WV-37317 targeting ACTB transcripts was included as a control. RNA was collected after 48h of incubation. RNA editing was quantified by Sanger sequencing. Error bars represent standard error of the mean (SEM). DETAILED DESCRIPTION

[0082] The presently disclosed technology may be understood more readily by reference to the following detailed description of certain embodiments. definition

[0083] As used herein, the following definitions shall apply unless otherwise indicated. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th ed. Additionally, general principles of organic chemistry are described in: "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5th ed., eds. Smith, MB and March, J., John Wiley & Sons, New York: 2001.

[0084] As used herein in this disclosure, unless the context clearly indicates otherwise, (i) the term "a" or "an" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising," "comprise," "including" (whether or not used with "not limited to") and "include" (whether or not used with "not limited to") may be understood to cover the itemized components or steps, whether shown alone or with one or more other components or steps; (iv) the term "another" may be understood to mean at least one additional / second one or more; (v) the terms "about" and "approximately" may be understood to allow for standard deviations, as would be understood by one of ordinary skill in the art; and (vi) where ranges are provided, the endpoints are inclusive.

[0085] Unless otherwise indicated, descriptions of oligonucleotides and their elements (e.g., base sequence, sugar modifications, internucleotide linkages, linkage phosphorus stereochemistry, patterns thereof, etc.) are from 5' to 3'. As will be understood by those skilled in the art, in some embodiments, oligonucleotides may be provided and / or used as salt forms, particularly pharmaceutically acceptable salt forms (e.g., sodium salts). As will be understood by those skilled in the art, in some embodiments, a single oligonucleotide in a composition may be considered to have the same composition and / or structure, even though in such a composition (e.g., a liquid composition), in particular, such an oligonucleotide may be in one or more different salt forms at a particular time (and, for example, when in a liquid composition, it may be dissolved and the oligonucleotide chain may be present in anionic form). For example, it will be understood by those skilled in the art that at a given pH, a single internucleotide linkage along an oligonucleotide chain may be in acid (H) form, or in 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 it will be understood that as long as their acid form (e.g., with H) is present, the oligonucleotides may be present in the same salt form. + Instead of all cations, if any) having the same composition and / or structure, such a single oligonucleotide may appropriately be considered to have the same composition and / or structure.

[0086] Aliphatic: As used herein, "aliphatic" means a linear (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units (but is not aromatic), or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more unsaturated units (but is not aromatic), or a combination thereof. In some embodiments, aliphatic groups contain 1-50 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0087] Alkenyl: As used herein, the term "alkenyl" refers to an aliphatic group as defined herein having one or more double bonds.

[0088] Alkyl: As used herein, the term "alkyl" is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight chain alkyls, branched chain alkyls, cycloalkyls (alicyclic groups), cycloalkyls substituted with alkyls, and alkyls substituted with cycloalkyls. In some embodiments, the alkyl group has 1-100 carbon atoms. In certain embodiments, the backbone of a straight chain or branched chain alkyl group has about 1-20 carbon atoms (e.g., a straight chain is C1-C 20 , the branch chain is C2-C 20 ), alternatively having about 1-10 carbon atoms. In some embodiments, the cycloalkyl ring has about 3-10 carbon atoms in its ring structure when such ring is monocyclic, bicyclic or polycyclic, alternatively having about 5, 6 or 7 carbons in the ring structure. In some embodiments, the alkyl group can be a lower alkyl group, wherein the lower alkyl group contains 1-4 carbon atoms (for example, a straight chain lower alkyl is C1-C4).

[0089] Alkynyl: As used herein, the term "alkynyl" refers to an aliphatic group as defined herein having one or more triple bonds.

[0090] Analog: The term "analog" includes any chemical moiety that is structurally different from a reference chemical moiety or class of moieties but that can perform at least one function of such referenced chemical moiety or class of moieties. As non-limiting examples, a nucleotide analog is structurally different from a nucleotide but can perform at least one function of a nucleotide; a nucleobase analog is structurally different from a nucleobase but can perform at least one function of a nucleobase; and so on.

[0091] Animal: As used herein, the term "animal" refers to any member of the kingdom Animalia. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0092] Aryl: As used herein, the term "aryl," used alone or as part of a larger moiety such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic, or polycyclic ring system having a total of five to thirty ring members, wherein at least one ring in the system is aromatic. In some embodiments, the aryl group is a monocyclic, bicyclic, or polycyclic ring 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 ring unit is aromatic. In some embodiments, the aryl group is a biaryl group. The term "aryl" is used interchangeably with the term "aryl ring." In certain embodiments of the present disclosure, "aryl" refers to an aromatic ring system including, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracenyl, and the like, which may have one or more substituents. Also included within the scope of the term "aryl," as used herein, are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthyridinyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0093] Characteristic portion: As used herein, the term "characteristic portion" refers, in its broadest sense, to a portion of a substance whose presence (or absence) correlates with 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 that is found in that substance and in related substances that share the particular characteristic, property, or activity, but not in substances that do not share the particular characteristic, property, or activity. In certain embodiments, a characteristic portion shares at least one functional property with the entire substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is a portion that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are unique to the protein or polypeptide. In some embodiments, each such continuous stretch 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 relevant entire substance. In some embodiments, a characteristic portion may be biologically active.

[0094] Chiral Control: As used herein, "chiral control" refers to controlling the stereochemical identity of the chiral phosphorus of a chiral internucleotide linkage within an oligonucleotide. As used herein, a chiral internucleotide linkage is an internucleotide linkage whose phosphorus is chiral. In some embodiments, control is achieved by a chiral element that is not present in the sugar and base portions of the oligonucleotide, for example, in some embodiments, by using one or more chiral auxiliaries during oligonucleotide preparation, which are typically part of the chiral phosphoramidites used during oligonucleotide preparation. In contrast to chiral control, one of ordinary skill in the art will recognize that if conventional oligonucleotide synthesis is used to form chiral internucleotide linkages, such conventional oligonucleotide synthesis without the use of chiral auxiliaries cannot control the stereochemistry at the chiral internucleotide linkage. In some embodiments, the stereochemical identity of each chiral phosphorus of each chiral internucleotide linkage within an oligonucleotide is controlled.

[0095] Chirality-controlled oligonucleotide composition: As used herein, the terms "chirality-controlled oligonucleotide composition," "chirality-controlled nucleic acid composition," and the like refer to a composition comprising a plurality of oligonucleotides (or nucleic acids) that share a common base sequence, wherein the plurality of oligonucleotides (or nucleic acids) share the same linkage phosphorus stereochemistry at one or more chiral internucleotide linkages (chirality-controlled or stereo-defined internucleotide linkages, wherein the chiral linkage phosphorus is either Rp or Sp ("stereo-defined") in the composition, rather than a random mixture of Rp and Sp as in achiral-controlled internucleotide linkages). In some embodiments, a chirality-controlled oligonucleotide composition comprises a plurality of oligonucleotides (or nucleic acids) that share 1) a common base sequence, 2) a common backbone linkage pattern, and 3) a common backbone phosphorus modification pattern, wherein the plurality of oligonucleotides (or nucleic acids) share the same linkage phosphorus stereochemistry at one or more chiral internucleotide linkages (chirality-controlled or stereo-defined internucleotide linkages, wherein the chiral linkage phosphorus is Rp or Sp ("stereo-defined") in the composition, rather than a random mixture of Rp and Sp as in achiral controlled internucleotide linkages). The levels of the plurality of oligonucleotides (or nucleic acids) in the chirality-controlled oligonucleotide composition are predetermined / controlled or enriched (e.g., prepared by chirality-controlled oligonucleotides to stereoselectively form one or more chiral internucleotide linkages) as compared to the random levels in the achiral controlled oligonucleotide composition. In some embodiments, 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%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 50%, 90%, 10 ... 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%) of the plurality of oligonucleotides.In some embodiments, 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%, 10%-100% of all oligonucleotides in a chirality-controlled oligonucleotide composition share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern. %, 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 plurality of oligonucleotides. In some embodiments, the level is of all oligonucleotides in the composition; or of all oligonucleotides in the composition that share a common base sequence (e.g., a base sequence of a plurality of oligonucleotides or a type of oligonucleotide); or of all oligonucleotides in the composition that share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern; or of about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%) of all oligonucleotides in the composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linkage type pattern, and / or a common internucleotide linkage modification pattern. %, 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 about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, 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 internucleotide linkages. In some embodiments, the plurality of oligonucleotides is present in an amount between 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%, about 5%, 10%, 15%, 20%, 25%, 30%, In some embodiments, the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at the chiral internucleotide linkages (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%). In some embodiments, the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at the chiral internucleotide linkages (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%). In some embodiments, the plurality of oligonucleotides (or nucleic acids) share the same sugar and / or nucleobase modification pattern. In some embodiments, the plurality of 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 plurality of 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% (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%-100%) of all oligonucleotides (or nucleic acids) in the composition having the same composition as the plurality of oligonucleotides (or nucleic acids). %, 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 internucleotide linkage is a chiral controlled internucleotide linkage, and the composition is a fully chiral controlled oligonucleotide composition. In some embodiments, a plurality of oligonucleotides (or nucleic acids) are structurally identical. In some embodiments, the chiral controlled internucleotide linkage has 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 internucleotide linkage has at least 95% diastereomeric purity. In some embodiments, the chiral controlled internucleotide linkage has at least 96% diastereomeric purity. In some embodiments, the chiral controlled internucleotide linkage has at least 97% diastereomeric purity. In some embodiments, the chirality-controlled internucleotide linkage has a diastereomeric purity of at least 98%. In some embodiments, the chirality-controlled internucleotide linkage has a diastereomeric purity of at least 99%. In some embodiments, the percentage of the level is or is at least (DS). nc , wherein DS is the diastereomeric purity as described herein (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more), and nc is the number of chiral linked phosphorus as described herein (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 , wherein DS is the diastereomeric purity as described herein (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more), and nc is the number of chirality-controlled internucleotide linkages as described herein (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 multiple oligonucleotides in the composition is expressed as the product of the diastereomeric purity of each chiral linkage phosphorus. In some embodiments, the level of multiple oligonucleotides in the composition is expressed as the product of the diastereomeric purity of each chiral-controlled internucleotide linkage chain in the oligonucleotide. In some embodiments, the diastereomeric purity of the internucleotide linkage connecting two nucleosides in an oligonucleotide (or nucleic acid) is represented by the diastereomeric purity of the internucleotide linkage of a dimer connecting the same two nucleosides, wherein comparable conditions, in some cases, the same synthesis cycle conditions are used to prepare the dimer (e.g., for the linkage between Nx and Ny in the oligonucleotide .... NxNy ..., the dimer is NxNy). In some embodiments, not all chiral internucleotide linkages are chirally controlled internucleotide linkages, and the composition is a partially chirally controlled oligonucleotide composition. In some embodiments, as typically observed in stereo random oligonucleotide compositions (e.g., as understood by those skilled in the art, from traditional oligonucleotide synthesis, such as phosphoramidite methods), the internucleotide linkages of achiral control have a diastereomeric purity of less than about 80%, 75%, 70%, 65%, 60%, 55% or about 50%. In some embodiments, a plurality of oligonucleotides (or nucleic acids) have the same type. In some embodiments, the oligonucleotide compositions of chirality control comprise each oligonucleotide type or nucleic acid type of non-random level or control level. For example, in some embodiments, the oligonucleotide compositions of chirality control comprise one and no more than one oligonucleotide type. In some embodiments, the oligonucleotide compositions of chirality control comprise more than one oligonucleotide type. In some embodiments, the oligonucleotide compositions of chirality control comprise multiple oligonucleotide types. In some embodiments, the oligonucleotide compositions of chirality control are compositions of oligonucleotides of one oligonucleotide type, said composition comprising multiple oligonucleotides of this oligonucleotide type of non-random level or control level.

[0096] Comparable: The term "comparable" is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to allow comparison of the results obtained or the phenomena observed. In some embodiments, the sets of comparable conditions or circumstances are characterized by a plurality of substantially identical features and one or a few features that vary. One of ordinary skill in the art will understand that sets of conditions are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in the results obtained or the phenomena observed under different sets of conditions or circumstances are caused or indicated by variations in those features that vary.

[0097] Cycloaliphatic: The terms "cycloaliphatic," "carbocycle," "carbocyclyl," "carbocyclic radical," and "carbocyclic ring" 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 from 3 to 30 ring members, unless otherwise indicated. Cycloaliphatic radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, the cycloaliphatic radical has 3-6 carbon atoms. In some embodiments, the cycloaliphatic radical is saturated and is a cycloalkyl radical. The term "cycloaliphatic" may also include an aliphatic ring fused to 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 C3-C6 monocyclic hydrocarbon or C8-C6 monocyclic hydrocarbon that is completely saturated or contains one or more unsaturated units but is not aromatic. 10 Bicyclic or polycyclic hydrocarbons having a single point of attachment to the rest of the molecule, or C9-C12-C16 hydrocarbons that are fully saturated or contain one or more unsaturated units but are not aromatic 16 A polycyclic hydrocarbon having a single point of attachment to the rest of the molecule.

[0098] Heteroaliphatic: As used herein, the term "heteroaliphatic" is given its ordinary 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 oxidized and / or substituted forms thereof). In some embodiments, the heteroaliphatic group is a heteroalkyl group. In some embodiments, the heteroaliphatic group is a heteroalkenyl group.

[0099] Heteroalkyl: As used herein, the term "heteroalkyl" is given its ordinary meaning in the art and refers to an alkyl 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.). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, and the like.

[0100] Heteroaryl: As used herein, the terms "heteroaryl" and "heteroar-", used alone or as part of a larger moiety such as "heteroaralkyl" or "heteroaralkoxy", refer to a monocyclic, bicyclic, or polycyclic ring 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, the heteroaryl group is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), and in some embodiments, 5, 6, 9, or 10 ring atoms. In some embodiments, each monocyclic ring unit is aromatic. In some embodiments, the heteroaryl group has 6, 10, or 14 pi electrons shared in the cyclic array; and has one to five heteroatoms in addition to the carbon atoms. Heteroaryl includes, but is not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, heteroaryl is a heterobiaryl, such as bipyridyl, etc. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, wherein the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothiophenyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic, bicyclic, or polycyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which includes optionally substituted rings. The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group, wherein the alkyl portion and the heteroaryl portion are independently and optionally substituted.

[0101] Heteroatom: As used herein, the term "heteroatom" means an atom that is not carbon or hydrogen. In some embodiments, the 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, forms in imine groups, etc.), phosphorus, sulfur, oxygen; etc.). In some embodiments, the heteroatom is silicon, phosphorus, oxygen, sulfur, or nitrogen. In some embodiments, the heteroatom is silicon, oxygen, sulfur, or nitrogen. In some embodiments, the heteroatom is oxygen, sulfur, or nitrogen.

[0102] Heterocycle: As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a monocyclic, bicyclic, or polycyclic moiety (e.g., 3-30 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, the heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more, preferably one to four, heteroatoms as defined above in addition to carbon atoms. The term "nitrogen," when used with respect to the ring atoms of the heterocycle, includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, and nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or + NR (as in N-substituted pyrrolidinyl). The heterocyclic ring may be attached to its side group at any heteroatom or carbon atom that results in a stable structure, and any ring atom may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolane, diazepine, oxazolidinyl ... Oxazolin thiazolinone The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be monocyclic, bicyclic, or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl group, wherein the alkyl portion and the heterocyclyl portion are independently and optionally substituted.

[0103] Identity: As used herein, the term "identity" refers to the overall relatedness between polymeric molecules, such as nucleic acid molecules (e.g., oligonucleotides, DNA, RNA, etc.) and / or polypeptide molecules. In some embodiments, if the sequences of the polymeric molecules are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical, then they are considered to be "substantially identical" to each other. For example, the calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed 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 for comparison purposes, non-identical sequences can be ignored). In certain 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. The nucleotides at the corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., 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 percent identity between the two sequences is a function of the number of identical positions shared by the sequences. Mathematical algorithms can be used to complete the comparison of sequences and the determination of the percent identity between the two sequences. For example, the percent identity between two nucleotide sequences can be determined using the Meyers and Miller algorithm (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, the nucleic acid sequence comparison performed using the ALIGN program uses 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 to determine the percent identity between two nucleotide sequences using the GAP program in the GCG software package.

[0104] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to the linkage that connects the nucleoside units of an oligonucleotide or nucleic acid. In some embodiments, the internucleotide linkage is a phosphodiester linkage, such as is widely found in naturally occurring DNA and RNA molecules (natural phosphate linkages (-OP(=O)(OH)O-), which can exist in salt form as understood by those skilled in the art). In some embodiments, the internucleotide linkage is a modified internucleotide linkage (not a natural phosphate linkage). In some embodiments, the internucleotide linkage is a "modified internucleotide linkage" in which at least one oxygen atom or -OH of the phosphodiester linkage 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 the present disclosure. In certain embodiments, the internucleotide linkage is a phosphotriester linkage, a thiophosphate linkage (or a thiophosphate diester linkage, i.e. -OP (=O) (SH) O-, as understood by those skilled in the art, it can exist in salt form), or a thiophosphate triester linkage. In certain embodiments, the internucleotide linkage is a phosphotriester linkage. In certain embodiments, the internucleotide linkage is one of, for example, a PNA (peptide nucleic acid) or a PMO (phosphorodiamidate morpholino oligomer) linkage. In certain embodiments, the internucleotide linkage is a non-negatively charged internucleotide linkage. In certain embodiments, the internucleotide linkage is a neutral internucleotide linkage (e.g., n001 in some provided oligonucleotides). One of ordinary skill in the art understands that, due to the presence of acid or base moieties in the linkage, the internucleotide linkage can exist as an anion or a cation under a given pH. In some embodiments, the modified internucleotide linkages are modified internucleotide linkages designated 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.

[0105] 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 cell culture, etc., rather than within an organism (eg, an animal, plant, and / or microorganism).

[0106] In vivo: As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, a plant, and / or a microorganism).

[0107] Phosphorus linkage: As defined herein, the phrase "phosphorus linkage" is used to indicate that the specific phosphorus atom mentioned is a phosphorus atom present in an internucleotide linkage that corresponds to a phosphorus atom in a phosphodiester internucleotide linkage as present in naturally occurring DNA and RNA. In some embodiments, the phosphorus linkage atom is located in a modified internucleotide linkage, wherein each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, the phosphorus linkage atom is chiral (e.g., as in a phosphorothioate internucleotide linkage). In some embodiments, the phosphorus linkage atom is achiral (e.g., as in a natural phosphate linkage).

[0108] Modified nucleobase: The terms "modified nucleobase," "modified base," and the like refer to a chemical moiety that is chemically distinct from a nucleobase but that can perform 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 part of a polymer capable of base pairing with a nucleic acid 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 an oligonucleotide, a modified nucleobase is a nucleobase that is not A, T, C, G, or U.

[0109] Modified nucleosides: The term "modified nucleoside" refers to a portion that is derived from a natural nucleoside or is chemically similar to a natural nucleoside but contains a chemical modification that distinguishes it from a natural nucleoside. Non-limiting examples of modified nucleosides include those that contain modifications at the base and / or sugar. Non-limiting examples of modified nucleosides include those that have a 2' modification at the sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (which lack a nucleobase). In some embodiments, the modified nucleoside can have at least one function of a nucleoside, for example, forming a portion in a polymer that can base pair with a nucleic acid comprising at least a complementary base sequence.

[0110] Modified nucleotide: The term "modified nucleotide" includes any chemical moiety that is structurally different from a natural nucleotide but that can perform at least one function of a natural nucleotide. In some embodiments, the modified nucleotide comprises modifications at the sugar, base, and / or internucleotide linkage. In some embodiments, the modified nucleotide comprises a modified sugar, a modified nucleobase, and / or a modified internucleotide linkage. In some embodiments, the modified nucleotide can possess at least one function of a nucleotide, for example, forming a subunit in a polymer capable of base pairing with a nucleic acid comprising at least a complementary base sequence.

[0111] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar. A modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical properties of a sugar. In some embodiments, as described herein, the modified sugar is a substituted ribose or deoxyribose. In some embodiments, the modified sugar comprises 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, wherein R is an optionally substituted C 1-10 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., a sugar used in LNA, BNA, etc.). In some embodiments, in the case of oligonucleotides, the modified sugar is a sugar that is not typically found in natural RNA or DNA, such as ribose or deoxyribose.

[0112] Nucleic acid: As used herein, the term "nucleic acid" includes any nucleotide and polymers thereof. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or a combination thereof. These terms refer to the primary structure of the molecule and include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. These terms include analogs of RNA or DNA as equivalents, which contain modified nucleotides and / or modified polynucleotides (such as, but not limited to, methylated, protected and / or blocked nucleotides or polynucleotides). These terms encompass polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotide linkages. The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified internucleotide linkages. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxyribose moieties, nucleic acids containing both ribose moieties and deoxyribose moieties, nucleic acids containing both ribose moieties and modified ribose moieties. Unless otherwise indicated, the prefix "poly-" refers to nucleic acids containing from 2 to about 10,000 nucleotide monomer units, and wherein the prefix "oligo-" refers to nucleic acids containing from 2 to about 200 nucleotide monomer units.

[0113] Nucleobase: The term "nucleobase" refers to the portion of a nucleic acid that participates in hydrogen bonding, which binds one nucleic acid strand to another complementary strand in a sequence-specific manner. The most common naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the naturally occurring nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally occurring nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase comprises a heteroaryl ring wherein the ring atoms are nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, the nucleobase comprises a heterocyclic ring wherein the ring atoms are nitrogen, and when in a nucleoside, the nitrogen is bonded to a sugar moiety. In some embodiments, the nucleobase is a "modified nucleobase," i.e., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobase is a substituted A, T, C, G, or U. In some embodiments, the modified nucleobase is a substituted tautomer of A, T, C, G, or U. In some embodiments, the modified nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical properties of the nucleobase and retains the property of hydrogen bonding to bind one nucleic acid strand to another in a sequence-specific manner. In some embodiments, the modified nucleobase can pair with all five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex. As used herein, the term "nucleobase" also encompasses structural analogs used to replace natural nucleotides or naturally occurring nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, the nucleobase is 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 in an oligonucleotide or nucleic acid (e.g., A, T, C, G, or U in an oligonucleotide or nucleic acid).

[0114] Nucleoside: The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently bound to a sugar or modified sugar. In some embodiments, a nucleoside is a natural nucleoside, such as adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, a 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, a 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, a "nucleoside" refers to a nucleoside unit in an oligonucleotide or nucleic acid.

[0115] Nucleotide: As used herein, the term "nucleotide" refers to the monomeric unit of a polynucleotide, which is composed of a nucleobase, a sugar, and one or more internucleotide linkages (e.g., phosphate linkages in natural 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 is understood that naturally occurring and non-naturally occurring base analogs are also included. Naturally occurring sugars are pentoses (five-carbon sugars), i.e., deoxyribose (which forms DNA) or ribose (which forms RNA), but it is understood that naturally occurring and non-naturally occurring sugar analogs are also included. Nucleotides are linked via internucleotide linkages to form nucleic acids, or polynucleotides. Many internucleotide linkages are known in the art (such as, but not limited to, phosphates, phosphorothioates, boranephosphates, etc.). Artificial nucleic acids include PNA (peptide nucleic acid), phosphotriester, phosphorothioate, H-phosphonate, phosphoramidate, borane phosphate, methylphosphonate, phosphonoacetate (phosphonoacetate), thiophosphonoacetate, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. In certain embodiments, natural nucleotides include naturally occurring bases, sugars, and internucleotide linkages. As used herein, the term "nucleotide" also encompasses structural analogs for replacing natural nucleotides or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs. In certain embodiments, "nucleotide" refers to a nucleotide unit in an oligonucleotide or nucleic acid.

[0116] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides and may comprise any combination of natural and unnatural nucleobases, sugars, and internucleotide linkages.

[0117] Oligonucleotide can be single-stranded or double-stranded.Single-stranded oligonucleotide can have double-stranded region (being formed by two parts of single-stranded oligonucleotide), and the double-stranded oligonucleotide comprising two oligonucleotide chains can have single-stranded region, for example, wherein two oligonucleotide chains are not complementary to each other.Example oligonucleotide includes but is not limited to structural gene, the gene comprising control region and terminator, self-replication system (such as viral DNA or plasmid DNA), single-stranded and double-stranded RNAi reagent and other RNA interference reagents (RNAi agent or iRNA agent), shRNA, antisense oligonucleotide, ribozyme, microRNA, microRNA mimics, supermir, fit, antimir, antagomir, Ul adapter, the oligonucleotide forming triplex, G-quadruplex oligonucleotide, RNA activator, immunostimulatory oligonucleotide and decoy oligonucleotide.

[0118] The oligonucleotides disclosed herein can have various lengths. In specific embodiments, the length of the oligonucleotide can be from about 2 to about 200 nucleosides. In a plurality of related embodiments, the length range of the oligonucleotide (single-stranded, double-stranded or triple-stranded) can be from about 4 to about 10 nucleosides, from about 10 to about 50 nucleosides, from about 20 to about 50 nucleosides, from about 15 to about 30 nucleosides, from about 20 to about 30 nucleosides. In certain embodiments, the length of the oligonucleotide is from about 9 to about 39 nucleosides. In certain embodiments, the length of the oligonucleotide is from about 25 to about 70 nucleosides. In certain embodiments, the length of the oligonucleotide is from about 26 to about 70 nucleosides. In certain embodiments, the length of the oligonucleotide is from about 27 to about 70 nucleosides. In certain embodiments, the length of the oligonucleotide is from about 28 to about 70 nucleosides. In certain embodiments, the length of the oligonucleotide is from about 29 to about 70 nucleosides. In certain embodiments, the length of the oligonucleotide is from about 30 to about 70 nucleosides. In certain embodiments, the length of the oligonucleotide is from about 31 to about 70 nucleosides. In some embodiments, the length of the oligonucleotide is from about 32 to about 70 nucleosides. In some embodiments, the length of the oligonucleotide is from about 25 to about 60 nucleosides. In some embodiments, the length of the oligonucleotide is from about 25 to about 50 nucleosides. In some embodiments, the length of the oligonucleotide is from about 25 to about 40 nucleosides. In some embodiments, the length of the oligonucleotide is from about 30 to about 40 nucleosides. In some embodiments, the length of the oligonucleotide is 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 nucleosides. In some embodiments, the length of the oligonucleotide is at least 4 nucleosides. In some embodiments, the length of the oligonucleotide is at least 5 nucleosides. In some embodiments, the length of the oligonucleotide is at least 6 nucleosides. In some embodiments, the length of the oligonucleotide is at least 7 nucleosides. In some embodiments, the length of the oligonucleotide is at least 8 nucleosides. In some embodiments, the length of the oligonucleotide is at least 9 nucleosides. In some embodiments, the length of the oligonucleotide is at least 10 nucleosides. In some embodiments, the length of the oligonucleotide is at least 11 nucleosides. In some embodiments, the length of the oligonucleotide is at least 12 nucleosides. In some embodiments, the length of the oligonucleotide is at least 15 nucleosides. In some embodiments, the length of the oligonucleotide is at least 15 nucleosides. In some embodiments, the length of the oligonucleotide is at least 16 nucleosides. In some embodiments, the length of the oligonucleotide is at least 17 nucleosides. In some embodiments, the length of the oligonucleotide is at least 18 nucleosides. In some embodiments, the length of the oligonucleotide is at least 19 nucleosides. In some embodiments, the length of the oligonucleotide is at least 20 nucleosides. In some embodiments, the length of the oligonucleotide is at least 25 nucleosides.In some embodiments, the oligonucleotide is at least 26 nucleosides in length. In some embodiments, the oligonucleotide is at least 27 nucleosides in length. In some embodiments, the oligonucleotide is at least 28 nucleosides in length. In some embodiments, the oligonucleotide is at least 29 nucleosides in length. In some embodiments, the oligonucleotide is at least 30 nucleosides in length. In some embodiments, the oligonucleotide is at least 31 nucleosides in length. In some embodiments, the oligonucleotide is at least 32 nucleosides in length. In some embodiments, the oligonucleotide is at least 33 nucleosides in length. In some embodiments, the oligonucleotide is at least 34 nucleosides in length. In some embodiments, the oligonucleotide is at least 35 nucleosides in length. In some embodiments, the oligonucleotide is at least 36 nucleosides in length. In some embodiments, the oligonucleotide is at least 37 nucleosides in length. In some embodiments, the oligonucleotide is at least 38 nucleosides in length. In some embodiments, the oligonucleotide is at least 39 nucleosides in length. In some embodiments, the oligonucleotide is at least 40 nucleosides in length. In some embodiments, the oligonucleotide is 25 nucleosides in length. In some embodiments, the oligonucleotide is 26 nucleosides in length. In some embodiments, the oligonucleotide is 27 nucleosides in length. In some embodiments, the oligonucleotide is 28 nucleosides in length. In some embodiments, the oligonucleotide is 29 nucleosides in length. In some embodiments, the oligonucleotide is 30 nucleosides in length. In some embodiments, the oligonucleotide is 31 nucleosides in length. In some embodiments, the oligonucleotide is 32 nucleosides in length. In some embodiments, the oligonucleotide is 33 nucleosides in length. In some embodiments, the oligonucleotide is 34 nucleosides in length. In some embodiments, the oligonucleotide is 35 nucleosides in length. In some embodiments, the oligonucleotide is 36 nucleosides in length. In some embodiments, the oligonucleotide is 37 nucleosides in length. In some embodiments, the oligonucleotide is 38 nucleosides in length. In some embodiments, the oligonucleotide is 39 nucleosides in length. In some embodiments, the oligonucleotide is 40 nucleosides in length. In some embodiments, each nucleoside counted in the length of the oligonucleotide independently comprises a nucleobase comprising a ring having at least one nitrogen ring atom. In some embodiments, each nucleoside counted in the length of the oligonucleotide independently comprises 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.

[0119] Oligonucleotide type: As used herein, the phrase "oligonucleotide type" is used to define an oligonucleotide having a specific base sequence, backbone linkage pattern (i.e., the pattern of internucleotide linkage types (e.g., phosphate, phosphorothioate, phosphorothioate triester, etc.), backbone chiral center pattern [i.e., linkage phosphorus stereochemistry pattern (Rp / Sp)], and backbone phosphorus modification pattern. In some embodiments, oligonucleotides of a common designated "type" are structurally identical to one another.

[0120] It will be appreciated by those skilled in the art that the synthetic method of the present disclosure provides a certain degree of control during the synthesis of oligonucleotide chain, so that each nucleotide unit of the oligonucleotide chain can be designed and / or selected in advance to have specific stereochemistry and / or specific modification and / or specific base and / or specific sugar at the linkage phosphorus. In certain embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of stereocenters at the linkage phosphorus. In certain embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the linkage phosphorus. In certain embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In certain embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of one or more above structural features. In certain embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotide molecules or being composed thereof (for example, an oligonucleotide composition controlled by chirality). In certain embodiments, all such molecules belong to the same type (that is, structurally identical to each other). However, in certain embodiments, the composition provided comprises a plurality of different types of oligonucleotides (usually with predetermined relative amounts).

[0121] Optionally substituted: As described herein, the compounds of the present disclosure (e.g., oligonucleotides) may contain optionally substituted moieties and / or substituted moieties. Generally, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the specified moiety are replaced by suitable substituents. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted by more than one substituent selected from a specified group, the substituents at each position may be the same or different. In some embodiments, the optionally substituted group is unsubstituted. The combination of substituents contemplated by the present disclosure is preferably a combination that results in the formation of a stable or chemically feasible compound. As used herein, the term "stable" refers to compounds that do not substantially change when subjected to conditions for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more purposes disclosed herein. Certain substituents are described below.

[0122] Suitable monovalent substituents on a substitutable atom (e.g., a suitable carbon atom) are independently halogen; -(CH2) 0-4 R o ; -(CH2) 0-4 OR o ;-O(CH2) 0-4 R o 、-O-(CH2) 0-4 C(O)OR o ; -(CH2) 0-4 CH(OR o )2;-(CH2) 0-4 Ph, which can be R o Substitution; -(CH2) 0-4 O(CH2) 0-1 Ph, which can be R o Substitution; -CH=CHPh, which can be R o Substitution; -(CH2) 0-4 O(CH2)0-1-pyridyl, which may be R o Substitution; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R o )2;-(CH2) 0-4 N(R o )C(O)R o ;-N(R o )C(S)R o ; -(CH2) 0-4 N(R o )C(O)NR o 2;-N(R o )C(S)NR o 2; -(CH2) 0-4 N(R o )C(O)OR o ;-N(R o )N(R o )C(O)R o ;-N(R o )N(R o )C(O)NR o 2;-N(R o )N(R o )C(O)OR o ; -(CH2) 0-4 C(O)R o ;-C(S)R o ; -(CH2) 0-4 C(O)OR o ; -(CH2) 0-4 C(O)SR o;-(CH2) 0-4 C(O)OSiR o 3;-(CH2) 0-4 OC(O)R o ;-OC(O)(CH2) 0- 4SR o ,-SC(S)SR o ;-(CH2) 0-4 SC(O)R o ;-(CH2) 0-4 C(O)NR o 2;-C(S)NR o 2;-C(S)SR o ;-(CH2) 0-4 OC(O)NR o 2;-C(O)N(OR o )R o ;-C(O)C(O)R o ;-C(O)CH2C(O)R o ;-C(NOR o )R o ;-(CH2) 0-4 SSR o ;-(CH2) 0-4 S(O)2R o ;-(CH2) 0-4 S(O)2OR o ;-(CH2) 0-4 OS(O)2R o ;-S(O)2NR o 2;-(CH2) 0-4 S(O)R o ;-N(R o )S(O)2NR o 2;-N(R o )S(O)2R o ;-N(OR o )R o ;-C(NH)NR o 2;-Si(R o )3;-OSi(R o )3;-B(R o )2;-OB(R o )2;-OB(OR o )2;-P(R o )2;-P(OR o )2;-P(R o )(OR o );-OP(R o )2;-OP(ORo )2;-OP(R o )(OR o );-P(O)(R o )2;-P(O)(OR o )2;-OP(O)(R o )2;-OP(O)(OR o )2;-OP(O)(OR o )(SR o );-SP(O)(R o )2;-SP(O)(OR o )2;-N(R o )P(O)(R o )2;-N(R o )P(O)(OR o )2;-P(R o )2[B(R o )3];-P(OR o )2[B(R o )3];-OP(R o )2[B(R o )3];-OP(OR o )2[B(R o )3];-(C 1-4 linear or branched alkylene)ON(R o )2; or -(C 1-4 linear or branched alkylene) C(O)ON(R o )2, where each R o may be substituted as defined herein and is independently hydrogen; C 1-20 aliphatic; C having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus 1-20 Heteroaliphatic; -CH2-(C 6-14 aryl); -O(CH2) 0-1 (C 6-14 aryl); --CH2-(5-14 membered heteroaryl ring); a 5-20 membered monocyclic, bicyclic or polycyclic saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus; or, regardless of the above definitions, two independent occurrences of R o Together with one or more intervening atoms, they form a 5-20 membered monocyclic, bicyclic or polycyclic saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below.

[0123] R o (or two independent occurrences of R oSuitable monovalent substituents on the ring formed by taking together with the atoms intervening therebetween are independently halogen, -(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 linear or branched alkylene)C(O)OR · or -SSR · , where each R · is unsubstituted or, in the case of "halo" preceding it, is substituted only with one or more halogens, and is independently selected from C 1-4 Aliphatic group, -CH2Ph, -O(CH2) 0-1 Ph, and a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur. o Suitable divalent substituents on a saturated carbon atom of include =0 and =S.

[0124] For example, suitable divalent substituents on suitable carbon atoms are independently the following: =0, =S, =NNR ＊ 2. =NNHC(O)R ＊ 、=NNHC(O)OR ＊ 、=NNHS(O)2R ＊ 、=NR ＊ 、=NOR ＊ 、-O(C(R ＊ 2)) 2-3O-, or -S(C(R ＊ 2)) 2-3 S-, where each independent occurrence of R ＊ is selected from hydrogen, C which may be substituted as defined below 1-6 aliphatic, and unsubstituted 5-6 membered saturated, partially unsaturated or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur. Suitable divalent substituents bound to the ortho-substitutable carbon of the "optionally substituted" group include: -O(CR ＊ 2) 2-3 O-, where each independent occurrence of R ＊ is selected from hydrogen, C which may be substituted as defined below 1-6 aliphatic, and unsubstituted 5-6 membered saturated, partially unsaturated, and aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0125] R ＊ Suitable substituents on the aliphatic group are independently halogen, -R · 、-(halogenated R · ), -OH, -OR · 、-O(halogenated R · )、-CN、-C(O)OH、-C(O)OR · 、-NH2、-NHR · 、-NR · 2 or -NO2, where each R · is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and is independently C 1-4 Aliphatic group, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0126] In some embodiments, suitable substituents on the substitutable nitrogen are independently or Each of these is independently hydrogen, and may be substituted as defined below 1-6 aliphatic, unsubstituted -OPh or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or, notwithstanding the above definition, two independent occurrences of and one or more of their intervening atoms together form an unsubstituted 3-12 membered saturated, partially unsaturated or aromatic monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0127] Suitable substituents on the aliphatic group are independently halogen, -R · 、-(halogenated R · ), -OH, -OR · 、-O(halogenated R · )、-CN、-C(O)OH、-C(O)OR · 、-NH2、-NHR · 、-NR · 2 or -NO2, where each R · is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and is independently C 1-4 Aliphatic group, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0128] P-modification: As used herein, the term "P-modification" refers to any modification other than a stereochemical modification at the phosphorus bond. In some embodiments, the P-modification comprises adding, substituting, or removing a pendant moiety covalently attached to the phosphorus bond.

[0129] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

[0130] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical compositions can be specially formulated for administration in solid or liquid form, including those suitable for: oral administration, e.g., a drench (aqueous or non-aqueous solution or suspension), tablet (e.g., those for oral, sublingual, and systemic absorption), bolus, powder, granules, paste (for application to the tongue); parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension or sustained-release formulation; topical application, e.g., as a cream, ointment, or controlled-release patch or spray to the skin, lungs, or oral cavity; intravaginal or intrarectal, e.g., as a vaginal suppository, cream, or foam; sublingual; ophthalmic; transdermal; or nasal, pulmonary, and application to other mucosal surfaces.

[0131] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0132] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting the subject compound from one organ (or part of the body) to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; 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 buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic, compatible substances employed in pharmaceutical formulations.

[0133] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of compounds that are suitable for use in a pharmaceutical setting, i.e., salts that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and that are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe 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 formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with 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, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. In some embodiments, provided compounds (e.g., oligonucleotides) contain 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 the present disclosure). Representative alkali metal or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, 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, pharmaceutically acceptable salts suitably include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having from 1 to 6 carbon atoms, sulfonates, and arylsulfonates.In some embodiments, provided compounds contain more than one acidic group, for example, an oligonucleotide can contain two or more acidic groups (e.g., natural phosphate linkages and / or modified internucleotide linkages). In some embodiments, pharmaceutically acceptable salts (or generally, salts) of such compounds contain two or more cations, which can be the same or different. In some embodiments, in pharmaceutically acceptable salts (or generally, salts), all ionizable hydrogens in the acidic groups (e.g., in aqueous solutions with a pKa of no more than about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2; in some embodiments, no more than about 7; in some embodiments, no more than about 6; in some embodiments, no more than about 5; in some embodiments, no more than about 4; in some embodiments, no more than about 3) are replaced with cations. In some embodiments, each phosphorothioate and phosphate group is independently present in its salt form (e.g., if it is a sodium salt, it is -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively). In some embodiments, each phosphorothioate and phosphate internucleotide linkage is independently present in its salt form (e.g., if a sodium salt, -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively). In some embodiments, the pharmaceutically acceptable salt is the sodium salt of the oligonucleotide. In some embodiments, the pharmaceutically acceptable salt is the sodium salt of the oligonucleotide, wherein each acidic phosphate and modified phosphate group (e.g., phosphorothioate, phosphate, etc.), if any, is present in salt form (all as sodium salts).

[0134] Predetermined: "predetermined" means intentionally selected or non-random or controlled, for example, in contrast to random occurrence, random or in the absence of control. Those of ordinary skill in the art who read this specification will understand that the present disclosure provides the following technologies, which allow the selection of specific chemical characteristics and / or stereochemical characteristics to be incorporated into the oligonucleotide composition and further allow the controlled preparation of oligonucleotide compositions with such chemical characteristics and / or stereochemical characteristics. Such provided compositions are "predetermined" as described herein. Because some oligonucleotides are accidentally produced by processes that are not controlled to intentionally produce specific chemical characteristics and / or stereochemical characteristics, compositions that may contain these oligonucleotides are not "predetermined" compositions. In certain embodiments, a predetermined composition is a composition that can be intentionally replicated (for example, by repetition of a controlled process). In certain embodiments, the predetermined level of a plurality of oligonucleotides in a composition means that the absolute amount and / or relative amount (ratio, percentage, etc.) of the plurality of oligonucleotides in the composition is controlled. In certain embodiments, the predetermined level of a plurality of oligonucleotides in the composition is obtained by oligonucleotide preparation controlled by chirality.

[0135] Protecting Group: As used herein, the term "protecting group" is well known in the art and includes those described 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. Also included are those protecting groups particularly useful in nucleoside and nucleotide chemistry, which are described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al., June 2012, the entire contents of Section 2 of which are incorporated herein by reference. Suitable amino protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthenyl)]methyl carbamate (DBD-Tmoc), 4-methoxybenzoylmethyl carbamate (Phenoc ...9-(2,7-dibromo)fluorenylmethyl carbamate, 9-(1,10-dioxo-10,10,10,10-tetrahydrothioxanthenyl)]methyl carbamate (DBD-Tmoc), 4-methoxybenzoylmethyl carbamate (Phenoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 9-(1 2,2,2-trichloroethyl ester (Troc), 2-trimethylsilylethyl 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), 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-dicyclohexylcarbamylamino)ethyl carbamate, tert-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate esters (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-aminocarbamic acid4-dichlorobenzyl ester, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylphenylthiocarbamate (Mtpc), 2,4-dimethylphenylthiocarbamate (Bmpc), 2-phosphonoethyl carbamate (Peoc), 2-triphenylphosphonoisopropyl carbamate (Ppoc), 1,1-dimethyl-2-thiophenecarbamate -cyanoethyl ester, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) methyl carbamate, phenothiazinyl-(10)-carbonyl derivatives, N′-toluenesulfonylaminocarbonyl derivatives, N′-phenylaminothiocarbonyl derivatives, tert-amyl carbamate, S-thiocarbamate benzyl ester, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropyl methyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl 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-furylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinoyl carbamate, p-(p-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-tert-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-Trimethylbenzyl ester, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, picolinamide, 3-pyridylformamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (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 amine, 3-methyl-3-nitrobutyramide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilazide adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl -1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N- 2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-pyridylmethylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)benzylideneamine, 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, diphenylphosphinamide (dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkylphosphoramidates, dibenzylphosphoramidates, diphenylphosphoramidates, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (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-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide and phenacylmethylsulfonamide.

[0136] 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, trityl, 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 (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), as well as 2-pyridylmethyl and 4-pyridylmethyl.

[0137] Suitable hydroxy protecting groups include methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-[[(2-chloroethoxy)methyl]-1- ... -bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-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-(phenylhydrogen selenyl)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-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p′-dinitrodiphenylmethyl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxybenzyl 1-Benzodiphenylmethyl, 1-(4-methoxyphenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-1-nitrophenylmethyl, 1-(4- ...2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(4-phenyl)-2-nitrophenylmethyl, 1-(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 , phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinic acid ester), 4,4-(ethylenedithio)pentanoate (levulinic acid acetal), pivalate, 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-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoic acid Ester, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, p-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphino, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate and toluenesulfonate (Ts). In order to protect 1,2-diol or 1,3-diol, the protecting group includes methylene acetal, ethylene acetal, 1-tert-butylethylene ketal, 1-phenylethylene ketal, (4-methoxyphenyl)ethylene acetal, 2,2,2-trichloroethylene acetal, acetone, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzyl acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester Esters, 1-ethoxyethylene orthoester, 1,2-dimethoxyethylene orthoester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylene derivatives, α-(N,N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene orthoester, di-tert-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxane-1,3-diylidene) derivative (TIPDS), tetra-tert-butoxydisiloxane-1,3-diylidene derivative (TBDS), carbonates, cyclic boronates, ethyl boronates, and phenyl boronates.

[0138] In some embodiments, the hydroxy 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 (trityl), 4,4′-dimethoxytrityl, trimethyl methylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butylbiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, trityl, monomethoxytrityl (MMTr), 4,4′-dimethoxytrityl (DMTr) and 4,4′-dimethoxytrityl (DMTr). ',4"-trimethoxytrityl (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-trimethylsilyl In some embodiments, the hydroxy protecting groups are each independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butylbiphenylsilyl and 4,4′-dimethoxytrityl. In some embodiments, the hydroxy protecting groups are selected from the group consisting of trityl, monomethoxytrityl and 4,4′-dimethoxytrityl. In certain embodiments, the phosphorus linkage protecting group is a group that is attached to the phosphorus linkage (e.g., internucleotide linkage) during the synthesis of the entire oligonucleotide. In certain embodiments, the protecting group is attached to the sulfur atom of a thiophosphate group. In certain embodiments, the protecting group is attached to the oxygen atom of a thiophosphate linkage between the nucleotides. In certain embodiments, the protecting group is attached to the oxygen atom of a phosphate linkage between the nucleotides.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-butylformamido)-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.

[0139] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a compound (e.g., oligonucleotide) or composition according to the present disclosure is administered, e.g., for experimental, diagnostic, prophylactic, 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 be suffering from and / or susceptible to a disease, disorder, and / or condition.

[0140] Substantially: as used herein, the term "substantially" refers to the overall or near-overall scope or degree qualitative state of a feature or characteristic of interest. The base sequence substantially identical or complementary to the second sequence is not completely identical or complementary to the second sequence, but is mostly or almost identical or complementary to the second sequence. In certain embodiments, the oligonucleotide with a sequence substantially complementary to another oligonucleotide or nucleic acid forms a duplex with a mode similar to the oligonucleotide with a complete complementary sequence with this oligonucleotide or nucleic acid. In addition, it will be appreciated that those skilled in the art of biology and / or chemistry seldom reach completion and / or proceed to completion or realization or avoid absolute result in biology and chemical phenomena (if any). Therefore, the term "substantially" is used herein to obtain the inherent completeness potentially lacking in many biology and / or chemical phenomena.

[0141] Sugar: The term "sugar" refers to a monosaccharide or polysaccharide in closed and / or open form. In some embodiments, the sugar is a monosaccharide. In some embodiments, the sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranoses, pentopyranoses, and hexopyranose moieties. As used herein, the term "sugar" also encompasses structural analogs used to replace conventional sugar molecules, such as ethylene glycol, polymers that form the backbone of nucleic acid analogs, glycol 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, the sugar is an RNA or DNA sugar (ribose or deoxyribose). In some embodiments, the sugar is a modified ribose or deoxyribose, such as 2'-modified, 5'-modified, etc. As described herein, in some embodiments, when used in oligonucleotides and / or nucleic acids, the modified sugar can provide one or more desired properties, activities, etc. In some embodiments, the sugar is an optionally substituted ribose or deoxyribose. In some embodiments, "sugar" refers to a sugar unit in an oligonucleotide or nucleic acid.

[0142] Predisposed to: An individual who is "predisposed" to a disease, disorder, and / or condition is one who has a higher risk of developing the disease, disorder, and / or condition than members of the general public. In some embodiments, an individual who is predisposed to a disease, disorder, and / or condition has a predisposition to develop the disease, disorder, and / or condition. In some embodiments, an individual who is predisposed to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is predisposed to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is predisposed to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is predisposed to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is predisposed to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0143] Therapeutic agent: As used herein, the term "therapeutic agent" generally refers to any agent that, when administered to a subject, elicits a desired effect (e.g., a desired biological, clinical, or pharmacological effect). In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across a suitable population. In some embodiments, a suitable population is a population of subjects suffering from and / or susceptible to a disease, disorder, or condition. In some embodiments, a suitable population is a population of model organisms. In some embodiments, a suitable population can be defined by one or more criteria, such as age group, sex, genetic background, or pre-existing clinical conditions prior to receiving therapy. In some embodiments, a therapeutic agent, when administered to a subject in an effective amount, is a substance that alleviates, ameliorates, relieves, 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 a subject. In some embodiments, a "therapeutic agent" is an agent that has been or needs to be approved by a governmental agency before it can be marketed for administration 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 provided compound, such as a provided oligonucleotide.

[0144] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a treatment regimen. 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 suffering from or susceptible to a disease, disorder, and / or condition. As one of ordinary skill in the art will appreciate, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, ameliorate, mitigate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0145] Treatment: As used herein, the term "treat," "treatment," or "treating" refers to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment can be administered to subjects who do not exhibit signs of the disease, disorder, and / or condition. In some embodiments, treatment can be administered to subjects who only exhibit early signs of the disease, disorder, and / or condition, for example, for the purpose of reducing the risk of pathology associated with the disease, disorder, and / or condition.

[0146] Unsaturated: As used herein, the term "unsaturated" means a moiety having one or more units of unsaturation.

[0147] Wild-type: As used herein, the term "wild-type" has its art-understood meaning, which refers to an entity having structure and / or activity as found in nature in a "normal" (as opposed to mutant, diseased, altered, etc.) state or background. One of ordinary skill in the art will understand that wild-type genes and polypeptides typically exist in a variety of different forms (e.g., alleles).

[0148] As will be appreciated by those skilled in the art, the methods and compositions described herein involving provided compounds (eg, oligonucleotides) are generally also applicable to the pharmaceutically acceptable salts of such compounds. Description of Certain Embodiments

[0149] Oligonucleotide can be used for multiple treatment, diagnosis and research application.The purposes of naturally occurring nucleic acid is subject to for example their restriction to the sensitivity of endo- and exonucleases.Like this, developed multiple synthetic counterparts to circumvent these shortcomings and / or further improve multiple characteristics and activity.These synthetic counterparts comprise synthetic oligonucleotides containing chemical modifications, and these chemical modifications are for example base-modified, sugar-modified, backbone-modified etc., and it especially makes these molecules not too easy to degrade and improves other characteristics and / or the activity of oligonucleotide.

[0150] From a structural perspective, modifications to internucleotide linkages can introduce chirality, and certain properties and activities may be affected by the configuration of the oligonucleotide's linker phosphorus atoms. For example, the chirality of the backbone phosphorus atoms can affect binding affinity, sequence-specific binding to complementary RNA, stability against nucleases, activity, delivery, and pharmacokinetics, among other properties.

[0151] In particular, the present disclosure utilizes techniques for controlling various structural elements, such as sugar modifications and patterns thereof, nucleobase modifications and patterns thereof, modified internucleotide linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, additional chemical moieties (parts that are typically not in the oligonucleotide chain) and patterns thereof, and the like. With the ability to fully control the structural elements of oligonucleotides, the present disclosure provides oligonucleotides with improved and / or new properties and / or activities for various applications (e.g., as therapeutic agents, probes, etc.). For example, as demonstrated herein, provided oligonucleotides and compositions thereof are particularly effective for editing a target adenosine in a target nucleic acid to correct a G to A mutation, in some embodiments, by converting an A to an I.

[0152] In some embodiments, the oligonucleotide comprises 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 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 consecutive bases are identical or completely or substantially complementary. In some embodiments, the nucleic acid is a target nucleic acid comprising one or more target adenosines. In some embodiments, the target nucleic acid comprises one and no more than one target adenosine. In some embodiments, the oligonucleotide can hybridize with the target nucleic acid. In some embodiments, such hybridization is conducive to the modification of A in the nucleic acid or its product by, for example, ADAR1, ADAR2, etc. (e.g., A is converted to I).

[0153] In some embodiments, the present disclosure provides oligonucleotides, wherein the oligonucleotide has 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, at least 34 consecutive bases of an oligonucleotide or nucleic acid disclosed herein (e.g., in a table), or a sequence complementary to a target RNA sequence gene, transcript, etc. disclosed herein, and wherein each T can be optionally and independently replaced by a U and vice versa. In some embodiments, the present disclosure provides oligonucleotides or oligonucleotide compositions as disclosed herein (e.g., in a table).

[0154] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide for site-specific editing of a nucleoside (eg, a target adenosine) in a target nucleic acid (eg, RNA).

[0155] As described herein, oligonucleotides can contain one or more modified internucleotide linkages (non-natural phosphate linkages). In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage, and its linkage phosphorus is chiral. In some embodiments, the modified internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, the oligonucleotide comprises one or more negatively charged internucleotide linkages (e.g., phosphorothioate internucleotide linkages, natural phosphate linkages, etc.). In some embodiments, the oligonucleotide comprises one or more non-negatively charged internucleotide linkages. In some embodiments, the oligonucleotide comprises one or more neutral internucleotide linkages.

[0156] In some embodiments, the oligonucleotide is chirally controlled. In some embodiments, the oligonucleotide is chirally pure (or "stereoisomer", "stereochemically pure"), wherein the oligonucleotide exists in a single stereoisomer form (in many cases a single diastereomeric (or "diastereomeric") form, because multiple chiral centers can be present in the oligonucleotide, for example at the linkage phosphorus, sugar carbon, etc.). As will be appreciated by those skilled in the art, a chirally pure oligonucleotide is separated from its other stereoisomeric forms (to the extent that some impurities may be present, because chemical and biological processes, selectivity and / or purification, etc., rarely (if ever) achieve absolute completeness). In a chirally pure oligonucleotide, each chiral center is independently defined with respect to its configuration (for a chirally pure oligonucleotide, each internucleotide linkage is independently stereo-defined or chirally controlled). In contrast to chirally controlled and chirally pure oligonucleotides containing a stereodefined phosphorus linkage, "racemic" (or "stereorandom," "achiral") oligonucleotides containing a chiral phosphorus linkage (e.g., from conventional phosphoramidite oligonucleotide synthesis in which there is no stereochemical control in the coupling step and in combination with conventional sulfurization (forming stereorandom phosphorothioate internucleotide linkages)) refer to random mixtures of various stereoisomers (typically diastereomers (or "diastereomers") due to the presence of multiple chiral centers in the oligonucleotide; e.g., from conventional oligonucleotide preparation using reagents that contain no chiral elements other than those in the nucleoside and the phosphorus linkage). For example, for A*A*A where * is a phosphorothioate internucleotide linkage (which contains a chiral phosphorus linkage), the racemic oligonucleotide preparation includes four diastereomers [2 2 =4, taking into account the two chiral linked phosphorus, 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 Sp phosphorothioate internucleotide linkage and *R represents Rp phosphorothioate internucleotide linkage. For a chirally pure oligonucleotide, such as A*SA*SA, it exists in a single stereoisomeric form and is separated from other stereoisomers (e.g., diastereomers A*SA*RA, A*RA*SA and A*RA*RA).

[0157] In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stereorandom internucleotide linkages (a mixture of Rp and Sp linked phosphorus at the internucleotide linkages, e.g., from traditional achiral controlled oligonucleotide synthesis). 55, 56, 57, 58, 59, 60 or more) chirally controlled internucleotide linkages (e.g., Rp or Sp linked phosphorus at the internucleotide linkage, e.g., from chirally controlled oligonucleotide synthesis). In some embodiments, the internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage is a stereo-random phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage is a chirally controlled phosphorothioate internucleotide linkage.

[0158] In particular, the present disclosure provides techniques for preparing chirally 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%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 50%, 90%, 100%, 150 ...50%, 50%, 50%, 50%, 50%, 50%, 50%, 50%, 50%, 50%, 50%, 50%, 50%, 5 %, 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.

[0159] In certain embodiments, present disclosure provides multiple oligonucleotide compositions.In certain embodiments, oligonucleotide compositions are stereo random or not chirality controlled.In certain embodiments, there is not chirality controlled internucleotide linkage in the oligonucleotide of provided compositions.In certain embodiments, the internucleotide linkage of oligonucleotide in compositions comprises one or more chirality controlled internucleotide linkages (for example, chirality controlled oligonucleotide compositions).

[0160] In certain embodiments, oligonucleotide composition comprises a plurality of oligonucleotides sharing a common base sequence, wherein the one or more internucleotides in the oligonucleotide are linked by chirality control, and the one or more internucleotides are linked by stereo random (achiral control). In certain embodiments, oligonucleotide composition comprises a plurality of oligonucleotides sharing a common base sequence, wherein the internucleotides comprising chiral linkage phosphorus in the oligonucleotide are linked by chirality control independently. In certain embodiments, a plurality of oligonucleotides share the same base sequence, and identical base and sugar are modified. In certain embodiments, a plurality of oligonucleotides share the same base sequence, and identical base, sugar and internucleotides are linked by modification. In certain embodiments, oligonucleotide composition comprises the oligonucleotides with identical composition, wherein the one or more internucleotides are linked by chirality control, and the one or more internucleotides are linked by stereo random (achiral control). In certain embodiments, oligonucleotide composition comprises the oligonucleotides with identical composition, wherein the internucleotides comprising chiral linkage phosphorus are linked by chirality control independently. 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.

[0161] In some embodiments, the present disclosure provides techniques for making, evaluating, and / or utilizing provided oligonucleotides and compositions thereof.

[0162] 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” is one. In some embodiments, “one or more” is two. In some embodiments, “one or more” is three. In some embodiments, “one or more” is four. In some embodiments, “one or more” is five. In some embodiments, “one or more” is six. In some embodiments, “one or more” is seven. In some embodiments, “one or more” is eight. In some embodiments, “one or more” is nine. In some embodiments, “one or more” is ten. In some embodiments, “one or more” is at least one. In some embodiments, “one or more” is at least two. In some embodiments, “one or more” is at least three. In some embodiments, “one or more” is at least four. In some embodiments, “one or more” is at least five. In some embodiments, “one or more” is at least six. In some embodiments, “one or more” is at least seven. In some embodiments, “one or more” is at least eight. In some embodiments, “one or more” is at least nine. In some embodiments, “one or more” is at least ten.

[0163] As used in this disclosure, in some embodiments, "at least one" is one or more.

[0164] As an example, variables such as R, R L , L, etc. Embodiments described for a variable (e.g., R) are generally applicable to all variables that can be such variables (e.g., R', R", R L 、R L1 wait). Oligonucleotides

[0165] In particular, the present disclosure provides oligonucleotides of various designs that may comprise various nucleobases and patterns thereof, sugars and patterns thereof, internucleotide linkages and patterns thereof, and / or additional chemical moieties and patterns thereof described in the present disclosure. In some embodiments, the oligonucleotides provided can direct A to I editing in a target nucleic acid. In some embodiments, the oligonucleotides of the present disclosure are single-stranded oligonucleotides capable of site-directed editing of adenosine (converting A to I) in a target RNA sequence.

[0166] 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 sufficiently complementary to the target nucleic acid to distinguish the target nucleic acid from other nucleic acids to reduce 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 activity (e.g., editing of adenosine).

[0167] In some embodiments, 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, 25-50, 25 In some embodiments, the oligonucleotide has a base sequence of about 10 to 60 nucleobases. In some embodiments, the base sequence has a base sequence of about 15 to 50 nucleobases. In some embodiments, the base sequence has a base sequence of about 15 to about 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 at least 35 nucleobases in length. In some other embodiments, the base sequence is at least 34 nucleobases in length. In some other embodiments, the base sequence is at least 33 nucleobases in length. In some other embodiments, the base sequence is at least 32 nucleobases in length. In some other embodiments, the base sequence is at least 31 nucleobases in length. In some other embodiments, the base sequence is at least 30 nucleobases in length. In some other embodiments, the base sequence is at least 29 nucleobases in length. In some other embodiments, the base sequence is at least 28 nucleobases in length.In some other embodiments, the base sequence is or is at least 27 nucleobases in length. In some other embodiments, the base sequence is or is at least 26 nucleobases in length. In some other embodiments, the base sequence of the complementary portion in the duplex 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, it is at least 18 nucleobases in length. In some other embodiments, it is at least 19 nucleobases in length. In some other embodiments, it is at least 20 nucleobases in length. In some other embodiments, it is at least 21 nucleobases in length. In some other embodiments, it is at least 22 nucleobases in length. In some other embodiments, it is at least 23 nucleobases in length. In some other embodiments, it is at least 24 nucleobases in length. In some other embodiments, the length is at least 25 nucleobases.In particular, the present disclosure provides oligonucleotides that have comparable or better properties and / or comparable or higher activity than previously reported adenosine editing oligonucleotides but are shorter in length.

[0168] In some embodiments, the base sequence of the oligonucleotide is complementary to the base sequence of the target nucleic acid (e.g., complementary to a portion of the target nucleic acid comprising the target adenosine) and has 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, 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.) mismatches that are not Watson-Crick base pairs (AT, AU, and CG). In some embodiments, there are no mismatches. In some embodiments, there is 1 mismatch. In some embodiments, there are 2 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 that are not designed for complementarity (e.g., loops, protein binding sequences, etc., for raising proteins, such as ADARs). As will be appreciated by those skilled in the art, such portions may be appropriately excluded when calculating mismatches and / or complementarity. In some embodiments, for example, the complementarity between the oligonucleotide and the target nucleic acid is about 50%-100% (e.g., about 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%). In some embodiments, the complementarity is at least about 60%. In some embodiments, the complementarity is at least about 65%. In some embodiments, the complementarity is at least about 70%. In some embodiments, the complementarity is at least about 75%. In some embodiments, the complementarity is at least about 80%. In some embodiments, the complementarity is at least about 85%. In some embodiments, the complementarity is at least about 80%. In some embodiments, the complementarity is at least about 85%. In some embodiments, the complementarity is at least about 90%.In certain embodiments, the complementarity is at least about 95%. In certain embodiments, the complementarity is 100% on the length of the oligonucleotide. In certain embodiments, the complementarity is 100% on the length of the oligonucleotide, except at the nucleoside opposite to the target nucleoside (e.g., adenosine). Typically, the complementarity is based on Watson-Crick base pairs AT, AU and CG. It will be appreciated by those skilled in the art that when assessing the complementarity of two sequences of different lengths (e.g., the oligonucleotide and target nucleic acid provided), the complementarity can be suitably based on the length of shorter sequence and / or the maximum complementarity between the two sequences. In many embodiments, the oligonucleotide and target nucleic acid have enough complementarity so that modification is selectively directed to the target adenosine site.

[0169] In some embodiments, one or more mismatches are independently wobbles. In some embodiments, each mismatch is a wobble. In some embodiments, there are 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, 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.) wobbles. 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, when I is the 3' adjacent nucleoside adjacent to the nucleoside opposite to the target nucleoside, IC can be considered a match. In some embodiments, the base forming the wobble pair (e.g., U that can form a GU wobble) can replace the base forming the matching pair (e.g., C that matches G) and can provide an oligonucleotide with editing activity.

[0170] In some embodiments, the duplex of the oligonucleotide and the target nucleic acid comprises one or more protrusions, each of which independently comprises one or more mismatches that are not wobble. In some embodiments, there are 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, 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.) protrusions. 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.

[0171] In some embodiments, the distance between two mismatches, between a mismatch and one or both ends of an oligonucleotide (or a portion thereof, e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain), and / or between a mismatch and the nucleoside opposite the target adenosine can independently be 0-50, 0-40, 0-30, 0-25, 0-20, 0-15, 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 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 nucleoside of an oligonucleotide is 0-20. In some embodiments, the distance between a mismatch and the 5′-terminal nucleoside of an oligonucleotide is 5-20. In some embodiments, the distance between a mismatch and the 3′-terminal nucleoside of an oligonucleotide is 0-40. In some embodiments, the distance between a mismatch and the 3′-terminal nucleoside of an oligonucleotide is 5-20. In some embodiments, the distance between a mismatch and the nucleoside opposite to the target adenosine is 0-20. In some embodiments, the distance between a mismatch and the nucleoside opposite to the target adenosine is 1-10. In some embodiments, for a certain distance, the number of nucleobases is 0. 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 an end of the first domain, the second domain, the first subdomain, the second subdomain, or the third subdomain, such as the 5′-end or the 3′-end. In some embodiments, the mismatch is located at the nucleoside opposite the target adenosine.

[0172] In some embodiments, provided oligonucleotides can direct adenosine editing in a target nucleic acid (e.g., converting an A to an I) and have a base sequence consisting of, comprising, or comprising a portion of (e.g., a span of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more consecutive bases) a base sequence of an oligonucleotide disclosed herein, wherein each T can be independently replaced by a U and vice versa, and the oligonucleotide comprises at least one non-naturally occurring modification of a base, a sugar, and / or an internucleotide linkage.

[0173] In certain embodiments, the oligonucleotide provided comprises one or more carbohydrate moieties. In certain embodiments, the oligonucleotide provided comprises one or more GalNAc moieties. In certain embodiments, the oligonucleotide provided comprises one or more targeting moieties. Non-limiting examples of such other chemical moieties that can be conjugated to an oligonucleotide chain are described herein.

[0174] In certain embodiments, the oligonucleotide provided can instruct the correction of G to A sudden change in target sequence or its product.In certain embodiments, the correction of G to A sudden change is or includes converting A into I, which can be read as G in translation or other biological processes.In certain embodiments, the oligonucleotide provided can instruct the correction of G to A sudden change in target sequence or its product via the deamination mediated by ADAR.In certain embodiments, the oligonucleotide provided can instruct the correction of G to A sudden change in target sequence or its product via the deamination mediated by ADAR by raising endogenous ADAR (for example, in target cells) and promoting the deamination mediated by ADAR.However, in any case, the disclosure is not limited to any particular mechanism.In certain embodiments, the disclosure provides oligonucleotides, compositions, methods etc. that can be operated via the combination of double-stranded RNA interference, single-stranded RNA interference, RNA enzyme H mediation, knocking down, the steric hindrance of translation, the deamination mediated by ADAR or two or more such mechanisms.

[0175] In some embodiments, the oligonucleotide comprises a structural element or portion thereof as described herein, for example, in the table. In some embodiments, the oligonucleotide has a base sequence comprising the base sequence (or portion thereof) (wherein each T can be independently substituted with a U), a chemical modification pattern (or a portion thereof), and / or the form of an oligonucleotide disclosed herein (e.g., in a table or figure, or otherwise disclosed herein). In some embodiments, such an oligonucleotide can guide the correction of a G to A mutation in a target sequence or its product.

[0176] In particular, the provided oligonucleotides can hybridize to their target nucleic acids (e.g., pre-mRNA, mature mRNA, etc.). In some embodiments, the oligonucleotides can hybridize to target RNA sequence nucleic acids (including but not limited to pre-mRNA or mature mRNA) at any stage of RNA processing. In some embodiments, the oligonucleotides can hybridize to any element of the oligonucleotide nucleic acid or its complementary sequence, including but not limited to: a promoter region, an enhancer region, a transcription termination region, a translation start signal, a translation termination signal, a coding region, a non-coding region, an exon, an intron, an intron / exon or an exon / intron junction, a 5'UTR or a 3'UTR.

[0177] In some embodiments, the oligonucleotide hybridizes to two or more variants of a transcript derived from the sense strand of a target site (eg, target sequence).

[0178] In some embodiments, the oligonucleotides provided contain one or more isotopes of increased levels. In some embodiments, the oligonucleotides provided are labeled with one or more isotopes of, for example, one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). In some embodiments, the oligonucleotides provided in the provided compositions (e.g., oligonucleotides of a plurality of compositions) comprise base modifications, sugar modifications, and / or internucleotide linkage modifications, wherein these oligonucleotides contain deuterium enrichment levels. In some embodiments, the oligonucleotides provided are labeled with deuterium at one or more positions (e.g., with - 2 H instead of - 1 H). In some embodiments, one or more of the oligonucleotide chain or any moiety conjugated to the oligonucleotide chain (e.g., a targeting moiety, etc.) 1 H is 2 Such oligonucleotides can be used in the compositions and methods described herein.

[0179] In some embodiments, the oligonucleotide comprises one or more modified nucleobases, one or more modified sugars, and / or one or more modified internucleotide linkages as described herein. In some embodiments, the oligonucleotide comprises a level of modified nucleobases, modified sugars, and / or modified internucleotide linkages, such as 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%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 60%-100%, 65%-85%, 65%-90%, 65%-95%, 65%-95%, 65%-95%, 65%-90%, 65%-95%, 65%-95%, 65%-95%, 65%-95%, 65%-10 ... 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%, etc.

[0180] In some embodiments, the oligonucleotide comprises one or more modified sugars. In some embodiments, the oligonucleotide comprises about 1-50 (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 1-50 (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, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc.) modified sugars having a 2'-F modification.In some embodiments, the oligonucleotide comprises about 2-50 (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, 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- In some embodiments, the oligonucleotide comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive modified sugars with a 2'-F modification. In some embodiments, the oligonucleotide comprises about 2 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises about 3 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises about 4 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises about 5 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises about 6 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises about 7 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises about 8 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 9 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises 10 consecutive 2'-F modified sugars. In some embodiments, the oligonucleotide comprises two or more 2'-F modified sugar segments, wherein each sugar in the 2'-F modified sugar segment is independently a 2'-F modified sugar. In some embodiments, each 2'-F modified sugar segment 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 segments are independently separated by a separating segment 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.) separating blocks. In some embodiments, the first domain 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.) separating blocks. In certain embodiments, each first domain block bonded to the first domain 2'-F block is a separation block. In certain embodiments, each first domain block bonded to the first domain separation block is a first domain 2'-F block. In certain embodiments, each sugar in the separation block is independently not modified by 2'-F. In certain embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) or all sugars in the separation block are independently not modified by 2'-F. In certain embodiments, the separation block includes one or more bicyclic sugars (e.g., LNA sugars, cEt sugars, etc.) and / or one or more 2'-OR modified sugars (wherein R is optionally substituted C. 1-6 In some embodiments, the separating block comprises one or more 2'-OR modified sugars, wherein R is an optionally substituted C 1-6 In some embodiments, two or more non-2'-F modified sugars are consecutive. In some embodiments, two or more 2'-OR modified sugars (wherein R is optionally substituted C 1-6In some embodiments, the separating block comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-OR modified sugars (wherein R is an optionally substituted C 1-6 In some embodiments, the separating block comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) consecutive 2'-OR modified sugars (wherein R is an optionally substituted C 1-6 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 separation block comprises one or more 2'-F modified sugars. In some embodiments, the 2'-F modified sugars in the separation block are not adjacent to each other. In some embodiments, the separation block does not contain a 2'-F modified sugar. In some embodiments, each sugar in the separation block is independently a 2'-OR modified sugar (wherein R is optionally substituted C 1-6 In some embodiments, each sugar in each separating block is independently a 2'-OR modified sugar (wherein R is an optionally substituted C 1-6 In some embodiments, each sugar in the separating segment is independently a 2'-OR modified sugar, wherein R is an optionally substituted C 1-6 In some embodiments, each sugar in each separating block is independently a 2'-OR modified sugar, wherein R is an optionally substituted C 1-6 Aliphatic group. In certain embodiments, each sugar in the separation block is independently a sugar modified by 2'-OMe or 2'-MOE. In certain embodiments, each sugar in each separation block is independently a sugar modified by 2'-OMe or 2'-MOE. In certain embodiments, each sugar in the separation block is independently a sugar modified by 2'-OMe. In certain embodiments, each sugar in the separation block is independently a sugar modified by 2'-MOE. In certain embodiments, the separation block comprises a sugar modified by 2'-OMe and 2'-MOE. In certain embodiments, each 2'-F block and each separation block independently contain 1, 2, 3, 4, or 5 nucleosides. In certain embodiments, each 2'-F block and each separation block independently contain 1, 2, or 3 nucleosides.

[0181] In some embodiments, 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%-85%, 70%-90%, 70%-95%, or about 10%-100% of all sugars. In some embodiments, the sugars are modified sugars, such as 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%. In some embodiments, 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%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85 %, 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., of all sugars are modified sugars independently selected from 2'-F modified sugars, 2'-OR modified sugars (wherein R is an optionally substituted C 1-6In 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%.

[0182] In some embodiments, 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%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-80%, 75%-90%, 75%-95%, 75%-10 ... %, 5%-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., are modified sugars independently selected from 2'-F modified sugars and 2'-OR modified sugars (wherein R is an optionally substituted C 1-6aliphatic groups). In some embodiments, 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%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-80%, 75%-90%, 75%-95%, 75%-10 ... 5%-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., are modified sugars independently selected from 2'-F modified sugars, 2'-OMe modified sugars, and 2'-MOE 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%.

[0183] In some embodiments, 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%-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., are modified sugars 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%.

[0184] In some embodiments, 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%-85%, 70%-90%, 70%-95% of all sugars. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. In some embodiments, the sugars are 2'-F modified. 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 (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.) sugars 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, separated by one or more separating blocks as described herein. In some embodiments, the 2'-F blocks have 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F modified sugars. In some embodiments, the 2'-F blocks have 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 four 2'-F modified sugars. In some embodiments, each block bonded to a 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 comprising a natural DNA or RNA sugar, a 2'-OR modified sugar (wherein R is an optionally substituted C. 1-6 In certain embodiments, each block bonded to the 2'-F block is independently a block of sugars comprising natural DNA or RNA sugars, 2'-OMe modified sugars, 2'-MOE modified sugars or bicyclic sugars. In certain embodiments, each block bonded to the 2'-F block is independently a block of sugars comprising natural DNA or RNA sugars, 2'-OMe modified sugars or 2'-MOE modified sugars. In certain embodiments, each nucleoside in the first domain bonded to the 2'-F block is independently a sugar (wherein R is optionally substituted C) modified by 2'-OR. 1-6 In some embodiments, each nucleoside in the first domain that is bonded to the 2'-F block in the first domain is independently a 2'-OR modified sugar, wherein R is an optionally substituted C 1-6 In some embodiments, each nucleoside in the first domain that is bonded to the 2'-F block in the first domain is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each nucleoside in the second domain that is bonded to the 2'-F block in the second domain is independently a 2'-OR modified sugar (wherein R is optionally substituted C 1-6 In some embodiments, each nucleoside in the second domain that is bonded to the 2'-F block in the second domain is independently a 2'-OR modified sugar, wherein R is an optionally substituted C 1-6 In some embodiments, each nucleoside in the second domain that is bonded to the 2'-F block in the second domain is independently a 2'-OMe or 2'-MOE modified sugar.

[0185] In some embodiments, 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%-85%, 70%-90%, 70%-95%, 70 ... %, 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% or more of the 2'-OR modified sugars wherein R is an optionally substituted C 1-6 In some embodiments, 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%-85%, 70%-90%, 70%-95%, 70%-90%, 70%-95%, 70%-100% of all sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. In some embodiments, the sugars are 2'-OMe or 2'-MOE modified sugars. 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%.

[0186] In some embodiments, 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%-85%, 70%-90%, 70%-95% of all sugars In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. In some embodiments, the sugars are 2'-OMe modified sugars. 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%.

[0187] In some embodiments, 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%-85%, 70%-90%, 70%-95%, 70 ... %, 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% or more of the 2'-OR modified sugars wherein R is an optionally substituted C1-6 In some embodiments, 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%-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%, etc. are 2'-MOE modified sugars.

[0188] In some embodiments, the first (5'-end) one or more (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 last (3 '-end) one or several (for example 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.) sugars of nucleoside are independently modified sugars. In certain embodiments, the first one or several sugars are independently modified sugars. In certain embodiments, the second one or several sugars are independently modified sugars. In certain embodiments, the first and second one or several sugars are independently modified sugars. In certain embodiments, the modified sugars are independently non-2 '-F modified sugars, for example bicyclic sugars, 2 '-OR modified sugars (wherein R is as described herein and is not -H (for example optionally substituted C 1-6 In some embodiments, it is independently selected from bicyclic sugars and 2'-OR modified sugars (wherein R is optionally substituted C 1-6 In some embodiments, it is independently a 2'-OR modified sugar, wherein R is an optionally substituted C1-6 In some embodiments, the first few sugars comprise one or more 2'-OR modified sugars (wherein R is an optionally substituted C 1-6 aliphatic) or bicyclic sugars (eg, LNA, cEt, etc.). In some embodiments, the first few sugars comprise one or more 2'-OR modified sugars, wherein R is an optionally substituted C 1-6 In some embodiments, the first sugars comprise one or more 2'-OMe modified sugars. In some embodiments, the first sugars comprise one or more 2'-MOE modified sugars. In some embodiments, the first sugars comprise one or more 2'-OMe modified sugars and one or more 2'-MOE modified sugars. In some embodiments, the last sugars comprise one or more 2'-OR modified sugars as described herein (wherein R is optionally substituted C 1-6 aliphatic) or bicyclic sugars (e.g., LNA, cEt, etc.). In some embodiments, the latter sugars comprise one or more 2'-OR modified sugars, wherein R is an optionally substituted C 1-6 In some embodiments, the latter sugars include one or more 2'-OMe modified sugars. In some embodiments, the latter sugars include one or more 2'-MOE modified sugars. In some embodiments, the latter sugars include one or more 2'-OMe modified sugars and one or more 2'-MOE modified sugars. In some embodiments, the latter sugars are independently 2'-OMe modified sugars. In some embodiments, the former sugars include two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) continuous bicyclic sugars or 2'-OR modified sugars (wherein R is optionally substituted C 1-6 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 an optionally substituted C 1-6Aliphatic group. In some embodiments, the first 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 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 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 last 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 substituted C 1-6 Aliphatic group. In some embodiments, the latter several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) continuous modified sugars, wherein each modified sugar is independently a 2'-OMe modified sugar or a 2'-MOE modified sugar. In some embodiments, the latter several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) continuous 2'-OMe modified sugars. In some embodiments, the latter several sugars comprise three or more continuous 2'-OMe modified sugars. In some embodiments, the latter several sugars comprise four or more continuous 2'-OMe modified sugars. In some embodiments, the latter several sugars comprise five or more continuous 2'-OMe modified sugars. In some embodiments, the latter several sugars comprise six or more continuous 2'-OMe modified sugars. In some embodiments, the latter several sugars comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) continuous 2'-MOE modified sugars.

[0189] In some embodiments, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the first (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are modified sugars. In some embodiments, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the first (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are modified sugars, each independently selected from a 2'-OR modified sugar (wherein R is an optionally substituted C 1-6 aliphatic groups) and bicyclic sugars (e.g., sugars comprising 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 2'-OR modified sugars (wherein R is optionally substituted C 1-6In some embodiments, three or more of the first few sugars are modified sugars, each independently selected from 2'-OR modified sugars (wherein R is optionally substituted C 1-6 In some embodiments, four or more of the first few sugars are modified sugars, each independently selected from 2'-OR modified sugars (wherein R is optionally substituted C 1-6 In some embodiments, one or more sugars are continuous. 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 a 2'-OR modified sugar (wherein R is an optionally substituted C 1-6 In some embodiments, the first three sugars are modified sugars, each independently selected from 2'-OR modified sugars (wherein R is optionally substituted C 1-6 In some embodiments, the first four sugars are modified sugars, each independently selected from 2'-OR modified sugars (wherein R is optionally substituted C 1-6 In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each bicyclic sugar is independently a LNA sugar or a cEt sugar. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) sugars in 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 an optionally substituted C 1-6Aliphatic group. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) sugars in the first sugars or the first sugars (e.g., 1, 2, 3, 4, or 5) are each independently 2'-OMe or 2'-MOE modified sugars. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) sugars in the first sugars or the first sugars (e.g., 1, 2, 3, 4, or 5) are each independently 2'-OMe modified sugars. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) sugars in the first sugars or the first sugars (e.g., 1, 2, 3, 4, or 5) are each independently 2'-MOE modified sugars. In some embodiments, the first, two, three, four, or more sugars are independently 2'-OMe modified sugars. In some embodiments, the first sugar is a 2'-OMe modified sugar. In some embodiments, the first two sugars are independently 2'-OMe modified sugars. In some embodiments, the first three sugars are independently 2'-OMe modified sugars. In certain embodiments, the first four sugars are independently 2'-OMe modified sugars. In certain embodiments, the first one, two, three, four or more sugars are independently 2'-MOE modified sugars. In certain embodiments, the first sugar is 2'-MOE modified sugars. In certain embodiments, the first two sugars are independently 2'-MOE modified sugars. In certain embodiments, the first three sugars are independently 2'-MOE modified sugars. In certain embodiments, the first four sugars are independently 2'-MOE modified sugars. In certain embodiments, each of such modified sugars is independently a sugar of a nucleoside in which its core base is 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 certain embodiments, one or more such sugars are independently bonded to a negatively charged internucleotide linkage. In certain embodiments, one or more such sugars are independently bonded to a neutral internucleotide linkage (such as n001). In certain embodiments, negatively charged internucleotide linkage or neutral internucleotide linkage (such as n001) is chirality controlled. In certain embodiments, it is Rp. In certain embodiments, one or more such sugars are independently bonded to a thiophosphate internucleotide linkage. In certain embodiments, the thiophosphate internucleotide linkage is chiral controlled. In certain embodiments, it is Sp. In certain embodiments, as described herein, the internucleotide linkage between the first and second nucleoside is a non-negatively charged internucleotide linkage. In certain embodiments, it is a neutral internucleotide linkage. In certain embodiments, it is a phosphorylguanidine internucleotide linkage. In certain embodiments, it is n001. In certain embodiments, it is chiral controlled. In certain embodiments, it is Rp.In certain embodiments, except the internucleotide linkage between the first and second nucleoside, linkage is independently phosphorothioate internucleotide linkage with each internucleotide linkage of the nucleoside bonding of one or more or first several modified sugars comprising the first several modified sugars.In certain embodiments, each is chiral controlled.In certain embodiments, each is Sp.In certain embodiments, the first nucleoside is optionally connected to other part, for example Mod001, by its 5 '-terminal carbon (in certain embodiments, by phosphate group) through a joint (for example L001).

[0190] In some embodiments, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the last several (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are modified sugars. In some embodiments, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the last several (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) sugars are modified sugars, each independently selected from a 2'-OR modified sugar (wherein R is an optionally substituted C 1-6 aliphatic groups) and bicyclic sugars (e.g., sugars comprising 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 2'-OR modified sugars (wherein R is optionally substituted C 1-6 In some embodiments, three or more of the latter sugars are modified sugars, each independently selected from 2'-OR modified sugars (wherein R is optionally substituted C 1-6 In some embodiments, four or more of the latter sugars are modified sugars, each independently selected from 2'-OR modified sugars (wherein R is optionally substituted C 1-6 In some embodiments, one or more sugars are continuous. 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 a 2'-OR modified sugar (wherein R is an optionally substituted C 1-6 In some embodiments, the last three sugars are modified sugars, each independently selected from 2'-OR modified sugars (wherein R is optionally substituted C 1-6 In some embodiments, the last four sugars are modified sugars, each independently selected from 2'-OR modified sugars (wherein R is optionally substituted C 1-6In some embodiments, each 2'-OR modified sugar is independently a 2'-OMe or 2'-MOE modified sugar. In some embodiments, each bicyclic sugar is independently a LNA sugar or a cEt sugar. In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) sugars in the latter several sugars or the latter several (e.g., 1, 2, 3, 4, or 5) sugars are each independently a 2'-OR modified sugar, wherein R is an optionally substituted C 1-6Aliphatic group. In certain embodiments, one or more (e.g., 1, 2, 3, 4, or 5) sugars in the last few sugars or the last few (e.g., 1, 2, 3, 4, or 5) sugars are each independently 2'-OMe or 2'-MOE modified sugars. In certain embodiments, one or more (e.g., 1, 2, 3, 4, or 5) sugars in the last few sugars or the last few (e.g., 1, 2, 3, 4, or 5) sugars are each independently 2'-OMe modified sugars. In certain embodiments, one or more (e.g., 1, 2, 3, 4, or 5) sugars in the last few sugars or the last few (e.g., 1, 2, 3, 4, or 5) sugars are each independently 2'-OMe modified sugars. In certain embodiments, the last one, two, three, four, or more sugars are independently 2'-OMe modified sugars. In certain embodiments, the last sugar is a 2'-OMe modified sugar. In certain embodiments, the last two sugars are independently 2'-OMe modified sugars. In certain embodiments, the last three sugars are independently 2'-OMe modified sugars. In certain embodiments, the last four sugars are independently 2'-OMe modified sugars. In certain embodiments, the last one, two, three, four or more sugars are independently 2'-MOE modified sugars. In certain embodiments, the last sugar is 2'-MOE modified sugars. In certain embodiments, the last two sugars are independently 2'-MOE modified sugars. In certain embodiments, the last three sugars are independently 2'-MOE modified sugars. In certain embodiments, the last four sugars are independently 2'-MOE modified sugars. In certain embodiments, each of such modified sugars is independently a sugar of a nucleoside in which its core base is 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 certain embodiments, one or more such sugars are independently bonded to a negatively charged internucleotide linkage. In certain embodiments, one or more such sugars are independently bonded to a neutral internucleotide linkage (such as n001). In certain embodiments, negatively charged internucleotide linkage or neutral internucleotide linkage (such as n001) is chirality controlled. In certain embodiments, it is Rp. In certain embodiments, one or more such sugars are independently bonded to a thiophosphate internucleotide linkage. In certain embodiments, the thiophosphate internucleotide linkage is chirality controlled. In certain embodiments, it is Sp. In certain embodiments, as described herein, the internucleotide linkage between the last and penultimate nucleoside is a non-negatively charged internucleotide linkage. In certain embodiments, it is a neutral internucleotide linkage. In certain embodiments, it is a phosphorylguanidine internucleotide linkage. In certain embodiments, it is n001. In certain embodiments, it is chirality controlled. In certain embodiments, it is Rp.In certain embodiments, except the internucleotide linkage between last and penultimate nucleoside, each internucleotide linkage with the nucleoside linkage of one or more or several modified sugars comprising the latter is independently a phosphorothioate internucleotide linkage. In certain embodiments, each is chirality controlled. In certain embodiments, each is Sp.

[0191] In some embodiments, the sugar at position +1 is a 2'-F modified sugar. In some embodiments, the sugar at position +1 is a natural DNA sugar. In some embodiments, the sugar at position 0 is a natural DNA sugar (the nucleoside at position 0 is opposite the target adenosine when aligned). 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 an optionally substituted C 1-6 aliphatic) or a bicyclic sugar (e.g., a sugar comprising 2'-O-CH2-4', wherein -CH2- is optionally substituted (e.g., an LNA sugar, a cET sugar (e.g., (S)-cEt)). In some embodiments, it is a 2'-OR modified sugar, wherein R is optionally substituted C 1-6 In some embodiments, the sugar at position -3 is a 2'-F modified sugar. In some embodiments, each sugar after position -3 (e.g., position -4, -5, -6, etc.) is independently a 2'-OR modified sugar (wherein R is optionally substituted C 1-6 aliphatic) or a bicyclic sugar (e.g., a sugar comprising 2'-O-CH2-4', wherein -CH2- is optionally substituted (e.g., an LNA sugar, a cET sugar (e.g., (S)-cEt)). In some embodiments, each is independently a 2'-OR modified sugar (wherein R is optionally substituted C 1-6In some embodiments, the sugars are 2'-OMe or 2'-MOE. ... In certain embodiments, it is n001.In certain embodiments, it is chirality controlled.In certain embodiments, it is Rp.In certain embodiments, each internucleotide linkage between a nucleoside and the 3 '-side of the nucleoside opposite to the target adenosine (except those internucleotide linkages between the nucleoside at position-1 and -2 and between the nucleoside at position-2 and -3 and between the last and penultimate nucleoside) is independently phosphorothioate internucleotide linkage.In certain embodiments, each internucleotide linkage is chirality controlled.In certain embodiments, each is Sp.

[0192] In some embodiments, the first and / or next sugar or sugars are modified sugars (e.g., bicyclic sugars and / or 2'-OR modified sugars) (wherein R is an optionally substituted C 1-6 In some embodiments, the sugar at the 5'- and / or 3'-terminus of the oligonucleotide is not a bicyclic sugar or a 2'-OR modified sugar (wherein R is an optionally substituted C 1-6 In some embodiments, the 5'-terminal sugar is a bicyclic sugar or a 2'-OR modified sugar (wherein R is optionally substituted C 1-6In some embodiments, such 5'-terminal sugars are not linked to another 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 another chemical moiety. In some embodiments, the 3'-terminal sugar is a bicyclic sugar or a 2'-OR modified sugar (wherein R is an optionally substituted C 1-6 In some embodiments, such 3'-terminal sugars are not linked to another chemical moiety. 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 another chemical moiety. In some embodiments, the latter sugars are 3'-side sugars relative to the nucleoside opposite the target adenosine (e.g., sugars of the 3'-side nucleoside, such as N -1 、N -2 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 (wherein R is optionally substituted C 1-6 In some embodiments, the 2'-OR modified sugar is a 2'-OMe modified sugar or a 2'-MOE modified sugar, as described herein; in some embodiments, it is a 2'-OMe modified sugar; in some embodiments, it is 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'-OR modified sugar, wherein R is an optionally substituted C 1-6Aliphatic group. In certain embodiments, the latter several sugars or 3'-side sugars comprise one or more or two or more continuous 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a sugar modified by 2'-OMe or a sugar modified by 2'-MOE. In certain embodiments, the latter several sugars or 3'-side sugars comprise one or more or two or more continuous 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a sugar modified by 2'-OMe. In certain embodiments, the latter several sugars or 3'-side sugars comprise one or more or two or more continuous 2'-F modified sugars, and the sugar of the last nucleoside of the oligonucleotide is a sugar modified by 2'-MOE. In certain embodiments, two and no more than two nucleosides of the 3'-side of the nucleoside opposite to adenosine independently have a sugar modified by 2'-F. In certain embodiments, it is at positions -4 and -5. In certain embodiments, it is the penultimate and third nucleoside of the oligonucleotide. In certain embodiments, one and no more than one nucleoside of the 3'-side of the nucleoside 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 nucleoside of the oligonucleotide.

[0193] In some embodiments, bicyclic sugars or 2'-OR modified sugars (wherein R is an optionally substituted C 1-6 In certain embodiments, the sugar of 2'-F modification comprises one or more (such as 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 modifications. In certain embodiments, most of sugars as described herein in such regions are sugars that 2'-F modifications modify. In certain embodiments, the sugars that two or more 2'-F modifications modify are continuous. In certain embodiments, the region is the first domain. In certain embodiments, dicyclic sugars are present in such regions. In certain embodiments, the sugar of 2'-OR modification (wherein R is optionally substituted C 1-6 In some embodiments, a 2'-OMe modified sugar is present in such a region. In some embodiments, a 2'-MOE modified sugar is present in such a region.

[0194] In some embodiments, positions -5, -4, -3, +1, +2, +4, +5, +6, +7, and +8 (position 0 is the position of the nucleoside opposite the target adenosine; "+" is extending from the nucleoside opposite the target adenosine toward the 5'-end of the oligonucleotide, and "-" is extending from the nucleoside opposite the target adenosine toward the 3'-end of the oligonucleotide; for example, in 5'-N1N0N -1-3', if N0 is the opposite nucleoside to the target adenosine, it is at position 0, and N1 is at position +1 and N -1 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 or more sugars at positions -5, -4, -3, +2, +4, +5, +6, +7, and +8 are independently 2'-F modified sugars.

[0195] 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.) natural DNA sugars. In some embodiments, one or more natural DNA sugars are located in the editing region, e.g., position +1, 0, and / or -1. In some embodiments, the natural DNA sugar is within the first few nucleosides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides) of the oligonucleotide. In some embodiments, the first, second, and / or third nucleoside of the oligonucleotide independently has a natural DNA sugar. In some embodiments, the natural DNA sugar is bonded to a modified internucleotide linkage (e.g., a non-negatively charged internucleotide linkage, a neutral internucleotide linkage, a phosphorylguanidine internucleotide linkage, n001, or a phosphorothioate internucleotide linkage (in various embodiments, Sp)).

[0196] Oligonucleotides can contain various types of internucleotide linkages. In some embodiments, the oligonucleotide comprises 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 phosphorothioate internucleotide linkage. In some embodiments, the modified internucleotide linkage is a non-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, the oligonucleotide comprises one or more natural phosphate linkages. In some embodiments, the natural phosphate linkage is linked to a nucleoside comprising a modified sugar that can improve stability (e.g., resistance to nucleases). In some embodiments, the natural phosphate linkage is linked to a bicyclic sugar. In some embodiments, the natural phosphate linkage is linked to a 2'-modified sugar. In some embodiments, the natural phosphate linkage is linked to a 2'-OR modified sugar (wherein R is optionally substituted C 1-6In some embodiments, the oligonucleotide comprises a linkage between thiophosphate nucleotides, a linkage between neutral nucleotides, and a natural phosphate ester linkage. In some embodiments, the oligonucleotide comprises a linkage between thiophosphate nucleotides, a linkage between phosphorylguanidine nucleotides, and a natural phosphate ester linkage. In some embodiments, the oligonucleotide comprises a linkage between thiophosphate nucleotides, a linkage between phosphorylguanidine nucleotides, and a natural phosphate ester linkage. In some embodiments, the oligonucleotide comprises a linkage between thiophosphate nucleotides, a linkage between phosphorylguanidine nucleotides, and a natural phosphate ester linkage. In some embodiments, the oligonucleotide comprises a linkage between thiophosphate nucleotides, a linkage between phosphorylguanidine nucleotides, and a natural phosphate ester linkage. In some embodiments, the oligonucleotide comprises a linkage between thiophosphate nucleotides, n001, and a natural phosphate ester linkage. In some embodiments, the linkage between each chiral nucleotide is independently chirality controlled. In some embodiments, the linkage between one or more chiral nucleotides is not chirality controlled. In some embodiments, the linkage between each thiophosphate nucleotide is independently chirality controlled. In some embodiments, the linkage between each chiral nucleotide is independently chirality controlled. In some embodiments, most or each phosphorothioate internucleotide linkage is Sp as described herein. In some embodiments, most or each non-negatively charged internucleotide linkage, e.g., n001, is Rp. In some embodiments, most or each non-negatively charged internucleotide linkage, e.g., n001, is Sp.

[0197] In certain embodiments, the oligonucleotide comprises a linkage between phosphorothioate nucleotides and a non-negatively charged nucleotide. In certain embodiments, the oligonucleotide comprises a linkage between phosphorothioate nucleotides and a neutral nucleotide. In certain embodiments, the oligonucleotide comprises a linkage between phosphorothioate nucleotides and a phosphorylguanidine nucleotide. In certain embodiments, the oligonucleotide comprises a linkage between phosphorothioate nucleotides and a phosphorylguanidine nucleotide. In certain embodiments, the oligonucleotide comprises a linkage between phosphorothioate nucleotides and n001. In certain embodiments, the linkage between each chiral nucleotide is independently chiral controlled. In certain embodiments, the linkage between one or more chiral nucleotides is not chiral controlled. In certain embodiments, the linkage between each phosphorothioate nucleotide is independently chiral controlled. In certain embodiments, the linkage between each chiral nucleotide is independently chiral controlled. In certain embodiments, the linkage between most or each phosphorothioate nucleotide is Sp as described herein. In certain embodiments, the linkage between one or more (e.g., 1, 2, 3, 4, or 5) phosphorothioate nucleotides is Rp. In certain embodiments, the linkage between most or each non-negatively charged nucleotides, e.g., n001 is Rp. In certain embodiments, most or each negatively charged internucleotide linkage, for example n001 is Sp.In certain embodiments, oligonucleotide does not comprise natural phosphate linkage.In certain embodiments, linkage is independently phosphorothioate or negatively charged internucleotide linkage between each Nucleotide.In certain embodiments, linkage is independently phosphorothioate or neutrally charged internucleotide linkage between each Nucleotide.In certain embodiments, linkage is independently phosphorothioate or phosphorylguanidine internucleotide linkage between each Nucleotide.In certain embodiments, linkage is independently phosphorothioate or n001 internucleotide linkage between each Nucleotide.In certain embodiments, linkage is independently phosphorothioate or n001 internucleotide linkage between each Nucleotide.In certain embodiments, linkage is independently non-negatively charged internucleotide linkage between the last Nucleotide of oligonucleotide, or linkage between neutral Nucleotide, or linkage between phosphorylguanidine internucleotide, or n001.

[0198] In some embodiments, the oligonucleotides of the present disclosure comprise one or more modified nucleobases. According to the present disclosure, a variety of modifications can be introduced into the sugar and / or nucleobase. For example, in some embodiments, the modification is a modification described in US 9006198. In some embodiments, the modification is described in US 9394333, US 9744183, US 9605019, US 9982257, US20170037399, US 20180216108, 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 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, and / or WO 2022 / 099159, their respective sugars, bases, and internucleotide linkages are independently incorporated herein by reference.

[0199] In some embodiments, the nucleobase in the nucleoside is or comprises cyclic BA having the structure 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 a tautomer of cyclic BA, wherein the nucleobase is optionally substituted or protected.

[0200] In some embodiments, the sugar is a modified sugar comprising a 2'-modification (e.g., 2'-F, 2'-OR (wherein R is an optionally substituted aliphatic group)), or a bicyclic sugar (e.g., an LNA sugar), or an acyclic sugar (e.g., a UNA sugar).

[0201] In some embodiments, as described herein, the oligonucleotides provided comprise one or more domains, each independently having certain lengths, modifications, linkage phosphorus stereochemistry, etc. as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising one or more modified sugars and / or one or more modified internucleotide linkages, wherein the oligonucleotide comprises a first domain and a second domain, each independently comprising one or more nucleobases. In some embodiments, the present disclosure provides oligonucleotides comprising one or more domains and / or subdomains as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising a first domain as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising a second domain as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising a first subdomain as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising a second subdomain as described herein. In some embodiments, the present disclosure provides oligonucleotides comprising a third subdomain as described herein. In some embodiments, the present 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, the present disclosure provides oligonucleotides comprising: first domain; and The second domain, in: The first domain comprises one or more 2′-F modifications; The second domain comprises one or more sugars that do not have a 2'-F modification.

[0202] In certain embodiments, oligonucleotide or its part (for example, first domain, second domain, first subdomain, second subdomain, third subdomain etc.) comprises certain level of modified sugar.In certain embodiments, modified sugar comprises 2 '-modification.In certain embodiments, modified sugar is bicyclic sugar.In certain embodiments, modified sugar is acyclic sugar (for example, by the C2-C3 key of fracture corresponding cyclic sugar).In certain embodiments, modified sugar comprises 5 '-modification.Typically, unless otherwise stated, for example, by context explanation, otherwise the oligonucleotide of present disclosure has free 5 '-OH at its 5 '-end and has free 3 '-OH at its 3 '-end.In certain embodiments, the 5 '-end sugar of oligonucleotide can comprise modified 5 '-OH.

[0203] In some embodiments, the level is about, 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%-85%, respectively, of all sugars in the oligonucleotide or portion thereof. 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%.

[0204] In some embodiments, the majority is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the majority is 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%. 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, a majority is about or at least about 80%. In some embodiments, a majority is about or at least about 85%. In some embodiments, a majority is about or at least about 90%. In some embodiments, a majority is about or at least about 95%.

[0205] 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 level of modified internucleotide linkages. 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 level of chiral internucleotide linkages. In some embodiments, the level is about, 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%-85%, 70%-90%, 70%-95%, 70 ... 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%.

[0206] 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 chiral controlled internucleotide linkages. 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 internucleotide linkages. In some embodiments, the level is about, 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%-85%, 70%-90%, 70%-95%, 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 level is about, 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%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 70%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 75 ... %-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%.

[0207] In some embodiments, an oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a level of Sp internucleotide linkages. In some embodiments, the level is about, 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%-90%, 70%-95%, 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 level is about, 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%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 70%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 75 ... 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 level is about, 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%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-80%, 80%-85%, 85%-90%, 85%-95%, 85%-100%, 80%-80%, 80 ...80%, 80%-80%, 80%-85%, 85%-90%, 85%-95%, 85%-100%, 80%-80%, 80%-80%, 80%-80%, 80%-80%, 80% 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, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 internucleotide linkages are independently Sp chiral internucleotide linkages. In many embodiments, it has been observed that a high percentage of Sp internucleotide linkages (for example, relative to Rp internucleotide linkages and / or natural phosphate linkages) in an oligonucleotide or some portion thereof can provide improved properties and / or activity, for example, high stability and / or high adenosine editing activity.

[0208] In some embodiments, an oligonucleotide or portion thereof (e.g., a first domain, a second domain, a first subdomain, a second subdomain, a third subdomain, etc.) comprises a level of Rp internucleotide linkages. In some embodiments, the level is about, 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%-90%, 70%-95%, 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 level is about, 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%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 70%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 75 ... 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 level is about, 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%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-80%, 80%-85%, 85%-90%, 85%-95%, 85%-100%, 80%-80%, 80 ...80%, 80%-80%, 80%-85%, 85%-90%, 85%-95%, 85%-100%, 80%-80%, 80%-80%, 80%-80%, 80%-80%, 80% 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, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 internucleotide linkages are independently Rp chiral internucleotide linkages. In some embodiments, the number is about or not greater than about 1. In some embodiments, the number is about or not greater than about 2. In some embodiments, the number is about or not greater than about 3.In some embodiments, the number is about or not greater than about 4. In some embodiments, the number is about or not greater than about 5. In some embodiments, the number is about or not greater than about 6. In some embodiments, the number is about or not greater than about 7. In some embodiments, the number is about or not greater than about 8. In some embodiments, the number is about or not greater than about 9. In some embodiments, the number is about or not greater than about 10.

[0209] While not wishing to be bound by theory, it is noted that in some cases, the Rp and Sp configurations of the internucleotide linkages can affect structural changes in the helical conformation of the double-stranded complex formed by the oligonucleotide and the target nucleic acid, such as RNA, and that the ADAR protein can recognize and interact with a variety of targets (e.g., double-stranded complexes formed by the oligonucleotide and the target nucleic acid, such as RNA) through multiple domains. In some embodiments, the oligonucleotides and compositions thereof provided promote and / or enhance the interaction spectrum of the oligonucleotide, target nucleic acid, and / or ADAR protein to provide effective adenosine modification of the ADAR protein by incorporation of a variety of modifications and / or control of stereochemistry.

[0210] In some embodiments, an oligonucleotide can have or comprise a base sequence; an internucleotide linkage, a base modification, a sugar modification, an additional chemical moiety or pattern thereof; and / or any other structural elements described herein, e.g., in the Tables.

[0211] In some embodiments, provided oligonucleotides or compositions are characterized in that, when contacted with a target nucleic acid comprising a target adenosine in a system (e.g., an ADAR-mediated deamination system), modification of the target adenosine (e.g., deamination of target A) is improved relative to 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 a reference oligonucleotide or composition, and combinations thereof). In some embodiments, the modification, e.g., ADAR-mediated deamination (e.g., endogenous ADAR-mediated deamination) is 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-fold or more.

[0212] In certain embodiments, oligonucleotide is provided in the form of salt.In certain embodiments, oligonucleotide is provided in the form of salt, and these salts comprise the negatively charged internucleotide linkage (for example, phosphorothioate internucleotide linkage, natural phosphate linkage etc.) that exists as salt form.In certain embodiments, oligonucleotide is provided in the form of pharmaceutically acceptable salt.In certain embodiments, oligonucleotide is provided in the form of metal salt.In certain embodiments, oligonucleotide is provided in the form of sodium salt.In certain embodiments, oligonucleotide is provided in the form of ammonium salt.In certain embodiments, oligonucleotide is provided in the form of metal salt such as sodium salt, wherein each negatively charged internucleotide linkage is independently in salt form (for example, for sodium salt, for phosphorothioate internucleotide linkage is-OP (O) (SNa)-O-, for natural phosphate linkage is-OP (O) (ONa)-O- etc.).

[0213] In certain embodiments, the oligonucleotide is chirality controlled, and it comprises one or more chirality controlled internucleotide linkages.In certain embodiments, the oligonucleotide provided is stereochemically pure.In certain embodiments, the oligonucleotide provided or its composition are substantially free of other stereoisomers.In certain embodiments, this disclosure provides the oligonucleotide composition of chirality controlled.

[0214] As described herein, the oligonucleotides of the present disclosure can be provided in high purity (e.g., 50%-100%). In some embodiments, the oligonucleotides of the present disclosure have high stereochemical purity (e.g., 50%-100%). In some embodiments, the oligonucleotides in the compositions provided have high stereochemical purity (e.g., a stereoisomer has a high percentage (e.g., 50%-100%) 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%.

[0215] In certain embodiments, the oligonucleotides of the present disclosure are at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% stereochemically pure at the linkage phosphorus of the chiral internucleotide linkage. In certain embodiments, the oligonucleotides of the present disclosure are stereoselectively prepared and are substantially free of stereoisomers. In certain embodiments, in the compositions of the multiple oligonucleotides comprising the same base sequence of the provided sharing identical chiral linkage phosphorus stereochemistry (e.g., comprising one or more of Rp and / or Sp, wherein each chiral linkage phosphorus is independently Rp or Sp) pattern, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of all oligonucleotides sharing the same base sequence with the multiple oligonucleotides in the composition share the same chiral linkage phosphorus stereochemistry pattern or are the multiple oligonucleotides. In some embodiments, in a provided composition comprising a plurality of oligonucleotides having the same base sequence sharing the same stereochemical pattern of chiral phosphorus linkages, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition sharing the same composition as the plurality of oligonucleotides share the same stereochemical pattern of chiral phosphorus linkages or are 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 , wherein DS is about 90%-100%, and nc is the number of chiral phosphorus bonds. 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 phosphorus bond. First domain

[0216] As described herein, in some embodiments, the oligonucleotide comprises a first domain and a second domain. In certain embodiments, the oligonucleotide consists of a first domain and a second domain. Certain embodiments are described below with examples.

[0217] In some embodiments, the first domain has a length of about 2-50 (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. In some embodiments, the first domain has a length of about 5-30 nucleobases. In some embodiments, the first domain has a length of about 10-30 nucleobases. In some embodiments, the first domain has a length of about 10-20 nucleobases. In some embodiments, the first domain has a length of about 13-16 nucleobases. In some embodiments, the first domain has a length of 10 nucleobases. In some embodiments, the first domain has a length of 11 nucleobases. In some embodiments, the first domain has a length of 12 nucleobases. In some embodiments, the first domain has a length of 13 nucleobases. In some embodiments, the first domain has a length of 14 nucleobases. In some embodiments, the first domain has a length of 15 nucleobases. In some embodiments, the first domain has a length of 16 nucleobases. In some embodiments, the first domain has a length of 17 nucleobases. In some embodiments, the first domain has a length of 18 nucleobases. In some embodiments, the first domain has a length of 19 nucleobases. In some embodiments, the first domain has a length of 20 nucleobases.

[0218] 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% of the oligonucleotide. 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%. 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%.

[0219] In some embodiments, when the oligonucleotide is aligned for complementarity with the target nucleic acid, there are one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) mismatches in the first domain. In some embodiments, there is 1 mismatch. In some embodiments, there are 2 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.

[0220] In some embodiments, when the oligonucleotide is aligned for complementarity with the target nucleic acid, one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobbles are present in the first domain. In some embodiments, there is 1 wobble. In some embodiments, there are 2 wobbles. In some embodiments, there are 3 wobbles. In some embodiments, there are 4 wobbles. In some embodiments, there are 5 wobbles. In some embodiments, there are 6 wobbles. In some embodiments, there are 7 wobbles. In some embodiments, there are 8 wobbles. In some embodiments, there are 9 wobbles. In some embodiments, there are 10 wobbles.

[0221] In certain embodiments, the duplex of the oligonucleotide and the target nucleic acid in the first domain region includes one or more protrusions, and each protrusion independently includes one or more mispairings that are not wobbling. In certain embodiments, there are 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, 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.) protrusions. In certain embodiments, the number is 0. In certain embodiments, the number is 1. In certain embodiments, the number is 2. In certain embodiments, the number is 3. In certain embodiments, the number is 4. In some embodiments, the number is 5.

[0222] In some embodiments, the first domain is fully complementary to the target nucleic acid.

[0223] In some embodiments, the first domain comprises one or more modified nucleobases.

[0224] In certain embodiments, the second domain comprises one or more sugars (e.g., natural DNA sugars) comprising two 2'-Hs. In certain embodiments, the second domain comprises one or more sugars (e.g., natural RNA sugars) comprising 2'-OHs. In certain embodiments, the first domain comprises one or more modified sugars. In certain embodiments, modified...

Claims

1. An oligonucleotide, wherein the oligonucleotide comprises 5'-N1N0N -1 -3', where N -1 , N0 and N1 are each independently a nucleoside; and wherein: (a) The nucleobase of N0 is BA, where BA is Alternatively, BA comprises cyclic BA or a tautomer thereof, wherein cyclic BA has the structure of formula BA-III-e: in: X 1 is -N(-)- or -C(-)=; W X2 and W X6 Each of is independently O, S or Se; R B4 Is halogen, -CN, -NO2 or -L B4 -R B41 , where R B41 It is R'; R B5 Is halogen, -CN, -NO2 or -L B5 -R B51 , where R B51 is -R', -N(R')2, -OR' or -SR', L B4 and L B5 Each of which is independently L B ; Each L B is independently a covalent bond, or an optionally substituted divalent C 1-10 a saturated or partially unsaturated chain in which one or more methylene units are optionally and independently replaced by -Cy-, -O-, -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, or -C(O)O-; Each -Cy- is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each R' is independently -R, -C(O)R, -C(O)OR, -C(O)N(R)2, or -SO2R; and Each R is independently -H, or an optionally substituted group selected from the following: C 1-20 Aliphatic, C with 1-10 heteroatoms 1-20 Heteroaliphatic, C 6-20 Aryl, C 6-20 Aryl aliphatic group, C 6-20 arylheteroaliphatic, 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-20 membered heterocyclyl having 1-10 heteroatoms, or Two R groups optionally and independently join together to form a covalent bond, or Two or more R groups on the same atom are optionally and independently taken together with that atom to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to that atom; or Two or more R groups on two or more atoms are optionally and independently taken together with the atoms intervening therebetween to form an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the atoms intervening therebetween; and (b) the sugar of N0 comprises a 2'-OR modification, wherein R is an optionally substituted C 1-6 Aliphatic group.

2. The oligonucleotide of claim 1, wherein the nucleobase of NO is 3. The oligonucleotide of claim 1, wherein the nucleobase of NO is 4. The oligonucleotide of claim 1, wherein the nucleobase of NO is 5. The oligonucleotide of claim 1, wherein the nucleobase of NO is 6. The oligonucleotide of claim 1, wherein R B4 Not -H.

7. The oligonucleotide of claim 2, wherein the sugar of NO is a 2'-OMe modified sugar.

8. The oligonucleotide of claim 2, wherein the sugar of NO is a 2'-MOE modified sugar.

9. The oligonucleotide of claim 1, wherein the oligonucleotide comprises one or more 2'-F modified sugars.

10. The oligonucleotide of claim 1, wherein when the oligonucleotide is contacted with a target nucleic acid comprising a target adenosine in a system, the target adenosine in the target nucleic acid is modified, wherein when the oligonucleotide is aligned with the target nucleic acid, NO is opposite to the target adenosine.

11. The oligonucleotide of claim 10, wherein the target adenosine is a G to A mutation.

12. The oligonucleotide of claim 1, 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, 25 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 100, 120, 150, 100, 150, 120, 150, 150, 100, 100, 100, 120, 150, 150, 100, 100, 100, 150, 120, 150, 150, 100, 100, 100, 150, 120, 150, 150, 100, 100, 100, 100, 150, 120, 150, 150 13. The oligonucleotide of claim 12, wherein the base sequence of the oligonucleotide is complementary to the base sequence of a portion of the target nucleic acid comprising the target adenosine, and has 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, 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.) mismatches that are not Watson-Crick base pairs.

14. The oligonucleotide of claim 13, wherein the complementarity is about 50%-100% (e.g., about 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%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc.).

15. The oligonucleotide of any one of claims 13-14, wherein the complementarity is 100% except at the nucleoside opposite the target adenosine.

16. An oligonucleotide as claimed in any one of the preceding claims, wherein N -1 The nucleobase is BA, wherein BA comprises cyclic BA or a tautomer thereof, wherein cyclic BA has the structure of formula BA-III-e.

17. An oligonucleotide as claimed in any one of the preceding claims, wherein N -1 The nucleobase is BA, where BA is 18. An oligonucleotide, wherein the oligonucleotide comprises a nucleotide sequence having about 2-50 (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. 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 in length.

19. An oligonucleotide, wherein the oligonucleotide comprises 5'-N1N0N -1 -3', where N -1 , N0 and N1 are each independently a nucleoside, wherein the nucleobase of N0 is BA, wherein BA comprises a ring BA or a tautomer thereof, wherein the ring BA has a structure of formula BA-III-e: in: X 1 is -N(-)- or -C(-)=; W X2 and W X6 Each of is independently O, S or Se; R B4 Is halogen, -CN, -NO2 or -L B4 -R B41 , where R B41 It is R'; R B5 Is halogen, -CN, -NO2 or -L B5 -R B51 , where R B51 is -R', -N(R')2, -OR' or -SR', L B4 and L B5 Each of which is independently L B ; Each L B is independently a covalent bond, or an optionally substituted divalent C 1-10 a saturated or partially unsaturated chain in which one or more methylene units are optionally and independently replaced by -Cy-, -O-, -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, or -C(O)O-; Each -Cy- is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each R' is independently -R, -C(O)R, -C(O)OR, -C(O)N(R)2, or -SO2R; and Each R is independently -H, or an optionally substituted group selected from the following: C 1-20 Aliphatic, C with 1-10 heteroatoms 1-20 Heteroaliphatic, C 6-20 Aryl, C 6-20 Aryl aliphatic group, C 6-20 arylheteroaliphatic, 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-20 membered heterocyclyl having 1-10 heteroatoms, or Two R groups optionally and independently join together to form a covalent bond, or Two or more R groups on the same atom are optionally and independently taken together with that atom to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to that atom; or Two or more R groups on two or more atoms are optionally and independently taken together with the atoms intervening therebetween to form an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the atoms intervening therebetween.

20. An oligonucleotide, wherein the oligonucleotide comprises a nucleoside NO, wherein the nucleobase of NO is BA, wherein BA comprises a ring BA or a tautomer thereof, wherein the ring BA has a structure of formula BA-III-e: in: X 1 is -N(-)- or -C(-)=; W X2 and W X6 Each of is independently O, S or Se; R B4 Is halogen, -CN, -NO2 or -L B4 -R B41 , where R B41 It is R'; R B5 Is halogen, -CN, -NO2 or -L B5 -R B51 , where R B51 is -R', -N(R')2, -OR' or -SR', L B4 and L B5 Each of which is independently L B ; Each L B is independently a covalent bond, or an optionally substituted divalent C 1-10 a saturated or partially unsaturated chain in which one or more methylene units are optionally and independently replaced by -Cy-, -O-, -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, or -C(O)O-; Each -Cy- is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each R' is independently -R, -C(O)R, -C(O)OR, -C(O)N(R)2, or -SO2R; and Each R is independently -H, or an optionally substituted group selected from the following: C 1-20 Aliphatic, C with 1-10 heteroatoms 1-20 Heteroaliphatic, C 6-20 Aryl, C 6-20 Aryl aliphatic group, C 6-20 arylheteroaliphatic, 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-20 membered heterocyclyl having 1-10 heteroatoms, or Two R groups optionally and independently join together to form a covalent bond, or Two or more R groups on the same atom are optionally and independently taken together with that atom to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to that atom; or Two or more R groups on two or more atoms are optionally and independently taken together with the atoms intervening therebetween to form an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the atoms intervening therebetween.

21. The oligonucleotide of claim 20, wherein when the oligonucleotide is aligned with a target nucleic acid, NO is opposite to the target adenosine.

22. An oligonucleotide, wherein the oligonucleotide comprises 5'-N1N0N -1 -3', where N -1 , N0 and N1 are each independently a nucleoside; and wherein the nucleobase of N0 is BA, wherein BA comprises cyclic BA or a tautomer thereof, wherein cyclic BA has a structure of formula BA-VI: in: Each are independently a single bond or a double bond; X 1’ is -N(-)- or -C(-)=; X 2’ Yes-C(W X2’ )-、-C(R B2’ )=、-C(OR B2’ )=, -N= or optionally substituted -CH= or -CH2-, wherein R B2’ Is halogen, -CN, -NO2 or -L B2’ -R', and W X2’ is O, S or Se; X 3’ Yes-N(R B3’ )-、-N=、-C(R B3’ )= or optionally substituted -NH- or -CH=, wherein R B3’ Is a halogen, -CN, -NO2, or -L B3’ -R'; X 4’ Yes-C(R B4’ )=,-C(OR B4’ )=,-C(-N(R B4’ )2)=,-C(R B4’ )2-,-C(W X4’ )-,-C(=NR B4’ )-,-N(R B4’ )-, -N= or optionally substituted -CH=, -NH- or -CH2-, wherein each R B4’ are independently halogen, -CN, -NO2, or -L B4’ -R B41’ , or two R on the same atom B4’ Combined to form =O, =C(-L B4’ -R B41’ )2, =NL B4’ -R B41’ , or optionally substituted =CH2 or =NH, wherein each R B41’ are independently -R', and W X4’ is O, S or Se; X 5’ Yes-C(R B5’ )2-,-N(R B5’ )-,-C(R B5’ )=,-C(W X5’ )-, -N= or optionally substituted -NH-, -CH2-, or -CH=, wherein each R B5’ are independently halogen, -CN, -NO2, or -L B5’ -R B51’ , where R B51’ is -R', -N(R')2, -OR', or -SR', and W X5’ is O, S, or Se; X 6’ Yes-C(R B6’ )=,-C(OR B6’ )=,-C(R B6’ )2-,-C(W X6’ )-,-C(-N(R B6’ )2)=, -N= or optionally substituted -NH-, -CH2- or -CH=, wherein each R B6’ are independently halogen, -CN, -NO2, or -L B6’ -R B61’ , or two R on the same atom B6’ Combined to form =O, =C(-L B6’ -R B61’ )2, =NL B6’ -R B61’ , or optionally substituted =CH2 or =NH, wherein each R B61’ are independently R', and W X6’ is O, S or Se; X 7’ is -C(R B7’ )=, -C(OR B7’ )=, -C(R B7’ )2-, -C(W X7’ )-, -C(-N(R B7’ )2)=,-N(R B7’ )-, -N= or optionally substituted -NH-, -CH2- or -CH=, where each R B7’ are independently halogen, -CN, -NO2, or -L B7’ -R B71’ , or two R on the same atom B7’ Combined to form =O, =C(-L B7’ -R B71’ )2, =NL B7’ -R B71’ , or optionally substituted =CH2 or =NH, wherein each R B71’ are independently R', and where W X7’ is O, S or Se; X 8’ and X 9’ Each of is independently C or N; L B2’ , L B3’ , L B4’ , L B5’ , L B6’ and L B7’ Each of which is independently L B ;and Each L B is independently a covalent bond, or an optionally substituted divalent C 1-10 a saturated or partially unsaturated chain in which one or more methylene units are optionally and independently replaced by -Cy-, -O-, -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, or -C(O)O-; Each -Cy- is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each R' is independently -R, -C(O)R, -C(O)OR, -C(O)N(R)2, or -SO2R; and Each R is independently -H, or an optionally substituted group selected from the following: C 1-20 Aliphatic, C with 1-10 heteroatoms 1-20 Heteroaliphatic, C 6-20 Aryl, C 6-20 Aryl aliphatic group, C 6-20 arylheteroaliphatic, 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-20 membered heterocyclyl having 1-10 heteroatoms, or Two R groups optionally and independently join together to form a covalent bond, or Two or more R groups on the same atom are optionally and independently taken together with that atom to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to that atom; or Two or more R groups on two or more atoms are optionally and independently taken together with the atoms intervening therebetween to form an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the atoms intervening therebetween.

23. The oligonucleotide of claim 22, wherein X 1’ It is -N(-)-.

24. The oligonucleotide of claim 23, wherein X 2’ It is -C(O)-.

25. The oligonucleotide of claim 24, wherein X 3’ It is -NH-.

26. The oligonucleotide of claim 22, wherein X 1’ yes 27. The oligonucleotide of claim 26, wherein the sugar of NO is a 2'-OMe modified sugar.

28. The oligonucleotide of claim 26, wherein the sugar of NO is a 2'-MOE modified sugar.

29. The oligonucleotide of claim 22, wherein when the oligonucleotide is aligned with a target nucleic acid, NO is opposite to the target adenosine.

30. The oligonucleotide of any one of claims 22-29, wherein N -1 The nucleobase is BA, where BA is Alternatively, BA comprises cyclic BA or a tautomer thereof, wherein cyclic BA has the structure of formula BA-III-e: BA-III-e in: X 1 is -N(-)- or -C(-)=; W X2 and W X6 Each of is independently O, S or Se; R B4 Is halogen, -CN, -NO2 or -L B4 -R B41 , where R B41 It is R'; R B5 Is halogen, -CN, -NO2 or -L B5 -R B51 , where R B51 is -R', -N(R')2, -OR' or -SR', L B4 and L B5 Each of which is independently L B ; Each L B is independently a covalent bond, or an optionally substituted divalent C 1-10 a saturated or partially unsaturated chain in which one or more methylene units are optionally and independently replaced by -Cy-, -O-, -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, or -C(O)O-; Each -Cy- is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each R' is independently -R, -C(O)R, -C(O)OR, -C(O)N(R)2, or -SO2R; and Each R is independently -H, or an optionally substituted group selected from the following: C 1-20 Aliphatic, C with 1-10 heteroatoms 1-20 Heteroaliphatic, C 6-20 Aryl, C 6-20 Aryl aliphatic group, C 6-20 arylheteroaliphatic, 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-20 membered heterocyclyl having 1-10 heteroatoms, or Two R groups optionally and independently join together to form a covalent bond, or Two or more R groups on the same atom are optionally and independently taken together with that atom to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to that atom; or Two or more R groups on two or more atoms are optionally and independently taken together with the atoms intervening therebetween to form an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the atoms intervening therebetween.

31. The oligonucleotide of claim 1, wherein the oligonucleotide comprises about 2-50 (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.) The invention also provides a first domain having a length of about 2-50 (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.

32. The oligonucleotide of any one of claims 1-31, wherein the oligonucleotide comprises about 1-50 (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) 1, 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.) have a 2'-F modified sugar, or wherein about 5%-100% (e.g., 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%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90 In some embodiments, the sugars of the present invention include 2'-F modifications (%, 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.).

33. The oligonucleotide of claim 32, wherein the oligonucleotide comprises about 1-50 (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, wherein about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc., have a 2'-OMe modified sugar, or wherein about 5%-100% (e.g., about 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-100%) of the oligonucleotides have a 2'-OMe modified sugar. 0%-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%, In some embodiments, the sugars of the invention may comprise a 2'-OMe modification, for example, 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.) of the sugars comprise a 2'-OMe modification.

34. The oligonucleotide of claim 33, wherein the oligonucleotide comprises about 1-50 (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, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc.) sugars with a 2'-MOE modification.

35. The oligonucleotide of claim 34, wherein about 5%-100% (e.g., 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%-85%, 70%- In some embodiments, the sugars of the present invention (e.g., 1'-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.) comprise a 2'-MOE modification.

36. The oligonucleotide of claim 33, wherein 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.) separating blocks, wherein each sugar in each 2'-F block is independently a 2'-F modified sugar, and wherein each sugar in each separating block is independently a sugar other than a 2'-F modified sugar.

37. The oligonucleotide of claim 36, wherein there are 2 or more 2'-F blocks in the oligonucleotide.

38. The oligonucleotide of claim 37, wherein there are 2 or more separated blocks in the oligonucleotide.

39. The oligonucleotide of claim 38, wherein each separation block independently comprises a 2'-OR modified sugar, wherein R is not -H.

40. The oligonucleotide of claim 38, wherein each sugar in each separating block is independently a 2'-OR modified sugar or a bicyclic sugar, wherein R is an optionally substituted C 1-6 Aliphatic group.

41. The oligonucleotide of claim 38, wherein each sugar in the separating block is independently a 2'-OMe or 2'-MOE modified sugar.

42. The oligonucleotide of claim 39, wherein there are about 1, 2, 3, 4, or 5 2'-F modified sugars in each 2'-F block.

43. The oligonucleotide of claim 39, wherein there are 5 or more (e.g., 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, or 30 or more) nucleosides on the 5' side of NO and 5 or more (e.g., 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, or 30 or more) nucleosides on the 3' side of NO.

44. The oligonucleotide of claim 39, wherein the oligonucleotide comprises 5'-N2N1N0N -1 N -2 -3', where N2, N1, N0, N -1 and N -2 Each of NO is independently a nucleoside, and wherein when the oligonucleotide is aligned with a target nucleic acid, NO is opposite to the target adenosine.

45. oligonucleotide as claimed in claim 44, wherein the sugar of NO is natural DNA sugar, 2'-F modified sugar, 2'-OR modified sugar or bicyclic sugar, wherein R is optionally substituted C 1-6 Aliphatic group.

46. ​​The oligonucleotide of claim 45, wherein the sugar of N1 is a natural DNA sugar, a 2'-F modified sugar, a 2'-OR modified sugar, or a bicyclic sugar, wherein R is an optionally substituted C 1-6 Aliphatic group.

47. The oligonucleotide of claim 46, wherein N -1 The sugar is a natural DNA sugar, a 2'-F modified sugar, a 2'-OR modified sugar, or a bicyclic sugar, wherein R is an optionally substituted C 1-6 Aliphatic group.

48. The oligonucleotide of claim 47, wherein N -1 The nucleobase is G.

49. The oligonucleotide of claim 47, wherein N -1 The nucleobase is hypoxanthine.

50. The oligonucleotide of claim 44, wherein the internucleotide linkage between N2 and N1 is a natural phosphate linkage, a PS internucleotide linkage (e.g., a phosphorothioate internucleotide linkage) or a PN internucleotide linkage (e.g., a phosphorylguanidine internucleotide linkage, such as n001).

51. The oligonucleotide of claim 44, wherein the internucleotide linkage between N1 and NO is a natural phosphate linkage, a PS internucleotide linkage (e.g., a phosphorothioate internucleotide linkage) or a PN internucleotide linkage (e.g., a phosphorylguanidine internucleotide linkage, such as n001).

52. The oligonucleotide of claim 44, wherein N0 and N -1 The internucleotide linkage between is a natural phosphate linkage, a PS internucleotide linkage (e.g., a phosphorothioate internucleotide linkage), or a PN internucleotide linkage (e.g., a phosphorylguanidine internucleotide linkage, such as n001).

53. The oligonucleotide of claim 44, wherein N -1 With N -2 The internucleotide linkage between is a natural phosphate linkage, a PS internucleotide linkage (e.g., a phosphorothioate internucleotide linkage), or a PN internucleotide linkage (e.g., a phosphorylguanidine internucleotide linkage, such as n001).

54. The oligonucleotide of claim 39, wherein the oligonucleotide comprises 5'-N2N1N0N -1 N -2 N -3 -3', where N -3 are independently nucleosides, and N -2 With N -3 The internucleotide linkage between is a natural phosphate linkage, a PS internucleotide linkage (e.g., a phosphorothioate internucleotide linkage), or a PN internucleotide linkage (e.g., a phosphorylguanidine internucleotide linkage, such as n001).

55. The oligonucleotide of claim 54, wherein N -2 With N -3 The internucleotide linkage between is a natural phosphate linkage or a PS internucleotide linkage (eg, a phosphorothioate internucleotide linkage).

56. The oligonucleotide of any one of the preceding claims, wherein each internucleotide linkage is independently selected from a PO linkage, a PN linkage, and a PS linkage.

57. An oligonucleotide as claimed in any one of the preceding claims, wherein the oligonucleotide comprises one or more PN linkages.

58. The oligonucleotide of claim 57, wherein each PN internucleotide linkage is independently n001.

59. An oligonucleotide wherein except for the positions of the modified internucleotide linkages there is a -O 5 -P L (R CA )-O 3 -, the oligonucleotide is otherwise identical to the oligonucleotide as claimed in any one of the preceding claims, wherein: P L is P or P(=W); W is O, S or W N ; R CA is or comprises an optionally substituted or blocked chiral auxiliary moiety, O 5 is the oxygen bonded to the 5'-carbon of the sugar, and O 3 It is the oxygen bonded to the 3'-carbon of the sugar.

60. The oligonucleotide of any one of the preceding claims, wherein each chiral internucleotide linkage is independently chirality controlled.

61. A phosphoramidite, wherein the nucleobase of the phosphoramidite is a nucleobase or tautomer thereof as claimed in any one of the preceding claims, wherein the nucleobase or tautomer thereof is optionally substituted or protected.

62. A phosphoramidite, wherein the nucleobase of the phosphoramidite is or comprises ring BA or a tautomer of ring BA, wherein ring BA has the following structure: 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, wherein the nucleobase is optionally substituted or protected.

63. The phosphoramidite of any one of claims 61-62, wherein the sugar of the phosphoramidite is the sugar of any one of claims 1-60, wherein the sugar is optionally protected.

64. The phosphoramidite of any one of claims 61-62, wherein the phosphoramidite has R NS -P(OR)N(R)2 structure, where R NS is an optionally protected nucleoside moiety, and each R is as described herein.

65. The phosphoramidite of any one of claims 61-62, wherein the phosphoramidite has R NS -P(OCH2CH2CN)N(i-Pr)2 structure.

66. The phosphoramidite of any one of claims 61-64, wherein the phosphoramidite comprises a chiral auxiliary moiety, wherein phosphorus is bonded to the oxygen and nitrogen atoms of the chiral auxiliary moiety.

67. The phosphoramidite of any one of claims 61-64, wherein the phosphoramidite has or the structure of a salt thereof.

68. A phosphoramidite having structure.

69. A method for preparing an oligonucleotide or composition, the method comprising coupling an -OH group of an oligonucleotide or nucleoside with a phosphoramidite or compound as described in any one of claims 61-68.

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

71. An oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides share: 1) common base sequence, and 2) identical linkage phosphorus stereochemistry at independently 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 internucleotide linkages (“chiral controlled internucleotide linkages”); wherein each of the plurality of oligonucleotides is independently an oligonucleotide as claimed in any one of the preceding claims, or an acid, base or salt form thereof.

72. The oligonucleotide of claim 71, wherein each of the plurality of oligonucleotides is independently in the form of a pharmaceutically acceptable salt.

73. The oligonucleotide of any one of claims 71-72, wherein the plurality of oligonucleotides are identical.

74. The oligonucleotide of any one of claims 71-73, wherein among the oligonucleotides that share the plurality of oligonucleotides in the composition, the percentage of the plurality of oligonucleotides is about or at least about (DS). nc , wherein 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.

75. The oligonucleotide of any one of claims 71-74, wherein the composition is a pharmaceutical composition and comprises a pharmaceutically acceptable carrier.

76. A method comprising: evaluating an agent or composition thereof in a cell, tissue or animal, wherein the cell, tissue or animal is or comprises a cell, tissue or organ associated with a condition, disorder or disease or a cell, tissue or organ of a condition, disorder or disease, and / or comprises a nucleotide sequence associated with a condition, disorder or disease; and An effective amount of an agent or composition for preventing or treating a condition, disorder or disease is administered to a subject susceptible to or suffering from the condition, disorder or disease.

77. A method for characterizing an oligonucleotide or a composition, the method comprising: The oligonucleotide or composition is administered to a cell or population thereof that contains or expresses an ADAR1 polypeptide or a characteristic portion thereof, or a polynucleotide encoding an ADAR1 polypeptide or a characteristic portion thereof.

78. 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 as claimed in any one of the preceding claims; or A method for deaminating a target adenosine in a target nucleic acid, the method comprising contacting the target nucleic acid with an oligonucleotide or composition as claimed in 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 as claimed in 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 an I or a 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 as claimed in any one of the preceding claims, wherein the target nucleic acid comprises a target adenosine; or A method for modulating a protein-protein interaction in a system, wherein a protein is translated from its encoding RNA, the method comprising contacting the encoding RNA with an oligonucleotide or composition as claimed in any one of the preceding claims, wherein adenosine in the encoding RNA is edited, wherein a protein is translated from the edited mRNA ("edited protein"), wherein the edited protein differs from the non-edited protein in the amino acid residues involved in the protein-protein interaction; or A method for modulating the interaction of a protein with an agent in a system, wherein the protein is translated from its encoding RNA, the method comprising contacting the encoding RNA with an oligonucleotide or composition as claimed in any one of the preceding claims, wherein adenosine in the encoding RNA is edited, wherein a protein is translated from the edited mRNA ("edited protein"), wherein the edited protein differs from the unedited protein in the amino acid residues involved in the protein-agent interaction; or A method for modulating the interaction of a protein with an agent in a system, wherein the protein is translated from its encoding RNA, the method comprising administering to the system an oligonucleotide or composition as claimed in any one of the preceding claims, wherein adenosine in the encoding RNA is edited, wherein a protein is translated from the edited mRNA ("edited protein"), wherein the edited protein differs from the unedited protein in the amino acid residues involved in the protein-agent interaction; or A method for modulating the interaction between a protein and its partner protein in a system, the method comprising administering to the system an oligonucleotide or composition as claimed in 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 differs from the protein encoded by the unedited nucleic acid in at least one amino acid residue involved in the interaction between the protein and its partner protein; or A method for modulating the level, structure and / or activity of a nucleic acid and / or a product encoded thereby in a system, the method comprising contacting the nucleic acid with an oligonucleotide or composition as claimed in any one of the preceding claims, wherein adenosine in the nucleic acid is edited; or A method for modulating the level, structure and / or activity of a nucleic acid and / or a product encoded thereby in a system, the method comprising administering to the system an oligonucleotide or composition as claimed in any one of the preceding claims, wherein adenosine in the nucleic acid is edited; or A method for editing a transcript in an immune cell, the method comprising administering to the immune cell an effective amount of an oligonucleotide or composition as claimed in any one of the preceding claims; or A method for delivering an oligonucleotide to a system, the method comprising administering to the system a conjugate of the oligonucleotide and an additional chemical moiety or a salt thereof; A method for increasing the level of editing of an oligonucleotide, the method comprising incorporating a structural element as described in any one of the preceding claims or the present disclosure; or A compound, oligonucleotide, composition, nucleobase, sugar, nucleoside, internucleotide linkage or method as described herein.

79. A method for preventing or treating a condition, disorder or disease, the method comprising administering or delivering to a subject susceptible to or suffering from the condition, disorder or disease an effective amount of an oligonucleotide or composition as claimed in any one of the preceding claims.

80. The method of claim 79, wherein the condition, disorder or disease is associated with a G to A mutation.

81. The method of claim 81, wherein the condition, disorder or disease is a liver condition, disorder or disease.

82. Use of an oligonucleotide or composition as claimed in any one of the preceding claims for altering mRNA splicing, wherein a target adenosine of the mRNA is edited; or Use of an oligonucleotide or composition as claimed in any preceding claim for silencing the expression of a protein, wherein a target adenosine of an mRNA encoding the protein is edited; or Use of an oligonucleotide or composition as claimed in any one of the preceding claims for repairing a nonsense mutation, wherein a target adenosine of an RNA is edited such that the nonsense mutation is repaired; Use of an oligonucleotide or composition as claimed in any one of the preceding claims for repairing a missense mutation, wherein the target adenosine of the RNA is edited such that the missense mutation is repaired; or Use of an oligonucleotide or composition as claimed in any preceding claim for editing a target adenosine in a codon; or Use of an oligonucleotide or composition as claimed in any preceding claim for editing a target adenosine in an upstream ORF.

83. An oligonucleotide, composition, phosphoramidite, compound, agent, method or use as described in any one of the specification or example examples 1-2597.

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