Oligonucleotide compositions and methods of use thereof

Abstract

To provide designed oligonucleotides and compositions thereof.SOLUTION: Provided is an oligonucleotide comprising a first domain and a second domain, where the first domain comprises one or more 2'-F modifications, and where the second domain comprises one or more sugars having no 2'-F modification. The provided oligonucleotides and compositions are useful for adenosine modification.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 911,334, filed October 6, 2019, U.S. Provisional Patent Application No. 62 / 959,917, filed January 11, 2020, U.S. Provisional Patent Application No. 63 / 022,559, filed May 10, 2020, and U.S. Provisional Patent Application No. 63 / 069,696, filed August 24, 2020, each of which is incorporated by reference herein in its entirety. [Background technology]

[0002] background Oligonucleotides are useful in a variety of applications, including therapeutic, diagnostic, and / or research applications. For example, oligonucleotides targeting various genes may be useful in treating conditions, disorders, or diseases associated with such target genes. Summary of the Invention [Means for solving the problem]

[0003] overview In particular, the present disclosure provides designed oligonucleotides and compositions thereof, wherein the oligonucleotides comprise modifications (e.g., modifications to the nucleobase sugar and / or internucleotide linkage, and patterns thereof) as described herein. In some embodiments, the techniques (compounds (e.g., oligonucleotides), compositions, methods, etc.) of the present disclosure (e.g., oligonucleotides, oligonucleotide compositions, methods, etc.) are particularly useful for editing nucleic acids, e.g., site-specific editing in nucleic acids (e.g., editing of targeted adenosines). In some embodiments, as demonstrated herein, the provided techniques can significantly improve the efficiency of nucleic acid editing, e.g., modifying one or more A residues, such as converting A to I. In some embodiments, the present disclosure provides techniques for editing in RNA (e.g., modifying an A residue, e.g., converting A to I). In some embodiments, the present disclosure provides techniques for editing in transcripts, e.g., mRNA (e.g., modifying an A residue, e.g., converting A to I). In particular, the provided technology offers the benefits of utilizing endogenous proteins, such as ADAR (adenosine deaminase acting on RNA) proteins (e.g., ADAR1 and / or ADR2), to edit nucleic acids, e.g., to modify A (resulting in a G to A mutation). Those skilled in the art will understand that such utilization of endogenous proteins can avoid some challenges and / or provide various advantages compared to those technologies that require delivery of foreign components (e.g., proteins (e.g., engineered to bind to an oligonucleotide (and / or its duplex with a target nucleic acid) that provides a desired activity), nucleic acids encoding proteins, viruses, etc.).

[0004] In particular, in some embodiments, the oligonucleotides of the provided technology include 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 linking phosphorus (Rp or Sp)), etc., which, when combined with one or more other structural elements (e.g., additional chemical moieties) described herein, can provide increased activity and / or various desirable properties, e.g., increased efficiency of nucleic acid editing, increased selectivity, increased stability, increased cellular uptake, reduced immune stimulation, reduced toxicity, improved distribution, improved affinity, etc. In some embodiments, the provided oligonucleotides can be, for example, , provide increased stability compared to oligonucleotides having a high percentage of natural RNA sugars utilized for adenosine editing. In some embodiments, provided oligonucleotides provide increased activity, e.g., adenosine editing activity. In some embodiments, provided oligonucleotides provide increased selectivity, e.g., in some embodiments, provided oligonucleotides provide selective modification of a target adenosine in a target nucleic acid over other adenosines in the same target nucleic acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-fold or more modification of the target adenosine over another adenosine, or all other adenosines in the target nucleic acid).

[0005] In some embodiments, the present disclosure provides an oligonucleotide comprising a first domain and a second domain, wherein the first domain comprises one or more 2'-F modifications and the second domain comprises one or more sugars without 2'-F modifications. In some embodiments, the provided oligonucleotide comprises one or more chiral modified internucleotide linkages. In some embodiments, the present disclosure provides (a) a first domain; and (b) Second Domain and providing an oligonucleotide comprising: 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 that comprise a 2'-F modification, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all sugars in the first domain comprise a 2'-F modification; 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 contain 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 contain a 2'-F modification.

[0006] In some embodiments, the second domain comprises or consists of a first subdomain, a second subdomain, and a third subdomain as described herein.

[0007] 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, and R is optionally substituted C 1~6 In some embodiments, R is aliphatic. In some embodiments, R is methyl. In some embodiments, R is CH2CH2OCH3. As described herein, other sugar modifications may also be utilized in accordance with the present disclosure, optionally in conjunction with the base modifications and / or internucleotide linkage modifications described herein.

[0008] In some embodiments, the base sequence of the provided oligonucleotide is substantially complementary to the base sequence of the target nucleic acid containing the target adenosine. In some embodiments, the provided oligonucleotide contains one or more mismatches (non-Watson-Crick base pairs) when aligned with the target nucleic acid. In some embodiments, the provided oligonucleotide contains one or more wobble (e.g., GU, IA, GA, IU, IC, etc.) when aligned with the target nucleic acid. In some embodiments, the mismatch and / or wobble may aid one or more proteins, such as ADAR1, ADAR2, etc., in recognizing the duplex formed by the provided oligonucleotide and the target nucleic acid. In some embodiments, the provided oligonucleotide forms a duplex with the target nucleic acid. In some embodiments, the ADAR protein recognizes and binds to such duplexes. In some embodiments, the nucleoside opposite the target adenosine is located in the middle of the provided oligonucleotide, e.g., with 5-50 nucleosides on the 5' side and 1-50 nucleosides on the 3' side. In some embodiments, the 5' side has more nucleosides than the 3' side. In some embodiments, the 5' side has fewer nucleosides than the 3' side. In some embodiments, the 5' side has the same number of nucleosides as the 3' side. In some embodiments, the provided oligonucleotides include oligonucleotides set forth in a table of 15-40, e.g., 15, 20, 25, 30, etc., consecutive bases. In some embodiments, the base sequence of the provided oligonucleotides is or includes the base sequence of an oligonucleotide set forth in a table.

[0009] In some embodiments, by utilizing a variety of structural elements (e.g., various modifications, stereochemistries, and patterns thereof), the present disclosure allows for the achievement of desirable properties and high activity with short oligonucleotides, e.g., about 20-40, 25-40, 25-35, 26-32, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length.

[0010] In some embodiments, the provided oligonucleotides comprise modified nucleobases. In some embodiments, the modified nucleobases facilitate modification of the target adenosine. In some embodiments, the nucleobase opposite the target adenine interacts less strongly with the target adenine than U (e.g., forms fewer hydrogen bonds) while maintaining interaction with an enzyme, e.g., ADAR, compared to when U is present. In some embodiments, the opposite nucleobase and / or its associated sugar provide flexibility (e.g., compared to U) in the ability of enzymes, e.g., ADAR1, ADAR2, etc., to modify the target adenosine. In some embodiments, the opposite nucleobase (relative to the target adenine), e.g., a nucleobase immediately 5' or 3' to the nucleobase of I and derivatives thereof, facilitates modification of the target adenine. In particular, the present disclosure recognizes that such nucleobases may provide less steric hindrance than G when a duplex of a provided oligonucleotide and its target nucleic acid interacts with a modifying enzyme, e.g., ADAR1 or ADAR2. In some embodiments, the base sequence of the oligonucleotide is selected (e.g., when multiple adenosine residues are suitable targets) and / or designed (e.g., through the use of various nucleobases described herein) so that steric hindrance can be reduced or eliminated (e.g., the nucleoside opposite the target A is not adjacent to G).

[0011] In some embodiments, oligonucleotides of the present disclosure provide modified internucleotide linkages (i.e., internucleotide linkages that are not natural phosphate linkages). In some embodiments, the linking phosphorus of a modified internucleotide linkage (e.g., a chiral internucleotide linkage) is chiral and can exist in different configurations (Rp and Rs). In particular, the present disclosure demonstrates that the incorporation of modified internucleotide linkages, particularly through control of the stereochemistry of the linking phosphorus center (enrichment of one configuration at such controlled centers compared to stereoirregular oligonucleotide preparations), can significantly improve properties (e.g., stability) and / or activity (e.g., adenosine-modifying activity (e.g., converting adenosine to inosine)). In some embodiments, provided oligonucleotides have significantly higher stereochemical purity than stereoirregular preparations. In some embodiments, provided oligonucleotides are chiral controlled.

[0012] In some embodiments, oligonucleotides of the present disclosure contain one or more chiral internucleotide linkages (e.g., phosphorothioate internucleotide linkages) in which the linked phosphorus is chiral. 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, or at least 50%, 60%, 70%, of all internucleotide linkages in the oligonucleotide are chiral. , 75%, 80%, 85%, 90%, 95%, or 99% are chiral internucleotide linkages. In some embodiments, at least one internucleotide linkage is a chiral internucleotide linkage. In some embodiments, at least one internucleotide linkage is a natural phosphate linkage. In some embodiments, each internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, at least one chiral internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, each is a phosphorothioate internucleotide linkage. The bound phosphorus can be either Rp or Sp. In some embodiments, at least one bound phosphorus is Rp. In some embodiments, at least one bound 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, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, of all chiral internucleotide linkages in the oligonucleotide 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, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, of all phosphorothioate internucleotide linkages in the oligonucleotide are Sp.

[0013] In some embodiments, the stereochemistry of one or more chiral internucleotide linkages of a provided oligonucleotide is controlled in the composition. In some embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotides, the plurality of oligonucleotides having a common base sequence and 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, 14, 16, 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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 2, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% chiral internucleotide linkages ("chiral controlled internucleotide linkages") independently share the same configuration of the linking phosphorus (e.g., all Rp or all Sp with respect to the chiral linking phosphorus). In some embodiments, they share the same stereochemistry at their respective chiral linking phosphorus. In some embodiments, multiple oligonucleotides share the same configuration. In some embodiments, multiple oligonucleotides are structurally identical except for the internucleotide linkage. In some embodiments, multiple oligonucleotides are structurally identical. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in a composition, or all oligonucleotides sharing a common base sequence, share the pattern of chiral centers in the backbones of multiple oligonucleotides. In some embodiments, at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all oligonucleotides in a composition, or all oligonucleotides sharing a common base sequence, are multiple oligonucleotides.

[0014] In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions of oligonucleotides, wherein at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all of the oligonucleotides in the composition, or all of the oligonucleotides having the same base sequence, or all of the oligonucleotides having the same base sequence and sugar and base modifications, or all of the oligonucleotides of the same composition, are chiral to the oligonucleotides, and at least 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, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 20, 21, 22, 23, 24, or 25 or more, or at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, of the chiral internucleotide linkages independently share the same configuration of the linking phosphorus (e.g., all Rp or all Sp with respect to the chiral linking phosphorus). In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions of oligonucleotides, wherein at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all of the oligonucleotides in the composition, or all of the oligonucleotides having the same base sequence, or all of the oligonucleotides having the same base sequence and sugar and base modifications, or all of the oligonucleotides of the same composition, are in one or more forms of the oligonucleotide (e.g., an acid form, a salt form (e.g., a pharmaceutically acceptable salt form; as will be understood by those skilled in the art, if the oligonucleotide is a salt, other salt forms of the corresponding acid or base form of the oligonucleotide), etc.).

[0015] In some embodiments, as demonstrated herein, chiral controlled oligonucleotide compositions provide several advantages over corresponding stereoirregular oligonucleotide compositions, e.g., greater stability, activity, etc. In some embodiments, chiral controlled oligonucleotide compositions have been observed to provide high levels of adenosine modifying (e.g., A to I conversion) activity with various isoforms of ADAR proteins (e.g., the p150 and p110 forms of ADAR1), while corresponding stereoirregular compositions have been observed to provide high levels of adenosine modifying (e.g., A to I conversion) activity with only certain isoforms of ADAR proteins (e.g., the p150 isoform of ADAR1).

[0016] In some embodiments, the provided oligonucleotides comprise an additional moiety, e.g., a targeting moiety, a carbohydrate moiety, etc. In some embodiments, the additional moiety is or comprises a ligand for the asialoglycoprotein receptor. In some embodiments, the additional moiety is or comprises GalNAc or a derivative thereof. In particular, the additional moiety may facilitate delivery to a particular target site, e.g., a cell, tissue, organ, etc. (e.g., a site comprising a receptor that interacts with the additional moiety). In some embodiments, the additional moiety facilitates delivery to the liver.

[0017] In some embodiments, the present disclosure provides techniques for preparing oligonucleotides and compositions thereof, particularly chiral-controlled oligonucleotide compositions. In some embodiments, the provided oligonucleotides and compositions thereof are highly pure. In some embodiments, the oligonucleotides of the present disclosure are at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% stereochemically pure at the linking phosphorus of the chiral internucleotide linkage. In some embodiments, the oligonucleotides of the present disclosure are stereoselectively prepared and are substantially free of stereoisomers. In some embodiments, in a provided composition comprising multiple oligonucleotides sharing the same base sequence with the same pattern of stereochemistry at the chiral linking phosphorus (e.g., each chiral linking phosphorus contains one or more Rp and / or Sp, independently of Rp or Sp), at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition sharing the same base sequence as the multiple oligonucleotides share the same pattern of stereochemistry at the chiral linking phosphorus. In some embodiments, in a provided composition comprising a plurality of oligonucleotides sharing the same base sequence with the same pattern of stereochemistry at the chiral linking phosphorus, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides in the composition sharing the same configuration as the plurality of oligonucleotides share the same pattern of stereochemistry at the chiral linking phosphorus or are a plurality of oligonucleotides.

[0018] In some embodiments, the present disclosure describes useful techniques for evaluating oligonucleotides and their compositions. For example, various techniques of the present disclosure are useful for evaluating adenosine modification. As will be understood by those skilled in the art, in some embodiments, adenosine modification / editing can be evaluated through sequencing, mass spectrometry, evaluation (e.g., level, activity, etc.) of products (e.g., RNA, protein, etc.) of modified nucleic acids (e.g., adenosine of target nucleic acids is converted to inosine), optionally in light of the presence of other components (e.g., ADAR proteins) in the modification system (e.g., in vitro system, ex vivo system, cell, tissue, organ, organism, subject, etc.). Those skilled in the art will understand that oligonucleotides that cause adenosine modification of target nucleic acids can also provide modified nucleic acids (e.g., target adenosine is converted to I) and one or more of their products (e.g., mRNA, protein, etc.). Certain useful techniques are described in the Examples.

[0019] As described herein, the oligonucleotides and compositions of the present disclosure may be provided / utilized in a variety of forms. In some embodiments, the present disclosure provides oligonucleotides in one or more forms, such as an acid form (e.g., a native phosphate linkage exists as -(P(O)(OH)-O- and a phosphorothioate internucleotide linkage exists as -O(P(O)(SH)-O-), a base form, a salt form (e.g., a native phosphate linkage exists as a salt form (e.g., a sodium salt (-O(P(O)(O-Na + )-O-), and phosphorothioate internucleotide linkages exist in salt form (e.g., sodium salt (-O(P(O)(S - Na +)-O-), 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), where the cation can dissociate from the anion. In some embodiments, the present disclosure provides pharmaceutical compositions comprising a provided oligonucleotide and / or one or more pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is a chiral controlled oligonucleotide composition.

[0020] The provided technology can be utilized for a variety of purposes. For example, those skilled in the art will understand that the provided technology is useful for many purposes, including modifying adenosines, e.g., correcting G to A mutations, modulating the levels of certain nucleic acids and / or products encoded thereby (e.g., reducing protein levels by introducing an A to G / I modification), modulating splicing, modulating translation (e.g., modulating translation start and / or stop sites by introducing an A to G / I modification), etc.

[0021] In some embodiments, the present disclosure provides techniques for preventing or treating conditions, disorders, or diseases susceptible to adenosine modifications, e.g., A to I or G conversion. As will be understood by one of skill in the art, I can perform one or more functions of G, e.g., in base pairing, translation, etc. In some embodiments, a G to A mutation can be corrected via A to I conversion so that one or more products of the G form of the nucleic acid, e.g., a protein, can be produced. In some embodiments, the present disclosure provides techniques for preventing or treating a condition, disorder, or disease associated with the mutation, comprising administering to a subject susceptible to or suffering from the mutation, a provided oligonucleotide or composition thereof, wherein the oligonucleotide or composition can edit the mutation. In some embodiments, the present disclosure provides techniques for preventing or treating a condition, disorder, or disease associated with a G to A mutation, comprising administering to a subject susceptible to or suffering from the mutation, a provided oligonucleotide or composition thereof, wherein the oligonucleotide or composition can modify the A. In some embodiments, the provided techniques modify A in a transcript, e.g., an RNA transcript. 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 is 1 compared to that encoded by its corresponding G form. In some embodiments, the A-form encodes one or more proteins having one or more higher desired activities and / or one or more better desired properties compared to its corresponding G-form. In some embodiments, the A-form encodes a product that is structurally different (e.g., a longer, in some embodiments, full-length protein) than that encoded by its corresponding G-form. In some embodiments, the A-form encodes a product (e.g., a protein) that is structurally identical compared to its corresponding G-form.

[0022] As one of skill in the art will appreciate, many conditions, disorders, or diseases are associated with mutations that can be altered by the provided techniques and can be prevented and / or treated using the provided techniques. For example, over 20,000 conditions, disorders, or diseases have been reported to be associated with G to A mutations and could benefit from A to I editing. [Brief explanation of the drawings]

[0023] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1a] The provided techniques with various sugar modification patterns can provide the desired activity. (a) ADAR1-mediated editing. All oligonucleotides have the same sequence targeting a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 and ADAR2, luciferase reporter constructs, and the indicated compositions, respectively. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 1b] The provided techniques with various sugar modification patterns can provide the desired activity. (b) ADAR2-mediated editing. All oligonucleotides have the same sequence targeting a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 and ADAR2, luciferase reporter constructs, and the indicated compositions, respectively. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 2a] The provided techniques, including various internucleotide linkage modifications, can provide the desired activity. (a) All compositions have the same sequence targeting a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 2b]The provided techniques, including various internucleotide linkage modifications, can provide the desired activity. (b) All compositions have the same sequence targeting a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 3] The provided techniques, including various sugar modifications, can provide the desired activity. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 4] The provided technology, including various glycoforms, can provide the desired activity. All compositions have the same sequence targeting the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 5a] The provided technology can provide the desired activity with a short sequence. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1, a luciferase reporter construct, and the indicated compositions. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 5b]The provided technology can provide the desired activity with a short sequence. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1, a luciferase reporter construct, and the indicated compositions. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 5c] The provided technology can provide the desired activity with a short sequence. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 5d] The provided technology can provide the desired activity with a short sequence. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 6] The provided technology can provide the desired activity in various cell types without exogenous ADARs. Figure 6 shows the editing of an endogenous target (TAG site in the 3'UTR of actin) without exogenous ADARs in different cell types. Cells were transfected with 50 nM of oligonucleotide, and editing was measured after 48 hours. (N=1 biological replicate for RPE and NHBE cells, N=2 biological replicates for hepatocytes.) [Figure 7]The provided techniques, containing varying numbers of mismatches, can provide the desired activity. All compositions target the premature UAG stop codon within the cLuc coding sequence and contain zero to two mismatches. 293T cells were transfected with ADAR1 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was measured at 48 and 96 hours and normalized to Gluc expression in mock-treated samples (n = 2 biological replicates). [Figure 8] The provided techniques, including various patterns of mismatches, can provide the desired activity. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 9] The provided techniques, including various patterns of mismatches, can provide the desired activity. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 10] Chiral oligonucleotide compositions can provide the desired activity. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 11]Chiral oligonucleotide compositions can provide the desired activity. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with the indicated compositions, including ADAR1 or ADAR2, a luciferase reporter construct, and various oligonucleotide concentrations. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 12] The chiral oligonucleotide compositions can provide significantly higher activity in various cell types without the need for exogenous ADARs. All compositions target the UAG motif in the 3'UTR of actin. Cells were treated with gymnosis at a dose of 10 μM oligonucleotides or transfected at a dose of 50 nM. RNA was collected 48 hours later, and the percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates). [Figure 13] The provided technology can provide the desired activity with short sequences without exogenous ADARs. Figure 13 shows editing in primary human retinal pigment epithelium (RPE) cells. All compositions target the UAG motif in the 3'UTR of actin. Primary human RPE cells were transfected with 50 nM of oligonucleotides. RNA was collected 48 hours later, and the percentage of edited transcripts was quantified by Sanger sequencing (n = 2 biological replicates). [Figure 14] Chiral oligonucleotide compositions can provide high activity in various cell types without exogenous ADARs. All compositions target the UAG motif in the 3'UTR of actin. Primary human bronchial epithelial cells were treated with gymnosis with 10 μM of oligonucleotides, while primary RPE cells were transfected with 50 μM of oligonucleotides. RNA was collected 48 hours later, and the percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates). [Figure 15]The provided technology, including various internucleotide linkage patterns, can provide the desired activity. All compositions have the same base sequence and target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 16] The provided technology, containing various internucleotide linkage patterns, can provide the desired activity without the need for exogenous ADARs. All compositions target the UAG motif in the 3'UTR of actin. Primary human hepatocytes were treated with 3.3 μM of the oligonucleotides by gymnosis. RNA was collected after 48 hours, and the percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates). [Figure 17] The provided technology, including various modifications and chiral control, can provide the desired activity without the need for exogenous ADARs. All compositions target the UAG motif in the 3'UTR of actin. Primary human hepatocytes were transfected with 50 nM of oligonucleotides. RNA was collected 48 hours later, and the percentage of edited transcripts was quantified by Sanger sequencing (n = 2 biological replicates). [Figure 18a] The provided technology, which includes an additional moiety, can provide high activity without exogenous ADAR. (a) All compositions target adenosines in the 3'UTR of beta-actin mRNA. Primary human hepatocytes were treated with gymnosis at various concentrations. Target editing was measured by Sanger sequencing (n = 2 biological replicates). [Figure 18b]The provided technology, which includes an additional moiety, can provide high activity without exogenous ADAR. (b) All compositions target adenosines in the 3'UTR of beta-actin mRNA. Primary human hepatocytes were treated with gymnosis at various concentrations. Target editing was measured by Sanger sequencing (n = 2 biological replicates). [Figure 19] The provided technology can provide the desired activity without the need for exogenous ADAR. Figure 19 shows the editing of SERPINA1 (PiZ allele) in primary mouse hepatocytes. All compositions target adenosines in mutant human SERPINA1 transcripts (PiZZ alleles). Primary hepatocytes (extracted from a mouse model expressing mutant human transcripts) were transfected with 50 nM of oligonucleotide. Target editing was measured by Sanger sequencing (n = 2 biological replicates). [Figure 20] The provided technology, including modified bases, can provide high activity without exogenous ADAR. Figure 20 shows the editing of SERPINA1 (PiZ allele) in primary mouse hepatocytes. All compositions target adenosines in mutant human SERPINA1 transcripts (PiZZ alleles). Primary hepatocytes (extracted from a mouse model expressing mutant human transcripts) were transfected and treated with 50 nM of oligonucleotides. Target editing was measured by Sanger sequencing (n = 2 biological replicates). As shown, the provided design, including modified nucleobases, can significantly improve activity. [Figure 21] The provided techniques, including modified bases, can provide the desired activity. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with the indicated compositions, including ADAR1 or ADAR2, a luciferase reporter construct, and various oligonucleotide concentrations. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 22]FIG. 22 shows an example of an oligonucleotide arrangement. [Figure 23] The provided technology, including modified bases, can provide high activity. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 24] The provided technology, which includes chiral oligonucleotide compositions, can provide higher activity compared to stereoirregular oligonucleotide compositions. The compositions use exogenous ADAR to target the UAG motif within a specified transcript. Primary human hepatocytes were transfected with the compositions at a 50 nM oligonucleotide concentration. The percentage of editing of the targeted transcript was determined by Sanger sequencing of RNA collected 48 hours after treatment (n=2 biological replicates). As demonstrated, the provided technology can provide high editing efficiency for various target transcripts. [Figure 25] The provided technology, which includes oligonucleotides with modified internucleotide linkages, can provide high activity. In some embodiments, the provided oligonucleotides include phosphorothioate linkages and non-negatively charged internucleotide linkages, such as n001. The compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR1-p150, a luciferase reporter construct, and the indicated compositions at a 3.3 nM oligonucleotide concentration. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 26]The provided technology, which includes oligonucleotides containing additional chemical moieties, can provide enhanced activity. The composition targets adenosines in the 3'UTR of beta-actin mRNA using exogenous ADAR. Primary monkey hepatocytes were treated with the indicated compositions at the indicated concentrations by gymnosis. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 27] The provided technology, which includes oligonucleotides with modified internucleotide linkages, sugar modifications, and / or additional chemical moieties, can provide enhanced activity. The composition targets adenosines in the 3'UTR of beta-actin mRNA using exogenous ADAR. Primary human hepatocytes were treated with the indicated compositions at the indicated oligonucleotide concentrations by gymnosis. The percentage of editing of the target transcript was determined by Sanger sequencing (n=2 biological replicates). [Figure 28] The provided technology, which includes oligonucleotides containing various modified internucleotide linkages, sugar modifications, and / or additional moieties, can provide high activity. The composition targets adenosines in the 3'UTR of beta-actin mRNA using exogenous ADAR. Primary human hepatocytes were treated with the indicated compositions at the indicated oligonucleotide concentrations by gymnosis. The percentage of editing of the target transcript was determined by Sanger sequencing (n=2 biological replicates). In some embodiments, certain structural elements, such as 2'-F modified sugars in the second subdomain, phosphorothioate linkages in Rp linked to the second subdomain nucleoside, the position and / or presence or absence of mismatches, and / or non-negatively charged internucleotide linkages such as n001 at certain positions, can improve editing efficiency. [Figure 29]The provided technology, which includes oligonucleotides with modified internucleotide linkages, sugar modifications, and / or additional chemical moieties, can provide enhanced activity. Primary human hepatocytes were treated with the indicated oligonucleotide compositions at the indicated concentrations by gymnosis. ADARs were endogenous. The compositions target adenosines in the 3'UTR of beta-actin mRNA. The percentage of edited transcripts was quantified by Sanger sequencing (n=2 biological replicates). [Figure 30] The provided technology, which includes oligonucleotides with modified internucleotide linkages, sugar modifications, and / or additional chemical moieties, can provide enhanced activity. Primary human (a; *: undetermined) or monkey (b) hepatocytes were treated with the indicated oligonucleotide compositions at the indicated concentrations by gymnosis. ADARs were endogenous. The compositions target adenosines in the 3'UTR of beta-actin mRNA. The percentage of edited transcripts was quantified by Sanger sequencing (n = 2 biological replicates). [Figure 31] Chirality control can improve editing efficiency. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p110 or -p150, luciferase reporter constructs, and the indicated oligonucleotide compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). Notably, increasing the number / level of chirality-controlled internucleotide linkages (e.g., Sp phosphorothioate internucleotide linkages) improved editing efficiency for both ADAR1-p110 and ADAR1-p150. [Figure 32]Chirality control can improve editing efficiency. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p110 or -p150, luciferase reporter constructs, and the indicated oligonucleotide compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). Notably, increasing the number / level of chirality-controlled internucleotide linkages (e.g., Sp phosphorothioate internucleotide linkages) improved editing efficiency for both ADAR1-p110 and ADAR1-p150. [Figure 33a] Chiral control and modified internucleotide linkages can improve editing efficiency. (a) All compositions target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with a plasmid encoding ADAR1-p110, a luciferase reporter construct, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 33b] Chiral control and modified internucleotide linkages can improve editing efficiency. (b) All compositions target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with a plasmid encoding ADAR1-p150, a luciferase reporter construct, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 34]Evaluation of Oligonucleotides Containing Natural Phosphate Linkages. In some embodiments, natural phosphate linkages can be utilized in accordance with the present disclosure (e.g., number, level, position, etc., in combination with other structural features (e.g., modifications, patterns, etc.)) to provide oligonucleotide compositions with a particular level of activity. In Figure 34, natural phosphate linkages can be utilized in oligonucleotides in accordance with the present disclosure. All compositions target a premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p150 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 35] Various sugar modifications can be utilized to provide oligonucleotide compositions with desired activity. All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p150 or ADAR2, luciferase reporter constructs, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 36a] Various sugar modifications can be utilized to provide oligonucleotide compositions with desired activity. (a) All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p150 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 36b]Various sugar modifications can be utilized to provide oligonucleotide compositions with desired activity. (b) All compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with plasmids encoding ADAR1-p150 or ADAR2, a luciferase reporter construct, and the indicated compositions. cLuc activity was normalized to Gluc expression in mock-treated samples (n=2 biological replicates). [Figure 37] The provided technology can provide efficient editing in primates. Non-human primates (NHPs) were administered several compositions (WV-37314, WV-37315, and WV-37330). All compositions target adenosines in the 3'UTR of beta-actin mRNA. Animals were administered the designated composition subcutaneously (5 mg / kg) once daily for five consecutive days (n = 2 animals per composition). Liver biopsies were taken two days after the last administration. Target editing was measured by Sanger sequencing. All three administered compositions provided significant levels of in vivo editing of ACTB mRNA (25-50% editing) without the administration of exogenous ADAR. [Figure 38a] The provided technology can provide sustained editing activity in vivo. The provided compositions were evaluated in non-human primates (NHPs). Specific data regarding the liver is shown. All compositions target adenosines in the 3'UTR of beta-actin mRNA. Animals were subcutaneously administered the designated compositions (5 mg / kg) once daily for five consecutive days (n=2 animals per oligonucleotide). Liver biopsies were taken 2 days and 45 days after the last dose, and kidney biopsies were taken 45 days after the last dose. Target editing was measured by Sanger sequencing. Oligonucleotides in tissues were measured by hybridization ELISA. [Figure 38b]The provided technology can provide sustained editing activity in vivo. The provided compositions were evaluated in non-human primates (NHPs). Specific data regarding the kidney is shown. All compositions target adenosines in the 3'UTR of beta-actin mRNA. Animals were subcutaneously administered the designated compositions (5 mg / kg) once daily for five consecutive days (n=2 animals per oligonucleotide). Liver biopsies were taken 2 days and 45 days after the last dose, and kidney biopsies were taken 45 days after the last dose. Target editing was measured by Sanger sequencing. Oligonucleotides in tissues were measured by hybridization ELISA. [Figure 39a] The provided technology can provide efficient editing in various systems, including neuronal cells. In some embodiments, compositions were evaluated in human iCell Neuronal cells (a). All compositions contain oligonucleotides targeting the UAG motif in the 3'UTR of ACTB and having the same base sequence. Cells were treated with the indicated compositions at the indicated concentrations by gymnosis, and RNA was collected after 6 and 5 days, respectively. Target editing was measured by Sanger sequencing (n = 2-3 biological replicates). [Figure 39b] The provided technology can provide efficient editing in various systems, including neuronal cells. In some embodiments, the compositions were evaluated in iCell astrocytes (b). All compositions contain oligonucleotides with the same base sequence targeting the UAG motif in the 3'UTR of ACTB. Cells were treated with the indicated compositions at the indicated concentrations by gymnosis, and RNA was collected after 6 and 5 days, respectively. Target editing was measured by Sanger sequencing (n = 2-3 biological replicates). [Figure 39c]The provided technology can provide efficient editing in a variety of systems, including neuronal cells. Certain initial results for certain additional targets are shown in (c) and (d). The compositions were evaluated in human iCell neurons and iCell astrocytes for editing of six different target sites. Each composition targets a UAG motif in the indicated transcript. Cells were treated with the indicated composition at the indicated concentration by gymnosis, and RNA was collected after 6 days. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 39d] The provided technology can provide efficient editing in a variety of systems, including neuronal cells. Certain initial results for certain additional targets are shown in (c) and (d). The compositions were evaluated in human iCell neurons and iCell astrocytes for editing of six different target sites. Each composition targets a UAG motif in the indicated transcript. Cells were treated with the indicated composition at the indicated concentration by gymnosis, and RNA was collected after 6 days. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 40a] Mice engineered to express human ADAR1 can provide an editing activity profile more similar to that of human cells compared to non-engineered mice. Compositions were evaluated in primary hepatocytes harvested from human ADAR1 transgenic mice, wild-type mice, and human primary hepatocytes. The administered oligonucleotides contain GalNAc moieties. Specific data are shown for editing of two distinct transcripts, UGP2. For each target, specific data are shown from three oligonucleotide compositions with identical base sequences but different modifications. Target editing was measured by Sanger sequencing (n=2-3 biological replicates). As noted, in some embodiments, chiral control and / or various modifications can be utilized to efficiently improve editing levels according to the present disclosure. For each cell type, from left to right: WV-38701, WV-38700, and WV-38702. [Figure 40b] Mice engineered to express human ADAR1 can provide an editing activity profile more similar to that of human cells compared to non-engineered mice. Compositions were evaluated in primary hepatocytes harvested from human ADAR1 transgenic mice, wild-type mice, and human primary hepatocytes. The administered oligonucleotides contain GalNAc moieties. Specific data are shown for editing of two distinct transcripts, EEF1A1. For each target, specific data are shown from three oligonucleotide compositions with identical base sequences but different modifications. Target editing was measured by Sanger sequencing (n=2-3 biological replicates). As noted, in some embodiments, chiral control and / or various modifications can be utilized to efficiently improve editing levels according to the present disclosure. For each cell type, from left to right: WV-38698, WV-38697, and WV-38699. [Figure 41a] The provided technology can provide editing in vivo. For example, specific in vivo data from the liver of a human ADAR1 transgenic mouse is shown. The animals were treated with a composition of oligonucleotides containing GalNAc. Wild-type (WT) mice were included as a control. Specific data is shown for editing of the UAG motif of two different transcripts, UGP2(a) and EEF1A1(b). Specific data is shown from two compositions of oligonucleotides with identical sequences but different modifications for each target. Three animals in each treatment group were administered PBS or 10 mg / kg of the indicated composition on days 1, 3, and 5, and liver biopsies were taken on day 8. (n=3 mice per group). As can be seen, in some embodiments, chiral-controlled oligonucleotide compositions of oligonucleotides containing non-negatively charged internucleotide linkages (e.g., n001) can be utilized to efficiently improve editing levels, including in vivo, according to the present disclosure. [Figure 41b]The provided technology can provide editing in vivo. For example, specific in vivo data from the liver of a human ADAR1 transgenic mouse is shown. The animals were treated with a composition of oligonucleotides containing GalNAc. Wild-type (WT) mice were included as a control. Specific data is shown for editing of the UAG motif of two different transcripts, UGP2(a) and EEF1A1(b). Specific data is shown from two compositions of oligonucleotides with identical sequences but different modifications for each target. Three animals in each treatment group were administered PBS or 10 mg / kg of the indicated composition on days 1, 3, and 5, and liver biopsies were taken on day 8. (n=3 mice per group). As can be seen, in some embodiments, chiral-controlled oligonucleotide compositions of oligonucleotides containing non-negatively charged internucleotide linkages (e.g., n001) can be utilized to efficiently improve editing levels, including in vivo, according to the present disclosure. [Figure 42a] The provided technology can provide in vivo editing in various tissues, including the central nervous system. The composition was evaluated in the CNS tissues of human ADAR1 transgenic mice. Specific data on editing of UGP2 transcripts was presented. Animals were treated with the composition via ICV injection. Five mice in each group were injected with PBS, two 50 μg doses of the oligonucleotide composition, or a single 100 μg dose on day 0 on days 0 and 2. Animals were necropsied on day 7. RNA from the designated tissues was collected, and editing was measured by Sanger sequencing (n=5 mice per group). Oligonucleotide distribution in different brain tissues was also measured by hybridization ELISA. In particular, it was confirmed that the provided oligonucleotides can be delivered to various tissues and provide editing activity there. [Figure 42b]The provided technology can provide in vivo editing in various tissues, including the central nervous system. The composition was evaluated in the CNS tissues of human ADAR1 transgenic mice. Specific data on editing of SRSF1 transcripts was presented. Animals were treated with the composition via ICV injection. Five mice in each group were injected with PBS, two 50 μg doses of the oligonucleotide composition, or a single 100 μg dose on day 0 on days 0 and 2. Animals were necropsied on day 7. RNA from the designated tissues was collected, and editing was measured by Sanger sequencing (n=5 mice per group). Oligonucleotide distribution in different brain tissues was also measured by hybridization ELISA. In particular, it was confirmed that the provided oligonucleotides can be delivered to various tissues and provide editing activity there. [Figure 43] Depletion of certain proteins using siRNA. Shown are ADAR1 p150 (top), ADAR1 p110 (middle), and vinculin loading control (bottom) in ARPE-19 cells treated with the indicated siRNA reagents with and without IFN-α. [Figure 44] Specific compilation data of endogenous ACTB observed in WV-23928 or WV-27395 with siRNA-mediated deletion of the indicated ADAR with and without IFN-α treatment. N≧3, mean ± SEM. ****P<0.0001 by Welch's two-way ANOVA followed by two-tailed post-hoc test. nd, not detected; NTC, non-targeting control. [Figure 45a] Figure 45. The provided technology can provide highly specific editing. (a) Scatter plot of variants detected in WV-30298 samples (top). On-target ACTB editing and off-target editing have LOD scores of >3 and >5% editing. LOD scores calculated by Mutect2 indicate the likelihood odds ratio that a variant is present in treated samples compared to mock samples. Genes with the highest percentage of editing and highest LOD scores are labeled. Total RNA coverage across replicates for all variants (potential editing sites) (bottom). [Figure 45b] Figure 45. The provided technology can provide highly specific editing. (b) Scatter plot of variants (top) and total RNA coverage (bottom) in the WV-27458 sample. [Figure 46a] The provided technology can provide multiple editing. Specific data in primary human hepatocytes is shown. (a) Percentage of editing observed on a designated transcript in the presence of 20 nM of each of a single (isolated) or multiple (multiplex) oligonucleotide compositions after transfection of primary human hepatocytes. [Figure 46b] The provided technology can provide multiplex editing. Specific data in primary human hepatocytes are shown. (b) Percentage of editing detected on the indicated transcript in the presence of 1.1 uM of each of a single (isolated) or multiple (multiplex) GalNAc-conjugated oligonucleotides. N=3, mean ± SEM. *P<0.05, ***P<0.001 by two-tailed Welch's t-test. For each target, from left to right: isolated, multiplex. [Figure 47] Mice engineered to express human ADAR1 can provide an editing activity profile more similar to that of human cells compared to non-engineered mice. Compositions were evaluated in primary hepatocytes harvested from human ADAR1 transgenic mice, wild-type mice, and human primary hepatocytes. The administered oligonucleotides contained GalNAc moieties. Specific data are shown for editing of two distinct transcripts, UGP2(ac) and EEF1A1(df). For each target, specific data are shown from three oligonucleotide compositions with identical base sequences but different modifications. Target editing was measured by Sanger sequencing (n=2-3 biological replicates). As can be seen, in some embodiments, chiral control and / or various modifications can be utilized to efficiently improve editing levels according to the present disclosure. [Figure 48]The provided technology provides editing in various cell types, including CD8+ T cells. Figure 48 shows editing in primary human CD8+ T cells by incorporation by gymnosis. All oligonucleotides have the same sequence, and all target the UAG motif in the 3'UTR of ACTB. Primary T cells were pre-stimulated for 24 or 96 hours as indicated, then treated with gymnosis with the indicated concentrations of the oligonucleotide compositions, and RNA was collected 4 days later. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 49] The provided technology provides editing in various cell types, including primary human fibroblasts. Figure 49 shows specific editing in primary human fibroblasts by gymnosis uptake and transfection. The oligonucleotide composition WV-37318 targets the UAG motif in the 3'UTR of ACTB. Three different primary human fibroblast cell lines were treated by transfection (50 nM) or gymnosis uptake (10 uM) as indicated, and RNA was collected 60 hours later. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 50] The provided technology provides editing in various cell types, including ex vivo retinal tissue isolated from non-human primate eyes. Figure 50 shows specific editing in ex vivo retinal tissue isolated from non-human primate eyes. The oligonucleotide composition targets the UAG motif in the 3'UTR of ACTB. In two independent experiments, eyes from NHPs were freshly dissected, and retinal tissue was treated with the oligonucleotide composition via gymnosis uptake. RNA was collected 48 hours later. Target editing was measured by Sanger sequencing (n = 4-5 biological replicates per experimental condition). [Figure 51]The provided techniques, including various modifications, can provide editing. Figure 51 ((a)-(d)) shows certain editing in primary human hepatocytes. The oligonucleotide compositions target specific adenosine residues (surrogate sites #1, 2, 3, or 4) in the coding sequence of the wild-type SERPINA1 (SA1) transcript. As can be seen, oligonucleotides containing various modifications, sequences, and / or additional chemical moieties can provide the desired editing. Primary human hepatocytes were treated with the indicated compositions and concentrations. RNA was collected after 48 hours. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 52] The provided techniques, including various modifications, can provide editing. Figure 52 shows a specific editing in primary human hepatocytes. Oligonucleotide compositions target specific adenosine residues (surrogate sites #1, 2, 3, or 4) in the coding sequence of the wild-type SERPINA1 (SA1) transcript. Primary human hepatocytes were treated with the indicated oligonucleotide compositions and concentrations. RNA was collected 48 hours later. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 53a] The provided techniques, including various modifications, can provide editing. Figure 53, (a) shows editing in primary human hepatocytes. Oligonucleotide compositions target specific adenosine residues (surrogate site #1) in the coding sequence of wild-type SERPINA1 (SA1) transcripts. Primary human hepatocytes were treated with the indicated oligonucleotide compositions and concentrations. RNA was collected after 48 hours. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 53b]The provided techniques, including various modifications, can provide editing. Figure 53, (b) shows editing in primary human hepatocytes. Oligonucleotide compositions target specific adenosine residues (surrogate site #2) in the coding sequence of wild-type SERPINA1 (SA1) transcripts. Primary human hepatocytes were treated with the indicated oligonucleotide compositions and concentrations. RNA was collected 48 hours later. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 53c] The provided techniques, including various modifications, can provide editing. Figure 53, (c) shows editing in primary human hepatocytes. Oligonucleotide compositions target specific adenosine residues (surrogate sites #1 or #2) in the coding sequence of wild-type SERPINA1 (SA1) transcripts. Primary human hepatocytes were treated with the indicated oligonucleotide compositions and concentrations. RNA was collected after 48 hours. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 54] Removing fluctuations and / or mismatches can improve editing levels. Figure 54 shows editing in primary human and NHP hepatocytes. Oligonucleotide compositions target specific adenosine residues (surrogate sites #1 or #2) in the coding sequence of the WT SERPINA1 (SA1) transcript. Primary human and NHP hepatocytes were treated with the indicated oligonucleotide compositions and concentrations. RNA was collected 48 hours later. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 55]The provided technology can provide editing in NHP and human cells at various concentrations. Figure 55 shows editing in primary human and NHP hepatocytes. Oligonucleotide compositions target specific adenosine residues (surrogate sites #1 or #2) in the coding sequence of the wild-type SERPINA1 (SA1) transcript. Both oligonucleotide compositions have a GU wobble relative to the NHP mRNA sequence. Primary human and NHP hepatocytes were treated with the indicated oligonucleotide compositions and concentrations. RNA was collected after 48 hours. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 56] The provided techniques, including various modifications, can provide editing. Figure 56 shows editing in primary NHP hepatocytes. Oligonucleotide compositions target specific adenosine residues (surrogate site #2) in the coding sequence of wild-type SERPINA1 (SA1) transcripts. Primary NHP hepatocytes were treated with the indicated oligonucleotide compositions and concentrations. RNA was collected 48 hours later. Target editing was measured by Sanger sequencing (n=2 biological replicates). [Figure 57] The provided techniques, including various modifications, including base modifications, can provide editing. All oligonucleotide compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR-p110 or ADAR1-p150, a luciferase reporter construct, and the indicated oligonucleotide compositions. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n = 2 biological replicates). [Figure 58]The provided techniques, including various modifications including abasic units, can provide editing. All oligonucleotide compositions target the premature UAG stop codon within the cLuc coding sequence. 293T cells were transfected with ADAR-p110 or ADAR1-p150, a luciferase reporter construct, and the indicated oligonucleotide compositions. cLuc activity was measured and normalized to Gluc expression in mock-treated samples (n = 2 biological replicates). [Figure 59] The provided techniques, including various modifications, including base modifications, can provide editing. Figure 59 shows editing with oligonucleotide compositions containing modified nucleobases across from the target site. All oligonucleotide compositions target the PiZ mutation in the SERPINA1 (SA1) transcript. ARPE cells stably expressing the SA1-PiZ allele from a lentiviral vector were transfected with the indicated oligonucleotide compositions. RNA was harvested after 3 days. RNA editing was quantified by Sanger sequencing (n=2 biological replicates). [Figure 60] The provided technology, containing various types of nucleobases and sugars, can provide editing. All oligonucleotide compositions target the PiZ mutation of the SERPINA1 (SA1) transcript. 293T cells were transfected with plasmids expressing the SA1-PiZ allele, ADAR1-p110, or ADAR1-p150, and the indicated oligonucleotide compositions. RNA was harvested 48 hours later. RNA editing was quantified by Sanger sequencing (n=2 biological replicates). [Figure 61] The provided technology contains various types of nucleobases and sugars that can provide editing. All oligonucleotide compositions target the PiZ mutation of the SERPINA1 (SA1) transcript. Freshly harvested primary hepatocytes from the SA1-PiZ- mouse model were treated with the indicated oligonucleotide compositions. RNA was harvested 48 hours later. RNA editing was quantified by Sanger sequencing (n=2 biological replicates). [Figure 62]The provided technology contains various nucleobase and sugar types and can provide editing. All oligonucleotide compositions target the PiZ mutation of the SERPINA1 (SA1) transcript. ARPE cells stably expressing the SA1-PiZ allele from a lentiviral vector were transfected with the indicated oligonucleotide compositions. RNA was harvested after 3 days. RNA editing was quantified by Sanger sequencing (n=2 biological replicates). [Figure 63] The provided techniques, including inosine, can provide editing. All oligonucleotide compositions target the PiZ mutation in the SERPINA1 (SA1) transcript. ARPE cells stably expressing the SA1-PiZ allele from a lentiviral vector were transfected with the indicated oligonucleotide compositions. RNA was harvested after 3 days. RNA editing was quantified by Sanger sequencing (n=2 biological replicates). [Figure 64] The techniques provided include varying nucleobases at sites opposite the target site to provide editing. Figure 64 shows editing of a premature UAG stop codon within the cLuc coding sequence. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description of Certain Embodiments The techniques of the present disclosure may be more readily understood by reference to the following detailed description of certain embodiments.

[0025] definition As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS system, 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., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York:2001.

[0026] As used herein in this disclosure, unless otherwise clear from the context, (i) the terms "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 in conjunction with "limited to"), and "include" (" (whether used in conjunction with "but not limited to") may be understood to include the itemized components or steps, whether presented by themselves or together with one or more additional components or steps; (iv) the term "another" may be understood to mean at least an additional / second one or more; (v) the terms "about" and "approximately" may be understood to allow for standard variations as understood by one of ordinary skill in the art; and (vi) when ranges are given, the endpoints are included.

[0027] Unless otherwise specified, descriptions of oligonucleotides and their elements (e.g., base sequence, sugar modifications, internucleotide linkages, stereochemistry of the linking phosphorus, their patterns, etc.) are in 5' to 3' order. As one of skill in the art will understand, in some embodiments, oligonucleotides may be provided and / or utilized as salt forms, particularly pharmaceutically acceptable salt forms, e.g., sodium salts. As one of skill in the art will also understand, in some embodiments, individual oligonucleotides within a composition may be considered to be of the same constitution and / or structure even within such a composition (e.g., a liquid composition), and a particular such oligonucleotide may be in a different salt form at a particular moment (and may be dissolved or may exist as an anionic form when the oligonucleotide chain is, for example, in a liquid composition). For example, one of skill in the art will understand that at a given pH, individual internucleotide bonds along an oligonucleotide chain may be in the acid (H) form or one of several 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 may be in their acid form (e.g., when all cations, if any, are H). + It will be understood that so long as the nucleotides (replaced by ) are of the same composition and / or structure, such individual oligonucleotides will be considered to be of the same composition and / or structure, as appropriate.

[0028] Aliphatic: As used herein, "aliphatic" refers to an alkyl group that is either fully saturated or unsaturated (e.g., "Aliphatic" refers to a linear (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain containing one or more units of unsaturation (but not aromatic), or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more units of unsaturation (but 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.

[0029] Alkenyl: As used herein, the term "alkenyl" refers to an aliphatic group, as defined herein, having one or more double bonds.

[0030] Alkyl: As used herein, the term "alkyl" has its ordinary meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, an alkyl has 1-100 carbon atoms. In certain embodiments, a straight-chain or branched-chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C for a straight chain). 20 , C2 to C for branched chains 20), or about 1-10. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure, and such rings are monocyclic, bicyclic, or polycyclic, or have about 5, 6, or 7 carbons in the ring structure. In some embodiments, an alkyl group can be a lower alkyl group, which lower alkyl group has from 1-4 carbon atoms (e.g., C1-C4 for a straight chain lower alkyl).

[0031] Alkynyl: As used herein, the term "alkynyl" refers to an aliphatic group, as defined herein, having one or more triple bonds.

[0032] 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 reference chemical moiety or class of moieties. Non-limiting examples include a nucleotide analog that is structurally different from a nucleotide but performs at least one function of a nucleotide; a nucleobase analog that is structurally different from a nucleobase but performs at least one function of a nucleobase, etc.

[0033] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals 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.

[0034] Aryl: The term "aryl," used alone or as part of a larger moiety of "aralkyl," "aralkoxy," or "aryloxyalkyl," as used herein, refers to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 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 5 to 14 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" may be 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, anthracyl, and the like, which may bear one or more substituents. As used herein, also included within the scope of the term "aryl" are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl.

[0035] Characteristic portion: As used herein, the term "characteristic portion," in its broadest sense, refers to a portion of a substance whose presence (or absence) correlates with the presence (or absence) of a particular characteristic, attribute, or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion found in the substance and related substances that share the particular characteristic, attribute, or activity, but not in those that do not share the particular characteristic, attribute, or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is one that contains a contiguous stretch of amino acids, or a set of contiguous stretches of amino acids, that together are characteristic of the protein or polypeptide. In some embodiments, each such contiguous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. Generally, a characteristic portion of a substance (e.g., a protein, antibody, etc.) is one that shares at least one functional characteristic with the related intact substance, in addition to the sequence and / or structural identity specified above. In some embodiments, a characteristic portion may be biologically active.

[0036] Chiral control: As used herein, "chiral control" refers to the control of the stereochemical assignment of the chiral linking phosphorus at a chiral internucleotide linkage within an oligonucleotide. As used herein, a chiral internucleotide linkage is an internucleotide linkage in which the linking phosphorus is chiral. In some embodiments, the control is achieved by a chiral element not present in the sugar and base moieties of the oligonucleotide; for example, in some embodiments, the control is achieved by the use of one or more chiral auxiliary agents during oligonucleotide preparation, which are often part of the chiral phosphoramidite used during oligonucleotide preparation. In contrast to chiral control, those skilled in the art will understand that conventional oligonucleotide synthesis without the use of a chiral auxiliary agent cannot control the stereochemistry at the chiral internucleotide linkage when such conventional oligonucleotide synthesis is used to form the chiral internucleotide linkage. In some embodiments, the stereochemical assignment of each chiral linking phosphorus at each chiral internucleotide linkage within an oligonucleotide is controlled.

[0037] Chirality-controlled oligonucleotide composition: The terms "chirality-controlled oligonucleotide composition," "chirality-controlled nucleic acid composition," and the like, as used herein, refer to a composition comprising multiple oligonucleotides (or nucleic acids) that share a common base sequence, where the multiple oligonucleotides (or nucleic acids) share the same linking phosphorus stereochemistry at one or more chiral internucleotide linkages (chirality-controlled or sterically-restricted internucleotide linkages, where the chiral linking phosphorus in the composition is Rp or Sp ("sterically-restricted"), rather than a random Rp and Sp mixture as in the case of non-chirality-controlled internucleotide linkages). In some embodiments, a chirality-controlled oligonucleotide composition comprises: 1) a common The chiral oligonucleotide composition includes a plurality of oligonucleotides (or nucleic acids) that share 1) a base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, where the plurality of oligonucleotides (or nucleic acids) share the same linking phosphorus stereochemistry at one or more chiral internucleotide linkages (chirally controlled or sterically restricted internucleotide linkages, where the chiral linking phosphorus is Rp or Sp ("sterically restricted") in the composition, rather than the random Rp and Sp mixture as in the case of non-chirally controlled internucleotide linkages). The level of the plurality of oligonucleotides (or nucleic acids) in the chiral controlled oligonucleotide composition is predefined / controlled or enhanced (e.g., by preparing chiral oligonucleotides that stereoselectively form one or more chiral internucleotide linkages) compared to the random level in a non-chirally controlled oligonucleotide composition. In some embodiments, about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5%, 10%, 20%, 30% or less) of all oligonucleotides in the chiral controlled oligonucleotide composition. , 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) are a plurality of oligonucleotides.In some embodiments, about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95 ... 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) are a plurality of oligonucleotides. In some embodiments, the level is between about 1% and 100% (e.g., between about 5% and 10%) of all oligonucleotides in a composition, or of all oligonucleotides in a composition (e.g., of multiple oligonucleotides or types of oligonucleotides) that share a common base sequence, or of all oligonucleotides in a composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications, or of all oligonucleotides in a composition that share a common base sequence, a common pattern of base modifications, a common pattern of sugar modifications, a common pattern of internucleotide linkage types, and / or a common pattern of internucleotide linkage modifications. 0%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 50% to 90%, or approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.In some embodiments, the plurality of oligonucleotides share the same stereochemistry at at least 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. Approximately 1% to 100% (e.g., approximately 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%) , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the oligonucleotides (or nucleic acids) share the same stereochemistry. In some embodiments, multiple oligonucleotides (or nucleic acids) all share the same pattern of sugar and / or nucleobase modifications. In some embodiments, multiple oligonucleotides (or nucleic acids) are various forms of the same oligonucleotide (e.g., acid and / or various salts of the same oligonucleotide). In some embodiments, multiple oligonucleotides (or nucleic acids) are of the same composition. In some embodiments, the level of the plurality of oligonucleotides (or nucleic acids) is between about 1% and 100% (e.g., between about 5% and 100%, 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 80% and 100%, 90% and 100%, 95% and 100%, or between about 1% and 100%, or between about 2% and 20%, 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 80% and 100%, 90% and 100%, 95 ... %, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, each chiral internucleotide linkage is a chiral controlled internucleotide linkage, and the composition is a completely chiral controlled oligonucleotide composition.In some embodiments, the multiple oligonucleotides (or nucleic acids) are structurally identical. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of 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%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 95%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 96%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 97%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 98%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 99%. In some embodiments, the percentage level is or is at least (DS)nc, where DS is 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-controlled internucleotide linkages as described herein (e.g., 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 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 level percentage is (DS)nc or at least (DS)nc, where DS is 95% to 100%. For example, when DS is 99% and nc is 10, the percentage is 90% or at least 90% ((99%)10≈0.90=90%). In some embodiments, the level of a plurality of oligonucleotides in a composition is expressed as the product of the diastereopurities of each chiral-controlled internucleotide linkage in the oligonucleotide.In some embodiments, two nucleotides in an oligonucleotide (or nucleic acid). The diastereopurity of an internucleotide linkage linking two nucleosides is expressed by the diastereopurity of the internucleotide linkage of a dimer linking the same two nucleosides, the dimers being prepared using comparable conditions, and in some instances, identical synthesis cycle conditions (e.g., for a linkage between Nx and Ny in an oligonucleotide...NxNy..., the dimer is NxNy). In some embodiments, not all chiral internucleotide linkages are chiral controlled internucleotide linkages, and the composition is a partially chiral controlled oligonucleotide composition. In some embodiments, the non-chiral controlled internucleotide linkages have a diastereopurity of less than about 80%, 75%, 70%, 65%, 60%, 55%, or about 50%, as typically observed in stereoirregular oligonucleotide compositions (e.g., from conventional oligonucleotide synthesis, e.g., phosphoramidite methodology, as will be appreciated by those skilled in the art). In some embodiments, the multiple oligonucleotides (or nucleic acids) are of the same type. In some embodiments, chiral controlled oligonucleotide compositions contain non-random or controlled levels of individual oligonucleotide or nucleic acid types. For example, in some embodiments, chiral controlled oligonucleotide compositions contain only one oligonucleotide type. In some embodiments, chiral controlled oligonucleotide compositions contain two or more oligonucleotide types. In some embodiments, chiral controlled oligonucleotide compositions contain multiple oligonucleotide types. In some embodiments, chiral controlled oligonucleotide compositions are compositions of oligonucleotides of a certain oligonucleotide type, and the composition contains non-random or controlled levels of multiple oligonucleotides of that oligonucleotide type.

[0038] 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 permit a comparison of the results obtained or the events observed. In some embodiments, comparable sets of conditions or circumstances are characterized by multiple substantially equivalent characteristics and one or a few varying characteristics. One skilled in the art will understand that sets of conditions are comparable to one another when they are characterized by a sufficient number and variety of substantially identical characteristics to warrant a reasonable conclusion that differences in the results obtained or the events observed under the different sets of conditions or circumstances are due to or represent changes in those altered characteristics.

[0039] Alicyclic: The terms "alicyclic," "carbocycle," "carbocyclyl," "carbocyclic group," and "carbocyclic ring" are used interchangeably and, as used herein, unless otherwise specified, refer to a saturated or partially unsaturated but non-aromatic cycloaliphatic monocyclic, bicyclic, or polycyclic ring system as described herein having 3 to 30 ring members. Alicyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, an alicyclic group has 3 to 6 carbons. In some embodiments, an alicyclic group is saturated and cycloalkyl. The term "alicyclic" can also include an alicyclic ring fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, an alicyclic group is bicyclic. In some embodiments, an alicyclic group is tricyclic. In some embodiments, an alicyclic group is polycyclic. In some embodiments, "alicyclic" refers to a C3-C6 monocyclic hydrocarbon having a single point of attachment to the rest of the molecule, or a C8-C6 monocyclic hydrocarbon having a single point of attachment to the rest of the molecule, which is fully saturated or contains one or more units of unsaturation, but is not aromatic. 10Bicyclic or polycyclic hydrocarbons, or C9-C, which are fully saturated or contain one or more units of unsaturation but are not aromatic, with a single point of attachment to the rest of the molecule 16 Refers to polycyclic hydrocarbons.

[0040] Heteroaliphatic: The term "heteroaliphatic," as used herein, refers to a heteroaliphatic group that is known in the art. " refers to an aliphatic group, as described herein, having its usual meaning of "a" or "b" and in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In some embodiments, one or more units selected from C, CH, CH, and CH are independently replaced with one or more heteroatoms (including oxidized and / or substituted forms thereof). In some embodiments, a heteroaliphatic group is a heteroalkyl. In some embodiments, a heteroaliphatic group is a heteroalkenyl.

[0041] Heteroalkyl: The term "heteroalkyl," as used herein, has 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 with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyl include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

[0042] Heteroaryl: The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroaralkyl" or "heteroaralkoxy," as used herein, refer to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, in which at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, heteroaryl groups are groups having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), in some embodiments, 5, 6, 9, or 10 ring atoms. In some embodiments, each monocyclic ring unit is aromatic. In some embodiments, heteroaryl groups have 6, 10, or 14 π-electrons, carbon atoms, plus 1 to 5 heteroatoms, shared in the cyclic arrangement. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group such as bipyridyl. The terms "heteroaryl" and "heteroar-," as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, and the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic, bicyclic, or polycyclic.The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl group, where the alkyl and heteroaryl portions independently are optionally substituted.

[0043] Heteroatom: As used herein, the term "heteroatom" refers to 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; nitrogen (e.g., quaternized forms, forms such as iminium groups, etc.), phosphorus, sulfur, charged forms of 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 embodiments, the heteroatom is oxygen, sulfur, or nitrogen.

[0044] Heterocycle: As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic group," and "heterocyclic ring" are used interchangeably and refer to a monocyclic, bicyclic, or polycyclic ring moiety (e.g., 3-30 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably 1 to 4, heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, and nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic group" are used interchangeably herein and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. Heterocyclyl groups can be monocyclic, bicyclic, or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0045] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., oligonucleotides, DNA, RNA, etc.) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, 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 for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. Nucleotides at 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. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the ALIGN program (version 2.0) The values ​​were determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) In some exemplary embodiments, the nucleic acid sequence comparison performed by the ALIGN program is Use the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences can be calculated using NWSgapdna.CMP This can be determined using the GAP program in the GCG software package using the matrix.

[0046] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to the bond connecting the nucleoside units of an oligonucleotide or nucleic acid. In some embodiments, the internucleotide linkage is a phosphodiester bond (a natural phosphate bond (-OP(=O)(OH)O-), which, as will be understood by those of skill in the art, may exist as a salt form) that is widely found in naturally occurring DNA and RNA molecules. In some embodiments, the internucleotide linkage is a modified internucleotide linkage (not a natural phosphate bond). In some embodiments, the internucleotide linkage is a "modified internucleotide linkage," in which at least one oxygen atom or -OH of the phosphodiester bond is replaced with 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')-, where each R' is independently as defined and described in this disclosure. In some embodiments, the internucleotide linkage is a phosphodiester linkage, a phosphorothioate linkage (or a phosphorothioate diester linkage, -OP(=O)(SH)O-, which may exist as a salt form as will be understood by those of skill in the art), or a phosphorothioate triester linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the internucleotide linkage is, for example, one of a PNA (peptide nucleic acid) or PMO (phosphorodiamidate morpholino oligomer) 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 (e.g., n001 in certain provided oligonucleotides). It will be understood by those of skill in the art that internucleotide linkages may exist as anions or cations at a given pH due to the presence of acidic or basic moieties in the linkage.In some embodiments, the modified internucleotide linkages are 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.

[0047] In vitro: As used herein, the term "in vitro" refers to events that take place not within an organism (e.g., an animal, plant, or microorganism) but in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc.

[0048] In vivo: As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, a plant, and / or a microorganism).

[0049] Bound phosphorus: As defined herein, the phrase "bound phosphorus" is used to indicate that the particular phosphorus atom being referenced is a phosphorus atom present in an internucleotide linkage, where the phosphorus atom corresponds to the phosphorus atom of a phosphodiester internucleotide linkage as it occurs in naturally occurring DNA and RNA. In some embodiments, the bound phosphorus atom is in a modified internucleotide linkage, where each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, the bound phosphorus atom is chiral (e.g., as in a phosphorothioate internucleotide linkage). In some embodiments, the bound phosphorus atom is achiral (e.g., as in a natural phosphate linkage).

[0050] Modified nucleobase: The terms "modified nucleobase," "modified base," and the like refer to a chemical moiety that is chemically different from a nucleobase but can perform at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase that includes a modification. In some embodiments, a modified nucleobase is capable of performing at least one function of a nucleobase, for example, forming a moiety in a polymer capable of base pairing to a nucleic acid comprising at least a complementary sequence of bases. 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, a modified nucleobase in the context of an oligonucleotide refers to a nucleobase that is not A, T, C, G, or U.

[0051] Modified nucleoside: The term "modified nucleoside" refers to a moiety derived from or chemically similar to a natural nucleoside, but containing a chemical modification that distinguishes it from a natural nucleoside. Non-limiting examples of modified nucleosides include those containing modifications at the base and / or sugar. Non-limiting examples of modified nucleosides include those having a 2' modification at the sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (lacking a nucleobase). In some embodiments, modified nucleosides are capable of forming moieties in polymers that retain at least one function of a nucleoside, e.g., capable of base pairing to a nucleic acid containing a complementary sequence of at least bases.

[0052] Modified Nucleotide: The term "modified nucleotide" includes any chemical moiety that is structurally different from a naturally occurring nucleotide but that can perform at least one function of a naturally occurring nucleotide. In some embodiments, a modified nucleotide comprises a modification at the sugar, base, and / or internucleotide linkage. In some embodiments, a modified nucleotide comprises a modified sugar, a modified nucleobase, and / or a modified internucleotide linkage. In some embodiments, a modified nucleotide is capable of forming a subunit in a polymer that is capable of at least one function of a nucleotide, e.g., capable of base pairing to a nucleic acid comprising a complementary sequence of at least bases.

[0053] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar. The modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of the sugar. In some embodiments, as described in this disclosure, 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 available in the art and described herein. In some embodiments, the 2'-modification is 2'-F. In some embodiments, the 2'-modification is 2'-OR (where R is an optionally substituted C 1~10 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., sugars used in LNA, BNA, etc.). In some embodiments, in the context of oligonucleotides, the modified sugar is a sugar that is not ribose or deoxyribose as typically found in natural RNA or DNA.

[0054] Nucleic Acid: The term "nucleic acid," as used herein, includes any nucleotide and polymers thereof. The term "polynucleotide," as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), or combinations thereof. These terms refer to the primary structure of the molecule and, thus, include double- and single-stranded DNA, as well as double- and single-stranded RNA. These terms also include, as equivalents, modified nucleotides, such as, but not limited to, via methylated, protected, and / or capped nucleotides or polynucleotides. The term includes analogs of either RNA or DNA, including nucleotides and / or modified polynucleotides. The term encompasses poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from nucleobases and / or modified nucleobase N-glycosides or C-glycosides; 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, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly- refers to a nucleic acid containing from 2 to about 10,000 nucleotide monomer units, and the prefix oligo- refers to a nucleic acid containing from 2 to about 200 nucleotide monomer units.

[0055] Nucleobase: The term "nucleobase" refers to the portion of a nucleic acid that participates in hydrogen bonding to link 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, naturally occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, naturally occurring nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a nucleobase comprises a heteroaryl ring, the ring atom is nitrogen, and in the case of a nucleoside, the nitrogen is linked to the sugar moiety. In some embodiments, a nucleobase comprises a heterocyclic ring, the ring atom is nitrogen, and in the case of a nucleoside, the nitrogen is linked to the sugar moiety. In some embodiments, the nucleobase is a "modified nucleobase," 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 property of the nucleobase and retains the hydrogen bonding properties that bind one nucleic acid strand to another in a sequence-specific manner. In some embodiments, the modified nucleobase is capable of pairing with all five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior or recognition by intracellular enzymes or activities of the oligonucleotide duplex. As used herein, the term "nucleobase" also encompasses modified nucleobases and structural analogs that are used in place of natural or naturally occurring nucleotides, such as nucleobase analogs. In some embodiments, the nucleobase is an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U.In some embodiments, "nucleobase" refers to a nucleobase unit in an oligonucleotide or nucleic acid (e.g., A, T, C, G, or U as in an oligonucleotide or nucleic acid).

[0056] Nucleoside: The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar. In some embodiments, the nucleoside is a natural nucleoside, e.g., adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, the nucleoside is a modified nucleoside, e.g., a substituted natural nucleoside selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, the nucleoside is a modified nucleoside, e.g., adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. A substituted tautomer of a natural nucleoside selected from adenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, "nucleoside" refers to a nucleoside unit in an oligonucleotide or nucleic acid.

[0057] Nucleotide: As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a nucleobase, a sugar, and one or more nucleotide 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, although it should be understood that naturally occurring and non-naturally occurring base analogs are also included. Naturally occurring sugars are the pentose (five-carbon sugar) deoxyribose (forming DNA) or ribose (forming RNA), although it should be 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, via phosphate, phosphorothioate, boranophosphate, etc.). Artificial nucleic acids include PNA (peptide nucleic acid), phosphotriester, phosphorothioate, H-phosphonate, phosphoramidate, boranophosphate, methylphosphonate, phosphonoacetate, thiophosphonoacetate, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. In some embodiments, natural nucleotides contain naturally occurring bases, sugars, and internucleotide linkages. As used herein, the term "nucleotide" also encompasses structural analogs that are used in place of natural or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs. In some embodiments, "nucleotide" refers to a nucleotide unit in an oligonucleotide or nucleic acid.

[0058] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides, which may contain any combination of natural and unnatural nucleobases, sugars, and internucleotide linkages.

[0059] Oligonucleotides can be single-stranded or double-stranded. Single-stranded oligonucleotides can have a double-stranded region (formed by two portions of the single-stranded oligonucleotide), and double-stranded oligonucleotides containing two oligonucleotide strands can have a single-stranded region, for example, in a region where the two oligonucleotide strands are not complementary to each other. Examples of oligonucleotides include, but are not limited to, structural genes, genes including regulatory and termination regions, self-replicating systems such as viruses or plasmid DNA, single-stranded and double-stranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, Ul adapters, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0060] Oligonucleotides of the present disclosure can be of various lengths. In certain embodiments, oligonucleotides can range from about 2 to about 200 nucleosides in length. In various related embodiments, single-stranded, double-stranded, or triple-stranded oligonucleotides can range in length from about 4 to about 10 nucleosides, about 10 to about 50 nucleosides, about 20 to about 50 nucleosides, about 15 to about 30 nucleosides, or about 20 to about 30 nucleosides in length. In some embodiments, oligonucleotides are about 9 to about 39 nucleosides in length. In some embodiments, oligonucleotides are at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 26 nucleosides in length. or 25 nucleosides in length. In some embodiments, the oligonucleotide is at least 4 nucleosides in length. In some embodiments, the oligonucleotide is at least 5 nucleosides in length. In some embodiments, the oligonucleotide is at least 6 nucleosides in length. In some embodiments, the oligonucleotide is at least 7 nucleosides in length. In some embodiments, the oligonucleotide is at least 8 nucleosides in length. In some embodiments, the oligonucleotide is at least 9 nucleosides in length. In some embodiments, the oligonucleotide is at least 10 nucleosides in length. In some embodiments, the oligonucleotide is at least 11 nucleosides in length. In some embodiments, the oligonucleotide is at least 12 nucleosides in length. In some embodiments, the oligonucleotide is at least 15 nucleosides in length. In some embodiments, the oligonucleotide is at least 15 nucleosides in length. In some embodiments, the oligonucleotide is at least 16 nucleosides in length. In some embodiments, the oligonucleotide is at least 17 nucleosides in length. In some embodiments, the oligonucleotide is at least 18 nucleosides in length. In some embodiments, the oligonucleotide is at least 19 nucleosides in length. In some embodiments, the oligonucleotide is at least 20 nucleosides in length. In some embodiments, the oligonucleotide is at least 25 nucleosides in length. In some embodiments, the oligonucleotide is at least 30 nucleosides in length. In some embodiments, each nucleoside counted in the oligonucleotide length independently comprises a nucleobase comprising a ring having at least one nitrogen ring atom. In some embodiments, each nucleoside counted in the oligonucleotide length independently comprises A, T, C, G, or U, or an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U.

[0061] Oligonucleotide Type: As used herein, the phrase "oligonucleotide type" is used to define oligonucleotides having a particular base sequence, backbone linkage pattern (i.e., pattern of internucleotide linkage types, e.g., phosphate, phosphorothioate, phosphorothioate triester, etc.), pattern of backbone chiral centers [i.e., pattern of phosphorus stereochemistry (Rp / Sp)], and pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides of a commonly designated "type" are structurally identical to each other.

[0062] Those skilled in the art will appreciate that the synthetic methods of the present disclosure provide a degree of control during the synthesis of an oligonucleotide chain, such that each nucleotide unit of the oligonucleotide chain can be designed and / or selected in advance to have a specific stereochemistry at the binding phosphorus and / or a specific modification at the binding phosphorus, and / or a specific base and / or a specific sugar. In some embodiments, the oligonucleotide chain is designed and / or selected in advance to have a specific combination of stereocenters at the binding phosphorus. In some embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the binding phosphorus. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of one or more of the above structural features. In some embodiments, the present disclosure provides compositions (e.g., chiral-controlled oligonucleotide compositions) comprising or consisting of a plurality of oligonucleotide molecules. In some embodiments, all such molecules are of the same type (i.e., structurally identical to one another). However, in some embodiments, the provided compositions typically comprise multiple oligonucleotides of different types in predetermined relative amounts.

[0063] Optionally substituted: As described herein, compounds of the present disclosure, e.g., oligonucleotides, may contain optionally substituted and / or substituted moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety have been replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and if more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at all positions. In some embodiments, an optionally substituted group is unsubstituted. Combinations of substituents envisioned by the present disclosure preferably result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that are substantially unchanged when subjected to conditions that allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Certain substituents are described below.

[0064] Suitable monovalent substituents on substitutable atoms, for example suitable carbon atoms, are independently halogen; -(CH) 0~4 R°;-(CH2) 0~4 OR°;-O(CH2) 0~4 R°, -O-(CH2) 0~4 C(O)OR°;-(CH2) 0~4 CH(OR°)2; R° can be substituted with -(CH2) 0~4 Ph; R° may be substituted with -(CH2) 0~4 O(CH2) 0~1 Ph; -CH=CHPh, which may be substituted with R°; -(CH2), which may be substituted with R° 0~4 O(CH2) 0~1 -pyridyl; -NO2; -CN; -N3; ​​-(CH2) 0~4 N(R°)2;-(CH2) 0~4 N(R°)C(O)R°;-N(R°)C(S)R°;-(CH2) 0~4N(R°)C(O)NR°2; -N(R°)C(S)NR°2; -(CH2) 0~4 N(R°)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2) 0~4 C(O)R°; -C(S)R°; -(CH2) 0~4 C(O)OR°; -(CH2) 0~4 C(O)SR°; -(CH2) 0~4 C(O)OSiR°3; -(CH2) 0~4 OC(O)R°; -OC(O)(CH2) 0~4 SR°, -SC(S)SR°; -(CH2) 0~4 SC(O)R°; -(CH2) 0~4 C(O)NR°2; -C(S)NR°2; -C(S)SR°; -(CH2) 0~4 OC(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2) 0~4 SSR°; -(CH2) 0~4 S(O)2R°; -(CH2) 0~4 S(O)2OR°; -(CH2) 0~4 OS(O)2R°; -S(O)2NR°2; -(CH2) 0~4 S(O)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2; -Si(R°)3; -OSi(R°)3; -B(R°)2; -OB(R°)2; -OB(OR°)2; -P(R°)2; -P(OR°)2; -P(R°)(OR°); -OP(R°)2; -OP(OR°)2; -OP(R°)(OR°); -P(O)(R°)2; -P(O)(OR°)2; -OP(O)(R°)2; -OP(O)(OR°)2; -OP(O)(OR°)(SR°); -SP(O)(R°)2; -SP(O)(OR°)2; -N(R°)P(O)(R°)2; -N(R°)P(O)(OR°)2; -P(R°)2[B(R°)3]; -P(OR°)2[B(R°)3]; -OP(R°)2[B(R°)3]; -OP(OR°)2[B(R°)3]; -(C 1~4 linear or branched alkylene)O-N(R°)2; or -(C1~4 linear or branched alkylene)C(O)ON(R°), where each R° may be optionally substituted as defined herein and independently represents hydrogen, C 1~20 C having 1 to 5 heteroatoms independently selected from aliphatic, 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 as defined above. Nevertheless, two independent occurrences of R° taken together with their intervening atoms 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.

[0065] Suitable monovalent substituents on R° (or the ring formed by combining two independent occurrences of R° with their intervening atoms) are independently halogen, —(CH) 0~2 R ● ,-(Halo R ● ), -(CH2) 0~2 OH, -(CH2) 0~2 OR ● , -(CH2) 0~2 CH(OR ● )2;-O(HaloR ● ), -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 ● and each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph is selected from a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0066] For example, suitable divalent substituents on suitable carbon atoms are, independently, the following: =O, =S, =NNR*, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)R*, =NR*, =NOR*, -O(C(R*)) 2~3 O- or -S(C(R*2)) 2~3 S-, and each independent occurrence of R* is 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. Preferred divalent substituents attached to adjacent substitutable carbons of an "optionally substituted" group include -O(CR*2) 2~3 and each independent occurrence of R* is hydrogen, C, which may be substituted as defined below. 1~6 It is selected from aliphatic and unsubstituted 5-6 membered saturated, partially unsaturated, and aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0067] Suitable substituents on the aliphatic groups of R* are independently halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, and each R ● is unsaturated or, if prefixed with "halo", is substituted only with one or more halogens, and independently, C 1~4 Aliphatic, -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.

[0068] In some embodiments, suitable substituents on a substitutable nitrogen are independently —R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † and each R † are independently hydrogen, C which may be substituted as defined below 1~6 an aliphatic, unsubstituted -OPh, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the above definitions, R † two independent occurrences of are taken together with their intervening atoms to form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0069] R † Suitable substituents on the aliphatic group are independently halogen, —R ● ,-(Halo R ● ), -OH, -OR ●, -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, and each R ● is unsubstituted or, if prefixed with "halo", is substituted only with one or more halogens, and independently, C 1~4 Aliphatic, -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.

[0070] P-modification: As used herein, the term "P-modification" refers to any modification at the bound phosphorus other than a stereochemical modification. In some embodiments, a P-modification includes the addition, substitution, or removal of a pendant moiety covalently attached to the bound phosphorus.

[0071] 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 with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as defined herein.

[0072] 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 dose suitable for administration in a treatment regimen that exhibits a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to an appropriate population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those suitable for: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeting systemic absorption, boluses, powders, granules, pastes 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 applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., as a suppository, cream, or foam; sublingual; ocular; transdermal; or intranasal, pulmonary, and other mucosal surfaces.

[0073] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0074] 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 that encapsulates a material, that is involved in carrying or transporting a compound of interest 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 which can function 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 glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and hydroxide Examples include aluminum; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic, compatible materials used in pharmaceutical formulations.

[0075] Pharmaceutically acceptable salts: The term "pharmaceutically acceptable salts," as used herein, refers to salts of such compounds that are suitable for use in a pharmaceutical context, i.e., salts that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, consistent with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. See, for example, SM Berge 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 of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or 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 adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, and 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 contain one or more acidic groups, e.g., oligonucleotides, and the pharmaceutically acceptable salt is an alkali, alkaline earth metal, or ammonium (e.g., an ammonium salt of N(R)3, where each R is independently defined and described in this disclosure) salt. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt.In some embodiments, the pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates, where appropriate. In some embodiments, provided compounds contain two or more acid groups; for example, oligonucleotides may contain two or more acid groups (e.g., in natural phosphate linkages and / or modified internucleotide linkages). In some embodiments, pharmaceutically acceptable salts, or salts of such compounds in general, contain two or more cations, which may be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or salt in general), all ionizable hydrogens in the acidic groups (e.g., in an aqueous solution having a pKa of about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or less, in some embodiments, about 7 or less; in some embodiments, about 6 or less; in some embodiments, about 5 or less; in some embodiments, about 4 or less; in some embodiments, about 3 or less) are replaced with a cation. In some embodiments, each phosphorothioate and phosphate group is independently present in its salt form (e.g., in the case of a sodium salt, -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively). In some embodiments, each phosphorothioate and phosphate group is independently present in its salt form (e.g., -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively). The thioate and phosphate internucleotide linkages are independently present in their salt form (e.g., -OP(O)(SNa)-O- and -OP(O)(ONa)-O-, respectively, in the case of sodium salts). In some embodiments, the pharmaceutically acceptable salt is a sodium salt of an oligonucleotide. In some embodiments, the pharmaceutically acceptable salt is a sodium salt of an oligonucleotide, and each acidic phosphate group and modified phosphate group (e.g., phosphorothioate, phosphate, etc.), if any, is present as a salt form (all sodium salts).

[0076] Predetermined: Predetermined (or pre-determined) means, for example, irregular "Predetermined" means deliberately selected or random or controlled, as opposed to randomly occurring, random, or achieved without control. Those skilled in the art will understand that the present disclosure provides techniques that allow for the selection of specific chemical and / or stereochemical features to be incorporated into oligonucleotide compositions and the controlled preparation of oligonucleotide compositions having such chemical and / or stereochemical features. Such provided compositions are "predetermined" as described herein. A composition that may contain a particular oligonucleotide is not a "predetermined" composition because it was accidentally generated through an uncontrolled process that intentionally produces specific chemical and / or stereochemical features. In some embodiments, a predetermined composition can be intentionally reproduced (e.g., by repeating a controlled process). In some embodiments, a predetermined level of a plurality of oligonucleotides in a composition means that the absolute and / or relative amounts (ratios, percentages, etc.) of the plurality of oligonucleotides in the composition are controlled. In some embodiments, a predetermined level of a plurality of oligonucleotides in a composition is achieved by the preparation of chiral-controlled oligonucleotides.

[0077] Protecting Group: The term "protecting group" as used herein includes those well known in the art and described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3rd edition, John Wiley & Sons, 1999, which is incorporated herein by reference in its entirety. Protecting Groups include those described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012, Chapter 2 of which is incorporated herein by reference in its entirety. Also included are those protecting groups specifically adapted for the nucleoside and nucleotide chemistry described herein. Suitable amino-protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (D BD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (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-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate butyl, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, vinyl carbamate benzyl (Cbz), p-methoxybenzyl carbamate (Moz), p-nitribenzyl carbamate (nitobenzyl), p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)carbamate (nyl)ethyl, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-methyl-2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate -Benzisoxazolylmethyl, 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'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives Conductors, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-Dimethylpropynyl, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, 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-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide , picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinamide, 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-Tetramethyldisilylazacyclopentane 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)-2-methyl-1,3,5-triazacyano-4-one, N-methyl-2-methyl-1,3,5-triazano-4-one, N-methyl-2-methyl-1,3,5-triazano-4-one, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl)-2-methyl-1,3,5-triazano-4-one ... Pyr-4-nitro-2-oxo-3-pyroolin-3-ylamine, quaternary ammonium salt, 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 -picolylamino N'-oxide, N -1,1-Dimethylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1 -cyclohexenyl)amine, N-borane derivatives, N-diphenylborinic acid derivatives, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate compounds, N-zinc chelate compounds, N-nitroamines, N-nitrosamines, amine N-oxides, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidate, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide amide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridine sulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methylbenzenesulfonamide thoxybenzenesulfonamide (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 phenacylsulfonamide.

[0078] 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, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and tetrahydropyran-2-yl. An example of a suitable alkenyl group is 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), and 2- and 4-picolyl.

[0079] Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl. (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-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-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxy Benzyl, 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'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-Tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (I PDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chloroacetate phenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamanoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, 2,2,2-trichloroethyl alkyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthothyl carbonate (napththyl), methyl dithiocarbonate, , 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 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, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene Examples of suitable hydroxyl groups include olefin orthoesters, 1-methoxyethylidene orthoesters, 1-ethoxyethylidene orthoesters, 1,2-dimethoxyethylidene orthoesters, α-methoxybenzylidene orthoesters, 1-(N,N-dimethylamino)ethylidene derivatives, α-(N,N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene orthoesters, di-t-butylsilylene groups (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivatives (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivatives (TBDS), cyclic carbonates, cyclic boronates, ethyl borons, and phenyl borons.

[0080] In some embodiments, the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl(trityl), 4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifiuoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, Triflate, trityl, monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr) and 4,4',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-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4"-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (pixyl), or 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, each of the hydroxyl protecting groups is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl group is selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups. In some embodiments, the phosphorus-linked protecting group is a group that is attached to a phosphorus linkage (e.g., an internucleotide linkage) throughout oligonucleotide synthesis. In some embodiments, the protecting group is attached to the sulfur atom of a phosphorothioate group. In some embodiments, the protecting group is attached to the oxygen atom of an internucleotide phosphorothioate linkage. In some embodiments, the protecting group is attached to the oxygen atom of an internucleotide phosphorus linkage. 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-butylcarboxamido)-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.

[0081] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a compound (e.g., an oligonucleotide) or composition is administered in accordance with the present disclosure, 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; parasites, 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.

[0082] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the entire or nearly full extent or degree of a desired characteristic or property. A base sequence that is substantially identical to or complementary to a second sequence is not completely identical to or complementary to the second sequence, but is largely or nearly identical to or complementary to the second sequence. In some embodiments, an oligonucleotide having a sequence that is substantially complementary to another oligonucleotide or nucleic acid will form a duplex with that oligonucleotide or nucleic acid in a manner similar to an oligonucleotide having a completely complementary sequence. Additionally, those skilled in the art of biology and / or chemistry will understand that biological and chemical events rarely, if ever, proceed to completion and / or perfection, or achieve or avoid absolute results. Thus, as used herein, the term "substantially" is used to capture the potential lack of completeness inherent in many biological and / or chemical events.

[0083] Sugar: The term "sugar" refers to closed and / or open monosaccharides or polysaccharides. In some embodiments, a sugar is a monosaccharide. In some embodiments, a sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term "sugar" also encompasses structural analogs used in place of traditional sugar molecules, such as glycols, polymers that form the backbone of nucleic acid analogs, glycol nucleic acids ("GNAs"). As used herein, the term "sugar" also encompasses structural analogs used in place of natural or naturally occurring nucleotides, such as modified sugars and nucleotide sugars. In some embodiments, a sugar is an RNA or DNA sugar (ribose or deoxyribose). In some embodiments, a sugar is a modified ribose or deoxyribose sugar, e.g., 2'-modified, 5'-modified, etc. As described herein, in some embodiments, modified sugars, when used in oligonucleotides and / or nucleic acids, can provide one or more desirable properties, activities, etc. In some embodiments, the sugar is an optionally substituted ribose or deoxyribose. In some embodiments, "sugar" refers to the sugar unit in an oligonucleotide or nucleic acid.

[0084] Susceptible to: A disease, disorder, and / or condition. An individual who is "susceptible to" a disease, disorder, and / or condition is an individual who is at higher risk than the general population of individuals for developing the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition has a predisposition to having the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible 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 susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible 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 susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0085] Therapeutic Agent: As used herein, the term "therapeutic agent" generally refers to any agent that induces a desired effect (e.g., a desired biological, clinical, or pharmacological effect) when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it exhibits a statistically significant effect across a relevant population. In some embodiments, the relevant population is a population of subjects suffering from and / or susceptible to a disease, disorder, or condition. In some embodiments, the relevant population is a population of model organisms. In some embodiments, the relevant population may be defined by one or more criteria, such as age group, sex, genetic background, pre-existing clinical conditions, etc., prior to receiving therapy. In some embodiments, a therapeutic agent is a substance that, when administered to a subject in an effective amount, relieves, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition in the subject. In some embodiments, a "therapeutic agent" is an agent that has been approved, or is required to be approved, by a government agency before it can be marketed for administration to humans. In some embodiments, a "therapeutic agent" is a drug for which a prescription is required for administration to a human. In some embodiments, a therapeutic agent is a provided compound, e.g., a provided oligonucleotide.

[0086] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to 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 that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be understood by one of skill in the art, 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 relieves, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the 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.

[0087] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, 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. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who show very early signs of the disease, disorder, and / or condition, e.g., to reduce the risk of developing pathologies associated with the disease, disorder, and / or condition.

[0088] Unsaturated: The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation.

[0089] Wild-type: As used herein, the term "wild-type" has its art-recognized meaning, which refers to an entity that has structure and / or activity as found in nature in a "normal" (as opposed to mutant, diseased, altered, etc.) state or context. Those of skill in the art will understand that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0090] As will be understood by those of skill in the art, the methods and compositions described herein relating to provided compounds (e.g., oligonucleotides) also generally apply to pharmaceutically acceptable salts of such compounds.

[0091] Description of Certain Embodiments Oligonucleotides are useful in a variety of therapeutic, diagnostic, and research applications. The use of naturally occurring nucleic acids is limited, for example, by their susceptibility to endo- and exonucleases. Therefore, various synthetic counterparts have been developed to circumvent these drawbacks and / or further improve various properties and activities. These include, among others, synthetic oligonucleotides that contain chemical modifications, e.g., base modifications, sugar modifications, backbone modifications, etc., that make these molecules less susceptible to degradation and improve other properties and / or activities.

[0092] From a structural standpoint, modifications to the internucleotide linkages can introduce chirality, and certain properties and activities can be influenced by the configuration of the backbone-bound phosphorus atoms of the oligonucleotide, such as binding affinity, sequence-specific binding to complementary RNA, stability against nucleases, activity, delivery, pharmacokinetics, and the like, among others, can be influenced by the chirality of the backbone-bound phosphorus atoms.

[0093] In particular, the present disclosure utilizes techniques for controlling various structural elements, such as sugar modifications and their patterns, nucleobase modifications and their patterns, modified internucleotide linkages and their patterns, stereochemistry of linked phosphorus and their patterns, additional chemical moieties (moieties not normally present in an oligonucleotide chain) and their patterns, etc. The ability to fully control the structural elements of oligonucleotides allows the present disclosure to provide oligonucleotides with improved and / or novel properties and / or activities for various applications, e.g., as therapeutic agents, probes, etc. For example, as demonstrated herein, the provided oligonucleotides and compositions thereof are particularly potent for editing target adenosines in target nucleic acids, in some embodiments correcting G to A mutations by converting A to I.

[0094] In some embodiments, the oligonucleotide is a fragment of a nucleic acid (e.g., DNA, pre-mRNA, mRNA, etc.) , 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. In some embodiments, the nucleic acid is a target nucleic acid containing one or more target adenosines. In some embodiments, the target nucleic acid contains only one target adenosine. In some embodiments, the oligonucleotide is capable of hybridizing to the target nucleic acid. In some embodiments, such hybridization promotes modification of A (e.g., conversion of A to I) in the nucleic acid or its product, e.g., by ADAR1, ADAR2, etc.

[0095] In some embodiments, the disclosure provides oligonucleotides, wherein the oligonucleotides have a base sequence that is or includes 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 contiguous 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, wherein each T may optionally and independently be replaced with a U, or vice versa. In some embodiments, the disclosure provides oligonucleotides or oligonucleotide compositions as disclosed herein, e.g., in a Table.

[0096] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide for site-specific editing of a nucleoside (e.g., a target adenosine) in a target nucleic acid, e.g., RNA.

[0097] As described herein, an oligonucleotide may contain one or more modified internucleotide linkages (non-natural phosphorus linkages). In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage in which the linked phosphorus is chiral. In some embodiments, the modified internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, an oligonucleotide comprises one or more negatively charged internucleotide linkages (e.g., phosphorothioate internucleotide linkages, natural phosphate linkages, etc.). In some embodiments, an oligonucleotide comprises one or more non-negatively charged internucleotide linkages. In some embodiments, an oligonucleotide comprises one or more neutral internucleotide linkages.

[0098] In some embodiments, the oligonucleotide is chirally controlled. In some embodiments, the oligonucleotide is chirally pure (or "stereically pure," "stereochemically pure"), and the oligonucleotide exists as a single stereoisomeric form (often a single diastereomeric (or "diastereomeric") form, since multiple chiral centers may exist in the oligonucleotide, for example, at the bond phosphorus, sugar, carbon, etc.). As will be understood by those skilled in the art, chirally pure oligonucleotides are separated from other stereoisomeric forms (to the extent that some impurities may be present, since chemical and biological processes, selectivity and / or purification, etc., are rarely, if ever, absolutely perfect). In chirally pure oligonucleotides, each chiral center is independently defined with respect to its configuration (for chirally pure oligonucleotides, each internucleotide linkage is independently stereorestricted or chiral controlled). In contrast to chiral controlled and chirally pure oligonucleotides containing sterically restricted linking phosphorus, racemic (or "sterically irregular," "chiral non-controlled") oligonucleotides containing chiral linking phosphorus, derived from conventional phosphoramidite oligonucleotide synthesis without stereochemical control during the coupling step in combination with conventional sulfurization (producing sterically irregular phosphorothioate internucleotide linkages), refer to an irregular mixture of various stereoisomers (typically diastereoisomers (or "diastereomers"), since multiple chiral centers are present in the oligonucleotide; for example, derived from conventional oligonucleotide preparations using reagents that do not contain chiral elements other than those in the nucleotides and linking phosphorus). For example, in the context of A*A*A (where * is a phosphorothioate internucleotide linkage (containing a chiral linking phosphorus)), racemic oligonucleotides The preparation of chiral oligonucleotides includes four diastereomers [22=4, considering the two chiral linking phosphorus, each of which can exist in either of two configurations (Sp or Rp)]: A*SA*SA, A*SA*RA, A*RA*SA, and A*RA*RA (*S represents the phosphorothioate internucleotide linkage of Sp, and *R represents the phosphorothioate internucleotide linkage of Rp). For chirally pure oligonucleotides, e.g., A*SA*SA, it exists in a single stereoisomeric form, which can be substituted with other stereoisomers (e.g., the diastereomers A*SA*RA, A*RA*SA, and A*RA*RA). A*RA).

[0099] In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sterically irregular internucleotide linkages (a mixture of Rp and Sp linked phosphorus at the internucleotide linkage, e.g., resulting from conventional, non-chiral oligonucleotide synthesis). In some embodiments, the oligonucleotide comprises one or more (e.g., 1-60, 1-50, 1-40, 1-30, 1-25, 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more) chirality-controlled internucleotide linkages (Rp or Sp binding phosphorus at the internucleotide linkage, e.g., derived from chirality-controlled oligonucleotide synthesis). In some embodiments, the internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage is a sterically disordered phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage is a chirality-controlled phosphorothioate internucleotide linkage.

[0100] In particular, the present disclosure provides techniques for preparing chiral controlled (and in some embodiments, stereochemically pure) oligonucleotides. In some embodiments, the oligonucleotides are stereochemically pure. In some embodiments, the oligonucleotides of the present disclosure have a chirality of about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5%, 10%, 15%, 20%, 25%, 30%, 35%. , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% stereochemically pure.

[0101] In some embodiments, the present disclosure provides various oligonucleotide compositions. In some embodiments, the oligonucleotide compositions are sterically disordered or chirality-uncontrolled. In some embodiments, the oligonucleotides of the provided compositions have no chirality-controlled internucleotide linkages. In some embodiments, the internucleotide linkages of the oligonucleotides in the compositions comprise one or more chirality-controlled internucleotide linkages (e.g., chirality-controlled oligonucleotide compositions).

[0102] In some embodiments, an oligonucleotide composition comprises a plurality of oligonucleotides sharing a common base sequence, wherein one or more internucleotide linkages in the oligonucleotides are chiral controlled and one or more internucleotide linkages are sterically irregular (chiral uncontrolled). In some embodiments, an oligonucleotide composition comprises a plurality of oligonucleotides sharing a common base sequence, wherein each internucleotide linkage containing a chiral phosphorus in the oligonucleotides is independently a chiral controlled internucleotide linkage. In some embodiments, the plurality of oligonucleotides share the same base sequence and the same base and sugar modifications. In some embodiments, the plurality of oligonucleotides share the same base sequence and the same base, sugar, and internucleotide linkage modifications. In some embodiments, the oligonucleotide composition comprises oligonucleotides of the same configuration, wherein one or more internucleotide linkages are chiral controlled and one or more internucleotide linkages are sterically irregular (chiral uncontrolled). In some embodiments, the oligonucleotide composition comprises oligonucleotides of the same configuration, wherein each internucleotide linkage containing a chiral phosphorus linkage is independently a chiral controlled internucleotide linkage. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or all, of all oligonucleotides of a common base sequence are a plurality of oligonucleotides.

[0103] In some embodiments, the present disclosure provides techniques for preparing, evaluating, and / or utilizing the provided oligonucleotides and compositions thereof.

[0104] As used in this disclosure, in some embodiments, "one or more" is 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 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 1. In some embodiments, "one or more" is 2. In some embodiments, "one or more" is 3. In some embodiments, "one or more" is 4. In some embodiments, "one or more" is 5. In some embodiments, "one or more" is 6. In some embodiments, "one or more" is 7. In some embodiments, "one or more" is 8. In some embodiments, "one or more" is 9. In some embodiments, "one or more" is 10. In some embodiments, "one or more" is at least 1. In some embodiments, "one or more" is at least 2. In some embodiments, "one or more" is at least 3. In some embodiments, "one or more" is at least 4. In some embodiments, "one or more" is at least 5. In some embodiments, "one or more" is at least 6. In some embodiments, "one or more" is at least 7. In some embodiments, "one or more" is at least 8. In some embodiments, "one or more" is at least 9. In some embodiments, "one or more" is at least ten.

[0105] As used in this disclosure, in some embodiments, "at least one" means one or more.

[0106] Oligonucleotides In particular, the present disclosure provides oligonucleotides of various designs, which may include various nucleobases and their patterns, sugars and their patterns, internucleotide linkages and their patterns, and / or additional chemical moieties and their patterns, as described herein. In some embodiments, the provided oligonucleotides are capable of inducing 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-specific editing (A to I conversion) of adenosines in a target RNA sequence.

[0107] In some embodiments, the oligonucleotides are of suitable length and specificity to the target nucleic acid. The oligonucleotide is of a complementary sequence to hybridize with the target nucleic acid. In some embodiments, the oligonucleotide is sufficiently long 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 sufficiently short to facilitate delivery and reduce manufacturing complexity and / or cost, while maintaining the desired properties and activity (e.g., adenosine editing).

[0108] In some embodiments, the oligonucleotides are about 10 to 200 (e.g., about 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 120, 10 to 150, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 80, 20 to 90, 20 to 100, 20 to 120, 20 to 150, 20 to 200, 25 to 30, 25 to 40, 25 to 50, The oligonucleotide has a length of nucleobases of 25 to 60, 25 to 70, 25 to 80, 25 to 90, 25 to 100, 25 to 120, 25 to 150, 25 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 70, 30 to 80, 30 to 90, 30 to 100, 30 to 120, 30 to 150, 30 to 200, 10, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, etc. In some embodiments, the oligonucleotide base sequence is about 10 to 60 nucleobases in length. In some embodiments, the base sequence is about 15 to 50 nucleobases in length. In some embodiments, the base sequence is about 15 to about 35 nucleobases in length. In some embodiments, the base sequence is about 25 to about 34 nucleobases in length. In some embodiments, the base sequence is about 26 to about 35 nucleobases in length. In some embodiments, the base sequence is about 27 to about 32 nucleobases in length. In some embodiments, the base sequence is about 29 to about 35 nucleobases in length. In some embodiments, 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 length. In some other embodiments, the base sequence is 35 nucleobases in length or at least 35 nucleobases in length. In some other embodiments, the base sequence is 34 nucleobases in length or at least 34 nucleobases in length.In some other embodiments, the base sequence is 33 nucleobases in length or at least 33 nucleobases in length. In some other embodiments, the base sequence is 32 nucleobases in length or at least 32 nucleobases in length. In some other embodiments, the base sequence is 31 nucleobases in length or at least 31 nucleobases in length. In some other embodiments, the base sequence is 30 nucleobases in length or at least 30 nucleobases in length. In some other embodiments, the base sequence is 29 nucleobases in length or at least 29 nucleobases in length. In some other embodiments, the base sequence is 28 nucleobases in length or at least 28 nucleobases in length. In some other embodiments, the base sequence is 27 nucleobases in length or at least 27 nucleobases in length. In some other embodiments, the base sequence is 26 nucleobases in length or at least 26 nucleobases in length. In some other embodiments, the base sequence of the complementary portions 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, it is at least 25 nucleobases in length. In particular, the present disclosure provides oligonucleotides of similar or better properties and / or similar or higher activity, but of shorter length, compared to previously reported adenosine-editing oligonucleotides.

[0109] 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 containing the target adenosine) with 0 to 10 mismatches that are not Watson-Crick base pairs (AT, AU, and CG) (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.). 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 not designed for complementarity (e.g., loops, protein binding sequences, etc., for recruitment of proteins, e.g., ADARs). As one of skill in the art will understand, such portions may be appropriately excluded when calculating mismatches and / or complementarity.In some embodiments, the complementarity, e.g., between the oligonucleotide and the target nucleic acid, is about 50% to 100% (e.g., about 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85 ... Complementarity may be 0% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, complementarity is at least about 60%. In some embodiments, complementarity is at least about 65%. In some embodiments, complementarity is at least about 70%. In some embodiments, complementarity is at least about 75%. In some embodiments, 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 some embodiments, the complementarity is at least about 95%. In some embodiments, the complementarity is 100% throughout the length of the oligonucleotide. In some embodiments, the complementarity is 100% except for the nucleoside opposite the target nucleoside (e.g., adenosine) throughout the length of the oligonucleotide. Typically, complementarity is based on Watson-Crick base pairs AT, AU, and CG. Those skilled in the art will understand that when assessing the complementarity of two sequences of different lengths (e.g., a provided oligonucleotide and a target nucleic acid), the complementarity can be appropriately based on the length and / or maximum complementarity of the shorter sequence between the two sequences. In many embodiments, the oligonucleotide and the target nucleic acid are sufficiently complementary so that modification is selectively directed to the target adenosine site.

[0110] In some embodiments, one or more mismatches are independently perturbations. In some embodiments, each mismatch is perturbation. In some embodiments, 0 to 10 (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0-10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc. In some embodiments, the number is 0. In some embodiments, the number is 1. In some embodiments, the number is 2. In some embodiments, the number is 3. In some embodiments, the number is 4. In some embodiments, the number is 5. In some embodiments, the perturbation is GU, IA, GA, IU, IC, IT, AA, or reverse AT. In some embodiments, the perturbation is GU, IA, GA, IU, or IC. In some embodiments, IC can be considered a match when I is the nucleoside immediately 3' to the nucleoside opposite the target nucleoside.

[0111] In some embodiments, the duplex of the oligonucleotide and the target nucleic acid contains one or more bulges, each independently containing one or more mismatches that are not wobble. In some embodiments, there are 0 to 10 bulges (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.). In some embodiments, the number is 0. In some embodiments, the number is 1. In some embodiments, the number is 2. In some embodiments, the number is 3. In some embodiments, the number is 4. In some embodiments, the number is 5.

[0112] In some embodiments, the distance between two mismatches, between the mismatch and one or both ends of the oligonucleotide (or portions thereof, e.g., the first domain, the second domain, the first subdomain, the second subdomain, the third subdomain), and / or between the mismatch and the nucleoside opposite the target adenosine, is independently 0 to 50, 0 to 40, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10 (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 8, 1 to 9, 1 to 10, 1 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6 ... In some embodiments, the number is 0 to 30. In some embodiments, the number is 0 to 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 to 20. In some embodiments, the distance between two mismatches is 1 to 10. In some embodiments, the distance between a mismatch and the 5'-terminal nucleoside of the oligonucleotide is 0 to 20. In some embodiments, the distance between a mismatch and the 5'-terminal nucleoside of the oligonucleotide is 0 to 20. In some embodiments, the distance between the mismatch and the 3'-terminal nucleoside of the oligonucleotide is 0 to 40. In some embodiments, the distance between the mismatch and the 3'-terminal nucleoside of the oligonucleotide is 5 to 20. In some embodiments, the distance between the mismatch and the nucleoside opposite the target adenosine is 0 to 20. In some embodiments, the distance between the mismatch and the nucleoside opposite the target adenosine is 1 to 10. In some embodiments, the number of nucleobases related to the distance 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 at an end, e.g., the 5'-end or 3'-end, of the first domain, second domain, first subdomain, second subdomain, or third subdomain. In some embodiments, the mismatch is at the nucleoside opposite the target adenosine.

[0113] In some embodiments, provided oligonucleotides are capable of inducing adenosine editing (e.g., A to I conversion) in a target nucleic acid, and consist of, comprise, or have a base sequence that includes a portion (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more consecutive bases) of the base sequence of an oligonucleotide disclosed herein, wherein each T may independently be replaced with a U, or vice versa, and the oligonucleotide includes at least one non-naturally occurring modification of the base, sugar, and / or internucleotide linkage.

[0114] In some embodiments, provided oligonucleotides comprise one or more carbohydrate moieties. In some embodiments, provided oligonucleotides comprise one or more GalNAc moieties. In some embodiments, provided oligonucleotides comprise one or more targeting moieties. Non-limiting examples of such additional chemical moieties that can be conjugated to oligonucleotide chains are described herein.

[0115] In some embodiments, provided oligonucleotides can induce correction of a G to A mutation in a target sequence or its product. In some embodiments, the correction of a G to A mutation is or includes conversion of A to I, which can be read as G during translation or other biological processes. In some embodiments, provided oligonucleotides can induce correction of a G to A mutation in a target sequence or its product via ADAR-mediated deamination. In some embodiments, provided oligonucleotides can induce correction of a G to A mutation in a target sequence or its product via ADAR-mediated deamination by recruiting endogenous ADARs (e.g., in target cells) and promoting ADAR-mediated deamination. Nevertheless, the present disclosure is not limited to any particular mechanism. In some embodiments, the present disclosure provides oligonucleotides, compositions, methods, etc. that can operate via double-stranded RNA interference, single-stranded RNA interference, RNase H-mediated knockdown, steric hindrance of translation, ADAR-mediated deamination, or a combination of two or more such mechanisms.

[0116] In some embodiments, the oligonucleotide comprises a structural element or portion thereof described herein, e.g., in a Table. In some embodiments, the oligonucleotide has a base sequence comprising a base sequence (or portion thereof), wherein each T is independently replaced with a U, a pattern of chemical modifications (or portions thereof) disclosed herein, e.g., in a Table or Figure, or otherwise disclosed herein, and / or the format of the oligonucleotide. In some embodiments, such oligonucleotides are capable of directing the correction of a G to A mutation in a target sequence or its product.

[0117] In particular, provided oligonucleotides can hybridize to their target nucleic acids (e.g., pre-mRNA, mature mRNA, etc.). In some embodiments, oligonucleotides can hybridize to nucleic acids of target RNA sequences at any stage of RNA processing, including, but not limited to, pre-mRNA or mature mRNA. In some embodiments, oligonucleotides can hybridize to any element of the oligonucleotide nucleic acid, or its complement, including, but not limited to, a promoter region, an enhancer region, a transcription termination region, a translation initiation signal, a translation termination signal, a coding region, a non-coding region, an exon, an intron, an intron / exon or exon / intron junction, a 5' UTR, or a 3' UTR.

[0118] In some embodiments, the oligonucleotide hybridizes to two or more variants of a transcript derived from the sense strand of a target site (eg, a target sequence).

[0119] In some embodiments, the provided oligonucleotides contain an increased level of one or more isotopes. In some embodiments, the provided oligonucleotides are labeled with, for example, one or more isotopes of one or more elements, such as hydrogen, carbon, nitrogen, etc. In some embodiments, the provided oligonucleotides in the provided compositions, e.g., multiple oligonucleotides of the composition, contain base modifications, sugar modifications, and / or internucleotide linkage modifications, and the oligonucleotides contain enriched levels of deuterium. In some embodiments, the provided oligonucleotides are labeled with deuterium at one or more positions (-1H replaced with -2H). In some embodiments, one or more 1H in the oligonucleotide strand or any moiety conjugated to the oligonucleotide strand (e.g., targeting moiety, etc.) is replaced with 2H. Such oligonucleotides can be used in the compositions and methods described herein.

[0120] In some embodiments, an 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, an oligonucleotide comprises, for example, about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, of all nucleobases, sugars, and internucleotide linkages, respectively, in the oligonucleotide. %, 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% modified nucleobases, modified sugars, and / or modified internucleotide linkages.

[0121] In some embodiments, the oligonucleotide comprises one or more modified sugars. In some embodiments, the oligonucleotides of the present disclosure comprise one or more modified nucleobases. Various modifications can be introduced into the sugar and / or nucleobase in accordance with the present disclosure. For example, in some embodiments, the modifications are those described in U.S. Patent No. 9,006,198. In some embodiments, the modifications are those described in U.S. Patent No. 9,394,333, U.S. Patent No. 9,774,474, each of which is incorporated herein by reference. 4183, U.S. Patent No. 9605019, U.S. Patent No. 9982257, U.S. Patent No. 20170037399, U.S. Patent No. 20180216108, U.S. Patent No. 20180216107, U.S. Patent No. 9598458, International Publication No. 2017 / 062862, International Publication No. 2018 / 067973, International Publication No. 2017 / 160741, International Publication No. 2017 / 192679, International Publication No. 2017 / 210647, International Publication No. 2018 / 098264, International Publication No. 2018 / 022473, and / or WO 2020 / 191252.

[0122] In some embodiments, the nucleobase in the nucleoside is or comprises a BA ring having the structure BA-I, BA-Ia, BA-Ib, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-Va, BA-Vb, or BA-VI, or a tautomer of the BA ring, wherein the nucleobase is optionally substituted or protected.

[0123] In some embodiments, the sugar is a modified sugar that includes a 2'-modification, e.g., 2'-F, 2'-OR, where R is an optionally substituted aliphatic, or bicyclic sugar (e.g., an LNA sugar), or an acyclic sugar (e.g., a UNA sugar).

[0124] In some embodiments, as described herein, provided oligonucleotides comprise one or more domains, each of which independently has a particular length, modification, stereochemistry of linked phosphorus, 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: the first domain; and Second Domain and providing an oligonucleotide comprising: where: 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.

[0125] In some embodiments, an oligonucleotide or portion thereof (e.g., first domain, second domain, first subdomain, second subdomain, third subdomain, etc.) comprises a certain level of modified sugars. In some embodiments, the modified sugar comprises a 2'-modification. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar is an acyclic sugar (e.g., by cleaving the C2-C3 bond of the corresponding cyclic sugar). In some embodiments, the modified sugar comprises a 5'-modification. Typically, oligonucleotides of the present disclosure have a free 5'-OH at their 5'-end and a free 3'-OH at their 3'-end, unless otherwise specified, e.g., by context. In some embodiments, the 5'-terminal sugar of the oligonucleotide may comprise a modified 5'-OH.

[0126] In some embodiments, the levels are the total number of nucleotides in the oligonucleotide or portion thereof, respectively. Approximately, for example, about 5% to 100%, about 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 85%, 70% to 90%, 70% to 90%, 70% to 100% % to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. 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%.

[0127] In some embodiments, the oligonucleotide or a portion thereof (e.g., the first domain, the second domain, the first subdomain, the second subdomain, the third subdomain, etc.) comprises a certain level of modified internucleotide linkages. In some embodiments, the oligonucleotide or a portion thereof (e.g., the first domain, the second domain, the first subdomain, the second subdomain, the third subdomain, etc.) comprises a certain level of chiral internucleotide linkages. In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 80% to 100%, 85% to 80%, 85% to 90%, 85% to 95%, 85% to 100%, 90 ... % to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. 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%.

[0128] In some embodiments, the oligonucleotide or portion thereof (e.g., the first domain, the second domain, the first subdomain, the second subdomain, the third subdomain, etc.) comprises a certain level of chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide or portion thereof (e.g., first domain, second domain, first subdomain, second subdomain, third subdomain, etc.) contains a certain level of Sp internucleotide linkages. In some embodiments, the level may be about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 85%, 70% to 95%, 80% to 100%, 85% to 100%, 90% to 100%, 90% to 100%, 95% to 100%, 10 ... % to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 85%, 75% to 90%, 75% to 95%, 75% to 10 ... 0% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.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%.

[0129] In some embodiments, an oligonucleotide or portion thereof (e.g., first domain, second domain, first subdomain, second subdomain, third subdomain, etc.) comprises a particular level of Sp internucleotide linkages. In some embodiments, the level may be about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 85%, 70% to 95%, 80% to 100%, 85% to 85%, 85% to 90%, 85% to 95%, 85% to 100%, 90 ... % to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of all chiral internucleotide linkages in the oligonucleotide or portion thereof, etc. In some embodiments, the levels are, respectively, approximately For example, about 5% to 100%, about 10% to 100%, 20 to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, or 65% to 100% of all chiral controlled internucleotide linkages in the oligonucleotide or portion thereof, respectively. %, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. 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 to 50, 1 to 40, 1 to 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 Sp chiral internucleotide linkages. In many embodiments, it has been observed that a high percentage of Sp internucleotide linkages (e.g., compared to Rp internucleotide linkages and / or natural phosphate linkages) in an oligonucleotide or particular portion thereof can result in improved properties and / or activity, e.g., increased stability and / or increased adenosine editing activity.

[0130] In some embodiments, an oligonucleotide or portion thereof (e.g., first domain, second domain, first subdomain, second subdomain, third subdomain, etc.) contains a particular level of Rp internucleotide linkages. In some embodiments, the level may be about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, or 65% of all internucleotide linkages in the oligonucleotide or portion thereof, respectively. ~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% of all chiral internucleotide linkages in the oligonucleotide or portion thereof, respectively. In some embodiments, the level is about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 85%, 75% to 90%, 65% to 95%, 65% to 100%, 75% to 100%, 80% to 85%, 80% to 95%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 95% to 100%, 10 ... 0% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the levels are about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, or 65% to 100% of all chiral controlled internucleotide linkages in the oligonucleotide or portion thereof, respectively. %, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. 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 5% or less than about 5%. In some embodiments, the percentage is about 10% or less than about 10%. In some embodiments, the percentage is about 15% or less than about 15%. In some embodiments, the percentage is about 20% or less than about 20%. In some embodiments, the percentage is about 25% or less. In some embodiments, the percentage is about 30% or less. In some embodiments, the percentage is about 35% or less. In some embodiments, the percentage is about 40% or less. In some embodiments, the percentage is about 45% or less. In some embodiments, the percentage is about 50% or less. In some embodiments, about 1 to 50, 1 to 40, 1 to 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 1 or less. In some embodiments, the number is about 2 or less. In some embodiments, that number is about 3 or less than about 3. In some embodiments, that number is about 4 or less than about 4. In some embodiments, that number is about 5 or less than about 5. In some embodiments, that number is about 6 or less than about 6.In some embodiments, the number is. , or about 7 or less. In some embodiments, that number is about 8 or about 8 or less. In some embodiments, that number is about 9 or about 9 or less. In some embodiments, that number is about 10 or about 10 or less.

[0131] Without wishing to be bound by any particular theory, in some examples, the Rp and Sp configurations of internucleotide bonds may affect the structural changes in the helical structure of the double-stranded complex formed by an oligonucleotide and a target nucleic acid such as RNA, and ADAR proteins may recognize and interact with various targets (e.g., double-stranded complexes formed by an oligonucleotide and a target nucleic acid such as RNA) through multiple domains. In some embodiments, the provided oligonucleotides and compositions thereof facilitate and / or enhance the interaction profile of the oligonucleotide, the target nucleic acid, and / or the ADAR protein, resulting in efficient adenosine modification by the ADAR protein through the incorporation of various modifications and / or stereochemical control.

[0132] In some embodiments, an oligonucleotide may have or include a base sequence; an internucleotide linkage, a base modification, a sugar modification, an additional chemical moiety, or a pattern thereof; and / or any other structural element described herein, e.g., in the Tables.

[0133] In some embodiments, a provided oligonucleotide or composition is characterized in that when it is linked to a target nucleic acid containing 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 compared to that observed under reference conditions (e.g., selected from the group consisting of the absence of the composition, the presence of the reference oligonucleotide or composition, and combinations thereof). In some embodiments, the modification, e.g., ADAR-mediated deamination (e.g., endogenous ADAR-mediated amination), 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 or more fold.

[0134] In some embodiments, oligonucleotides are provided as salt forms. In some embodiments, oligonucleotides are provided as salts that contain negatively charged internucleotide linkages (e.g., phosphorothioate internucleotide linkages, natural phosphate linkages, etc.) present in their salt forms. In some embodiments, oligonucleotides are provided as pharmaceutically acceptable salts. In some embodiments, oligonucleotides are provided as metal salts. In some embodiments, oligonucleotides are provided as sodium salts. In some embodiments, oligonucleotides are provided as ammonium salts. In some embodiments, oligonucleotides are provided as metal salts, e.g., sodium salts, and each negatively charged internucleotide linkage is independently present in salt form (e.g., -OP(O)(SNa)-O- for a phosphorothioate internucleotide linkage, -OP(O)(ONa)-O- for a natural phosphate linkage, etc., for the sodium salt).

[0135] In some embodiments, the oligonucleotides are chiral controlled and contain one or more chiral controlled internucleotide linkages. In some embodiments, the provided oligonucleotides are stereochemically pure. In some embodiments, the provided oligonucleotides or compositions thereof are stereochemically pure from other stereoisomers. In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions.

[0136] As described herein, oligonucleotides of the present disclosure can be provided with high purity (e.g., 50%-100%). In some embodiments, oligonucleotides of the present disclosure are of high stereochemical purity (e.g., 50%-100%). In some embodiments, the oligonucleotides in the provided compositions are of high stereochemical purity (e.g., a high percentage (e.g., 50%-100%) of a stereoisomer compared to other stereoisomers of the same oligonucleotide). In some embodiments, the percentage is at least or about 50%. In some embodiments, the percentage is at least or about 60%. In some embodiments, the percentage is at least or about 70%. In some embodiments, the percentage is at least or about 75%. In some embodiments, the percentage is at least or about 80%. In some embodiments, the percentage is at least or about 85%. In some embodiments, the percentage is at least or about 90%. In some embodiments, the percentage is at least or about 95%.

[0137] First Domain As described herein, in some embodiments, the oligonucleotide comprises a first domain and a second domain. In some embodiments, the oligonucleotide consists of a first domain and a second domain. Certain embodiments are described below as examples.

[0138] In some embodiments, the first domain has a length of about 2 to 50 nucleobases (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc.). In some embodiments, the first domain has a length of about 5 to 30 nucleobases. In some embodiments, the first domain has a length of about 10 to 30 nucleobases. In some embodiments, the first domain has a length of about 10 to 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.

[0139] 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%, or 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%. 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%.

[0140] In some embodiments, when the oligonucleotide is aligned with the target nucleic acid in terms of complementarity, 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 one mismatch. In some embodiments, there are two mismatches. In some embodiments, there are three mismatches. In some embodiments, there are four mismatches. In some embodiments, there are five mismatches. In some embodiments, there are six mismatches. In some embodiments, there are seven mismatches. In some embodiments, there are eight mismatches. In some embodiments, there are nine mismatches. In some embodiments, there are ten mismatches.

[0141] In some embodiments, when the oligonucleotide is aligned with the target nucleic acid in terms of complementarity, there are one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobbles in the first domain. In some embodiments, there is one wobble. In some embodiments, there are two wobbles. In some embodiments, there are three wobbles. In some embodiments, there are four wobbles. In some embodiments, there are five wobbles. In some embodiments, there are six wobbles. In some embodiments, there are seven wobbles. In some embodiments, there are eight wobbles. In some embodiments, there are nine wobbles. In some embodiments, there are ten wobbles.

[0142] In some embodiments, the duplex of the oligonucleotide and the target nucleic acid in the first domain region contains one or more bulges, each independently containing one or more mismatches that are not wobble. In some embodiments, there are 0 to 10 bulges (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.). In some embodiments, the number is 0. In some embodiments, the number is 1. In some embodiments, the number is 2. In some embodiments, the number is 3. In some embodiments, the number is 4. In some embodiments, the number is 5.

[0143] In some embodiments, the first domain is perfectly complementary to the target nucleic acid.

[0144] In some embodiments, the first domain comprises one or more modified nucleobases.

[0145] In some embodiments, the second domain comprises one or more sugars containing two 2'-H (e.g., natural DNA sugars). In some embodiments, the second domain comprises one or more sugars containing a 2'-OH (e.g., natural RNA sugars). In some embodiments, the first domain comprises one or more modified sugars. In some embodiments, the modified sugar comprises a 2'-modification. In some embodiments, the modified sugar is a bicyclic sugar, e.g., , LNA sugar. In some embodiments, the modified sugar is an acyclic sugar (e.g., by cleavage of the C2-C3 bond of the corresponding cyclic sugar).

[0146] In some embodiments, the first domain comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 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, such as 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) modified sugars. In some embodiments, the first domain comprises about 1 to 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 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, such as 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) modified sugars having 2'-F modifications.

[0147] In some embodiments, between about 5% and 100% (e.g., between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90% , 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc., is independently a modified sugar. In some embodiments, between about 5% and 100% (e.g., between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90 ... % of the 2'-F modified sugars (e.g., 0% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) are independently 2'-F modified sugars. 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%.

[0148] In some embodiments, the first domain does not include a bicyclic sugar or a 2'-OR modified sugar (wherein R is not -H). In some embodiments, the first domain includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) bicyclic sugars and / or 2'-OR modified sugars (wherein R is not -H). In some embodiments, the first domain includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) 2'-OR modified sugars (wherein R is not -H). In some embodiments, the first domain includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) 2'-OR modified sugars (wherein R is optionally substituted C 1~10In some embodiments, the levels of bicyclic sugars and / or 2'-OR modified sugars (where R is not -H), individually or collectively, are relatively low compared to the levels of 2'-F modified sugars. In some embodiments, about 1% to 95% or less (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) of the sugars in the first domain comprise 2'-OMe. In some embodiments, about 50% or less of the sugars in the first domain comprise 2'-OMe. In some embodiments, between about 1% and 95% or less (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) of the sugars in the first domain are 2′-OR (where R is an optionally substituted C 1~6 In some embodiments, no more than about 50% of the sugars in the first domain are 2'-OR (where R is an optionally substituted C 1~6 In some embodiments, no more than about 40% of the sugars in the first domain are 2'-OR (where R is an optionally substituted C 1~6 In some embodiments, no more than about 30% of the sugars in the first domain are 2'-OR (where R is an optionally substituted C 1~6 In some embodiments, no more than about 25% of the sugars in the first domain are 2'-OR (where R is an optionally substituted C 1~6 In some embodiments, no more than about 20% of the sugars in the first domain are 2'-OR (where R is an optionally substituted C 1~6 In some embodiments, no more than about 10% of the sugars in the first domain are 2'-OR (where R is an optionally substituted C 1~6In some embodiments, the 2'-OR is 2'-MOE, as described herein. In some embodiments, the 2'-OR is 2'-MOE or 2'-OMe, as described herein. In some embodiments, the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars that comprise a 2'-N(R)2 modification. In some embodiments, the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) modified sugars that comprise a 2'-NH2 modification. In some embodiments, the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) bicyclic sugars, e.g., LNA sugars. In some embodiments, the first domain comprises one or more (e.g., about 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) acyclic sugars (e.g., UNA sugars). In some embodiments, some 5'-terminal sugars in the first domain are independently 2'-OR modified sugars (wherein R is not -H). In some embodiments, several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 5'-terminal sugars in the first domain are independently 2'-OR modified sugars (wherein R is independently an optionally substituted C 1~6 In some embodiments, about the first 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, sugars from the 5' end in the first domain are independently 2'-OR modified sugars (wherein R is independently an optionally substituted C 1~6 aliphatic In some embodiments, the first one is 2'-OR modified. In some embodiments, the first two are independently 2'-OR modified. In some embodiments, the first three are independently 2'-OR modified. In some embodiments, the first four are independently 2'-OR modified. In some embodiments, the first five are independently 2'-OR modified. In some embodiments, all 2'-OR modifications in a domain (e.g., the first domain), subdomain (e.g., the first subdomain), or oligonucleotide are the same. In some embodiments, a 2'-OR is 2'-MOE. In some embodiments, a 2'-OR is 2'-OMe.

[0149] In some embodiments, no sugar in the first domain comprises a 2'-OR. In some embodiments, no sugar in the first domain comprises a 2'-OMe. In some embodiments, no sugar in the first domain comprises a 2'-MOE. In some embodiments, no sugar in the first domain comprises a 2'-MOE or a 2'-OMe. In some embodiments, no sugar in the first domain comprises a 2'-OR (where R is an optionally substituted C 1~6 In some embodiments, each sugar in the first domain comprises a 2'-F.

[0150] In some embodiments, the first domain comprises between about 1 and 50 (e.g., between 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 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, such as 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) modified internucleotide linkages. In some embodiments, between about 5% and 100% (e.g., between about 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 80%, 50% and 85%, 50% and 90%, 50% and 95%, 60% and 80%, 60% and 85%, 60% and 90%, 60% and 95%, 60% and 100%, 65% and 80%, 65% and 85%, 65% and 90%, 65% and 95%, 65% and 100%, 70% and 80%, 70% and 85%, 70% and 90% In some embodiments, at least one of the internucleotide linkages in the first domain is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, the modified or chiral internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, the modified or chiral internucleotide linkage is a non-negatively charged internucleotide linkage. In some embodiments, the modified or chiral internucleotide linkage is a neutral internucleotide linkage, e.g., n001.In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a non-negatively charged internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage or a neutral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, at least about 1 to 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50) modified internucleotide linkages in the first domain are present. The chiral internucleotide linkages of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, are chiral controlled. In some embodiments, at least 5% to 100% (e.g., about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85% %, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc., is chiral controlled.In some embodiments, at least 5% to 100% (e.g., about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% up to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (e.g., each independently chiral controlled). In some embodiments, at least about 1 to 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 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, such as about ...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 the first domain are Sp.In some embodiments, at least about 1 to 50 (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, such as 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, such as 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) phosphorothioate internucleotide linkages in the first domain are Sp. In some embodiments, at least 5% to 100% (e.g., about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70%~85%, 70%~90%, 70%~95%, 70%~100%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 75%~100%, 80%~85%, 80%~90%, 80%~95%, 80%~100%, 85%~90%, 85%~95%, 85%~100%, 90%~95%, 90%~100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, at least 5% to 100% (e.g., about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 70% to 80%, 75% to 10 ... In some embodiments, the number is 1 or greater. In some embodiments, the number is 2 or greater. In some embodiments, the number is 3 or greater. In some embodiments, the number is 4 or greater. In some embodiments, the number is 5 or greater. In some embodiments, the number is 6 or greater. In some embodiments, the number is 7 or greater. In some embodiments, the number is 8 or greater. In some embodiments, the number is 9 or greater. In some embodiments, the number is 1 or greater. In some embodiments, the number is 2 or greater. In some embodiments, the number is 3 or greater. In some embodiments, the number is 4 or greater. In some embodiments, the number is 5 or greater. In some embodiments, the number is 6 or greater. In some embodiments, the number is 7 or greater. In some embodiments, the number is 8 or greater. In some embodiments, the number is 9 or greater. In some embodiments, the number is 10 or greater. In some embodiments, the number is 11 or greater. In some embodiments, the number is 12 or greater. In some embodiments, the number is 13 or greater. In some embodiments, the number is 14 or greater. In some embodiments, the number is 15 or greater. 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, each internucleotide linkage connecting two first domain nucleosides is independently a modified internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a chiral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotide linkage is independently a phosphorothioate internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently an Sp chiral internucleotide linkage. In some embodiments, each modified internucleotide linkage is independently an Sp phosphorothioate internucleotide linkage. In some embodiments, each chiral internucleotide linkage is independently an Sp phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage of the first domain is linked to two nucleosides of the first domain. In some embodiments, the internucleotide linkages linked to the nucleosides in the first domain and the nucleosides in the second domain can be properly considered to be the internucleotide linkages of the first domain. In some embodiments, the internucleotide linkages linked to the nucleosides in the first domain and the nucleosides in the second domain are modified internucleotide linkages; in some embodiments, they are chiral internucleotide linkages; in some embodiments, they are chiral internucleotide linkages; in some embodiments, they are chiral; in some embodiments, they are Rp; and in some embodiments, they are Sp.In many embodiments, a high percentage of Sp internucleotide linkages (eg, compared to Rp internucleotide linkages and / or natural phosphate linkages) are improved. It has been observed that these confer improved properties and / or activity, for example, increased stability and / or increased adenosine editing activity.

[0151] In some embodiments, the first domain comprises a certain level of Rp internucleotide linkages. In some embodiments, the level may be about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, or 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the level is about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80% of all chiral internucleotide linkages in the first domain. , 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.In some embodiments, the level is about, e.g., about 5% to 100%, about 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 65% to 80%, 65% to 85%, 65% to 90%, 65% to 95%, 65% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 100%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the percentage is about 50% or less. 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 5% or less than about 5%. In some embodiments, the percentage is about 10% or less than about 10%. In some embodiments, the percentage is about 15% or less than about 15%. In some embodiments, the percentage is about 20% or less than about 20%. In some embodiments, the percentage is about 25% or about. In some embodiments, the percentage is about 25% or less. In some embodiments, the percentage is about 30% or about 30% or less. In some embodiments, the percentage is about 35% or about 35% or less. In some embodiments, the percentage is about 40% or about 40% or less. In some embodiments, the percentage is about 45% or about 45% or less. In some embodiments, the percentage is about 50% or about 50% or less. In some embodiments, about 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 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 1 or about 1 or less. In some embodiments, that number is about 2 or less. In some embodiments, that number is about 3 or less. In some embodiments, that number is about 4 or less. In some embodiments, that number is about 5 or less. In some embodiments, that number is about 6 or less. In some embodiments, that number is about 7 or less. In some embodiments, that number is about 8 or less. In some embodiments, that number is about 9 or less. In some embodiments, that number is about 10 or less.

[0152] In some embodiments, each phosphorothioate internucleotide linkage in the first domain is independently chiral-controlled. In some embodiments, each is independently Sp or Rp. In some embodiments, the high level is Sp as described herein. In some embodiments, each phosphorothioate internucleotide linkage in the first domain is chiral-controlled and is Sp.

[0153] In some embodiments, as shown in certain examples, the first domain comprises one or more non-negatively charged internucleotide linkages, each of which is optionally and independently chiral-controlled. In some embodiments, each non-negatively charged internucleotide linkage is independently n001. In some embodiments, the chiral non-negatively charged internucleotide linkage is not chiral-controlled. In some embodiments, each chiral non-negatively charged internucleotide linkage is not chiral-controlled. In some embodiments, the chiral non-negatively charged internucleotide linkage is chiral-controlled. In some embodiments, the chiral non-negatively charged internucleotide linkage is chiral-controlled and is Rp. In some embodiments, the chiral non-negatively charged internucleotide linkage is chiral-controlled and is Sp. In some embodiments, each chiral non-negatively charged internucleotide linkage is chiral-controlled. In some embodiments, the number of non-negatively charged internucleotide linkages in the first domain is about 1-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, it is about 1. In some embodiments, it is about 2. In some embodiments, it is about 3. In some embodiments, it is about 4. In some embodiments, it is about 5. In some embodiments, two or more non-negatively charged internucleotide linkages are contiguous. In some embodiments, no two non-negatively charged internucleotide linkages are contiguous. In some embodiments, all non-negatively charged internucleotide linkages in the first domain are contiguous (e.g., three contiguous non-negatively charged internucleotide linkages). In some embodiments, the non-negatively charged internucleotide linkage or two or more contiguous non-negatively charged internucleotide linkages is at the 5' end of the first domain. In some embodiments, the internucleotide linkage connecting the last two nucleosides of the first domain is a non-negatively charged internucleotide linkage.In some embodiments, the internucleotide linkage linking the last two nucleosides of the first domain is a non-negatively charged internucleotide linkage of Sp. In some embodiments, the internucleotide linkage linking the last two nucleosides of the first domain is a non-negatively charged internucleotide linkage of Rp. In some embodiments, the internucleotide linkage linking the last two nucleosides of the first domain is a phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage linking the last two nucleosides of the first domain is a phosphorothioate internucleotide linkage of Sp. In some embodiments, the internucleotide linkage linking the first two nucleosides of the first domain is a non-negatively charged internucleotide linkage. In some embodiments, the internucleotide linkage linking the first two nucleosides of the first domain is a non-negatively charged internucleotide linkage of Sp. In some embodiments, the internucleotide linkage linking the first two nucleosides of the first domain is a non-negatively charged internucleotide linkage of Rp. In some embodiments, the internucleotide linkage linking the first two nucleosides of the first domain is a phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkage linking the first two nucleosides of the first domain is a phosphorothioate internucleotide linkage of Sp. In some embodiments, the non-negatively charged internucleotide linkage is a neutral internucleotide linkage, such as n001. In some embodiments, the first two nucleosides in the first domain are the first two nucleosides of an oligonucleotide.

[0154] In some embodiments, the first domain comprises one or more native phosphate linkages. In some embodiments, the first domain does not contain any native phosphate linkages.

[0155] In some embodiments, the first domain recruits, facilitates, or contributes to the recruitment of a protein such as an ADAR protein (e.g., ADAR1, ADAR2, etc.). In some embodiments, the first domain recruits, or facilitates or contributes to the interaction with, a protein such as an ADAR protein. In some embodiments, the first domain contacts the RNA-binding domain (RBD) of an ADAR. In some embodiments, the first domain does not substantially contact the second RBD domain of an ADAR. In some embodiments, the first domain does not substantially contact the catalytic domain of an ADAR that has deaminase activity. In some embodiments, various nucleobases, sugars, and / or internucleotide linkages may interact with one or more residues of a protein, e.g., an ADAR protein.

[0156] Second Domain As described herein, in some embodiments, the oligonucleotide comprises a first domain and a second domain from 5' to 3'. In some embodiments, the oligonucleotide consists of a first domain and a second domain. Certain embodiments of the second domain are described below by way of example. In some embodiments, the second domain comprises the nucleoside opposite the target adenosine to be modified (e.g., converted to I).

[0157] In some embodiments, the second domain has a length of about 2 to 50 nucleobases (e.g., about 5, 6, 7, 8, 9, or 10 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50, etc.). In some embodiments, the second domain has a length of about 5 to 30 nucleobases. In some embodiments, the second domain has a length of about 10 to 30 nucleobases. In some embodiments, the second domain has a length of about 10 to 20 nucleobases. In some embodiments, the second domain has a length of about 5-15 nucleobases. In some embodiments, the second domain has a length of about 13-16 nucleobases. In some embodiments, the second domain has a length of about 1-7 nucleobases. In embodiments, the second domain has a length of 10 nucleobases. In some embodiments, the second domain has a length of 11 nucleobases. In some embodiments, the second domain has a length of 12 nucleobases. In some embodiments, the second domain has a length of 13 nucleobases. In some embodiments, the second domain has a length of 14 nucleobases. In some embodiments, the second domain has a length of 15 nucleobases. In some embodiments, the second domain has a length of 16 nucleobases. In some embodiments, the second domain has a length of 17 nucleobases. In some embodiments, the second domain has a length of 18 nucleobases. In some embodiments, the second domain has a length of 19 nucleobases. In some embodiments, the second domain has a length of 20 nucleobases.

[0158] In some embodiments, the second domain comprises 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%, or 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%.

[0159] In some embodiments, when the oligonucleotide is aligned with the target nucleic acid in terms of complementarity, there are one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) mismatches in the second domain. In some embodiments, there is one mismatch. In some embodiments, there are two mismatches. In some embodiments, there are three mismatches. In some embodiments, there are four mismatches. In some embodiments, there are five mismatches. In some embodiments, there are six mismatches. In some embodiments, there are seven mismatches. In some embodiments, there are eight mismatches. In some embodiments, there are nine mismatches. In some embodiments, there are ten mismatches.

[0160] In some embodiments, when the oligonucleotide is aligned with the target nucleic acid in terms of complementarity, there are one or more (e.g., 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobbles in the second domain. In some embodiments, there is one wobble. In some embodiments, there are two wobbles. In some embodiments, there are three wobbles. In some embodiments, there are four wobbles. In some embodiments, there are five wobbles. In some embodiments, there are six wobbles. In some embodiments, there are seven wobbles. In some embodiments, there are eight wobbles. In some embodiments, there are nine wobbles. In some embodiments, there are ten wobbles.

[0161] In some embodiments, the duplex of the oligonucleotide and the target nucleic acid in the second domain region contains one or more bulges, each independently containing one or more mismatches that are not wobble. In some embodiments, there are 0 to 10 bulges (e.g., 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 0 to 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.). In some embodiments, the number is 0. In some embodiments, the number is 1. In some embodiments, the number is 2. In some embodiments, the number is 3. In some embodiments, the number is 4. In some embodiments, the number is 5.

[0162] In some embodiments, the second domain is perfectly complementary to the target nucleic acid.

[0163] In some embodiments, the second domain comprises one or more modified nucleobases.

[0164] In some embodiments, the second domain comprises the nucleoside opposite the target adenosine, for example, when the oligonucleotide forms a duplex with the target nucleic acid. In some embodiments, the opposite nucleobase is an optionally substituted or protected U, or a tautomer of an optionally substituted or protected U. In some embodiments, the opposite nucleobase is U.

[0165] In some embodiments, the opposing nucleobase has a weaker hydrogen bond with the target adenine of the target adenosine compared to U. In some embodiments, the opposing nucleobase forms fewer hydrogen bonds with the target adenine of the target adenosine compared to U. In some embodiments, the opposing nucleobase forms one or more hydrogen bonds with one or more amino acid residues of a protein, e.g., ADAR, and these residues form one or more hydrogen bonds with the U opposite the target adenosine. In some embodiments, the opposing nucleobase forms one or more hydrogen bonds with each amino acid residue of the ADAR that forms one or more hydrogen bonds with the U opposite the target adenosine. In some embodiments, certain opposing nucleobases facilitate and / or promote adenosine modification by ADAR proteins, e.g., ADAR1 and ADAR2, by weakening hydrogen bonds with the target A and / or maintaining or enhancing interactions with proteins such as ADAR1 and ADAR2.

[0166] In some embodiments, the opposite nucleobase is an optionally substituted or protected C or a tautomer of an optionally substituted or protected C. In some embodiments, the opposite nucleobase is C. In some embodiments, the opposite nucleobase is an optionally substituted or protected A or a tautomer of an optionally substituted or protected A. In some embodiments, the opposite nucleobase is A. In some embodiments, the opposite nucleobase is an optionally substituted or protected pseudoisocytosine nucleobase or a tautomer of an optionally substituted or protected pseudoisocytosine nucleobase. In some embodiments, the opposite nucleobase is a pseudoisocytosine nucleobase.

[0167] In some embodiments, a nucleoside, eg, a nucleoside opposite an abasic as described herein (eg, having a structure such as L010, L012, L028, etc.).

[0168] For example, many useful embodiments of modified nucleobases relative to the opposing nucleobase are also , described below. In some embodiments, as described herein (e.g., in various oligonucleotides), the present disclosure provides oligonucleotides comprising a nucleobase on the nucleoside opposite a target nucleoside, such as A, that is or comprises, e.g., C, A, aC, b007U, b001U, b001A, b002U, b001C, b003U, b002C, b004U, b003C, b005U, b002I, b006U, b003I, b008U, b009U, b002A, b003A, b001G, or zdnp. In some embodiments, the nucleobase is C. In some embodiments, the nucleobase is A. In some embodiments, the nucleobase is aC. In some embodiments, the nucleobase is b007U. In some embodiments, the nucleobase is b001U. In some embodiments, the nucleobase is b001A. In some embodiments, the nucleobase is b002U. In some embodiments, the nucleobase is b001C. In some embodiments, the nucleobase is b003U. In some embodiments, the nucleobase is b002C. In some embodiments, the nucleobase is b004U. In some embodiments, the nucleobase is b003C. In some embodiments, the nucleobase is b005U. In some embodiments, the nucleobase is b002I. In some embodiments, the nucleobase is b006U. In some embodiments, the nucleobase is b003I. In some embodiments, the nucleobase is b008U. In some embodiments, the nucleobase is b009U. In some embodiments, the nucleobase is b002A. In some embodiments, the nucleobase is b003A. In some embodiments, the nucleobase is b001G. In some embodiments, the nucleobase is zdnp. In some embodiments, as will be appreciated by those of skill in the art, the nucleobases are protected, e.g., for oligonucleotide synthesis. For example, in some embodiments, the nucleobases are: [ka] wherein R' is as described herein. In some embodiments, R' is -C(O)R. In some embodiments, R' is -C(O)Ph.

[0169] Certain modified nucleobases In some embodiments, BA is or includes ring BA or a tautomer thereof, where ring BA is an optionally substituted 5-20 membered, monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms. In some embodiments, ring BA is or includes an optionally substituted 5-20 membered, monocyclic, bicyclic, or polycyclic ring having 1-10 heteroatoms, where at least one heteroatom is nitrogen. In some embodiments, ring BA is saturated. In some embodiments, ring BA includes one or more unsaturations. In some embodiments, ring BA is partially unsaturated. In some embodiments, ring BA is aromatic.

[0170] In some embodiments, BA is or includes ring BA, where ring BA is an optionally substituted 5-20 membered, monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms. In some embodiments, ring BA is or includes an optionally substituted 5-20 membered, monocyclic, bicyclic, or polycyclic ring having 1-10 heteroatoms, where at least one heteroatom is nitrogen. In some embodiments, ring BA is saturated. In some embodiments, ring BA includes one or more unsaturations. In some embodiments, ring BA is saturated. In some embodiments, ring BA includes one or more unsaturations. In some embodiments, Ring BA is partially unsaturated. In some embodiments, Ring BA is aromatic.

[0171] In some embodiments, BA is or includes ring BA. In some embodiments, BA is ring BA. In some embodiments, BA is or includes a tautomer of ring BA. In some embodiments, BA is a tautomer of ring BA.

[0172] In some embodiments, the structures of the present disclosure contain one or more optionally substituted rings (e.g., ring BA, -Cy-, ring BAA, R, etc., formed together with the R groups). In some embodiments, the ring is an optionally substituted C ring having 0-10 (e.g., 1-10, 1-5, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) heteroatoms. 3~30 , C 3~20 , C 3~15 , C 3~10 , C 3~9 , C 3~8 , C 3~7 , C 3~6 , C 5~50 , C 5~20 , C 5~15 , C 5~10 , C 5~9 , C 5~8 , C 5~7 , C 5~6or 3-30 (e.g., 3-30, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 5-50, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, etc.) membered monocyclic, bicyclic, or polycyclic ring. In some embodiments, the ring is an optionally substituted 3-10 membered monocyclic or bicyclic saturated, partially saturated, or aromatic ring having 0-3 heteroatoms. In some embodiments, the ring is substituted. In some embodiments, the ring is unsubstituted. In some embodiments, the ring is 3, 4, 5, 6, 7, 8, 9, or 10 membered. In some embodiments, the ring is 5, 6, or 7-membered. In some embodiments, the ring is 5-membered. In some embodiments, the ring is 6-membered. In some embodiments, the ring is 7-membered. In some embodiments, the ring is monocyclic. In some embodiments, the ring is bicyclic. In some embodiments, the ring is polycyclic. In some embodiments, the ring is saturated. In some embodiments, the ring contains at least one unsaturation. In some embodiments, the ring is partially unsaturated. In some embodiments, the ring is aromatic. In some embodiments, the ring has 0-5 heteroatoms. In some embodiments, the ring has 1-5 heteroatoms. In some embodiments, the ring has 1 or more heteroatoms. In some embodiments, the ring has 1 heteroatom. In some embodiments, the ring has 2 heteroatoms. In some embodiments, the ring has 3 heteroatoms. In some embodiments, the ring has 4 heteroatoms. In some embodiments, the ring has 5 heteroatoms. In some embodiments, the heteroatom is nitrogen. In some embodiments, the heteroatom is oxygen. In some embodiments, the ring is substituted, for example, with one or more alkyl groups and optionally one or more other substituents as described herein, hi some embodiments, the substituent is methyl.

[0173] In some embodiments, each monocyclic ring unit of a monocyclic, bicyclic, or polycyclic ring (e.g., Ring BA, -Cy-, Ring BAA, R, etc., formed in combination with an R group) of the present disclosure is independently an optionally substituted 5-7 membered, saturated, partially unsaturated, or aromatic ring having 0-5 heteroatoms. In some embodiments, one or more monocyclic units independently contain one or more unsaturations. In some embodiments, one or more monocyclic units are saturated. In some embodiments, one or more monocyclic units are partially saturated. In some embodiments, one or more monocyclic units are aromatic. In some embodiments, one or more monocyclic units independently have 1-5 heteroatoms. In some embodiments, one or more monocyclic units independently have at least one nitrogen atom. In some embodiments, each monocyclic unit is independently 5- or 6-membered. In some embodiments, In some embodiments, the monocyclic unit is 5-membered. In some embodiments, the monocyclic unit is 5-membered and has 1-2 nitrogen atoms. In some embodiments, the monocyclic unit is 6-membered. In some embodiments, the monocyclic unit is 6-membered and has 1-2 nitrogen atoms. The ring and its monocyclic unit are optionally substituted unless otherwise specified.

[0174] While not intending to be limited by any particular theory, the present disclosure recognizes that in some embodiments, the structure of a nucleobase (e.g., BA) can affect interaction with a protein (e.g., an ADAR protein such as ADAR1 or ADAR2). In some embodiments, provided oligonucleotides include a nucleobase that can promote interaction of the oligonucleotide with an enzyme, e.g., ADAR1. In some embodiments, provided oligonucleotides include a nucleobase that can reduce the strength of base pairing (e.g., compared to AT / U or CG). In some embodiments, the present disclosure recognizes that by maintaining and / or enhancing the interaction (e.g., hydrogen bonding) between a protein (e.g., an enzyme such as ADAR1) and a first nucleobase and / or reducing the interaction (e.g., hydrogen bonding) between the first nucleobase and its corresponding nucleobase (e.g., A) on the other strand of the duplex, modification of the corresponding nucleobase by a protein (e.g., an enzyme such as ADAR1) can be significantly improved. In some embodiments, the present disclosure provides oligonucleotides including such first nucleobases (e.g., various embodiments of BA described herein). The exemplary embodiment of such first nucleobase is as described herein.In some embodiments, when the oligonucleotide comprising such first nucleobase is aligned with another nucleic acid in terms of maximum complementarity, the first nucleobase is opposite to A.In some embodiments, such A on the opposite side of the first nucleobase as illustrated in many embodiments of the present disclosure can be effectively modified using the technology of the present disclosure.

[0175] In some embodiments, ring BA is the moiety [ka] wherein each variable is independently as described herein. In some embodiments, ring BA comprises the moiety [ka] wherein each variable is independently as described herein. In some embodiments, ring BA comprises the moiety [ka] wherein each variable is independently as described herein. In some embodiments, ring BA comprises the moiety [ka] (each variable independently as described herein). In some embodiments, X 1 is attached to the sugar. In some embodiments, X 1 is -N(-)- In some embodiments, X 1 is -C(=)-. In some embodiments, X 2 is —C(O)—. In some embodiments, X 3 is —NH—. In some embodiments, X 4 is not —C(O)—. In some embodiments, X 4 is -C(O)-, e.g., as part of the same nucleotide unit (e.g., within the same BA unit (e.g., X 5 In some embodiments, X forms an intramolecular hydrogen bond with a hydrogen bond donor (e.g., —OH, SH, etc.). 4 is -C(=NH)-. In some embodiments, ring BA is the moiety [ka] (each variable independently as described herein). In some embodiments, X 4 In some embodiments, X′ is —C(O)—. 5 ' is -NH-.

[0176] In some embodiments, BA is optionally substituted or protected C or a tautomer thereof. In some embodiments, BA is optionally substituted or optionally protected C. In some embodiments, BA is a tautomer of optionally substituted or optionally protected C. In some embodiments, BA is C; in some embodiments, BA is substituted C. In some embodiments, BA is protected C. In some embodiments, BA is a tautomer of substituted C. In some embodiments, BA is a tautomer of protected C.

[0177] In some embodiments, ring BA has the structure of formula BA-I: [ka] (In the formula, Ring BA is an optionally substituted 5-20 membered monocyclic, bicyclic or polycyclic saturated, partially saturated or aromatic ring having 1-10 heteroatoms; each [ka] are independently a single bond or a double bond; X 1 is -N(-)- or -C(-); X 2 -C(O)-, -C(R B2 )=, or -C(OR B2 )=(wherein, R B2 -L B2 -R') and X 3 is -N(R B3 )- or -N=(wherein R B3 -L B3 -R') and X 4 is -C(R B4)=, -C(-N(R B4 )2)=, -C(R B4 )2-, -C(O)-, or -C(=NR B4 )-(in the formula, each R B4 independently, -L B4 -R B41 or two R on the same atom B4 Combined, =O, =C(-L B4 -R B41 )2, =NL B4 -R B41 or optionally substituted =CH or =NH, wherein each R B41 are independently R')); L B2 , L B3 , and L B4 each of which is independently LB; Each LB independently represents 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- to 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), or -SOR; and Each R is independently -H or C 1~20 Aliphatic, C with 1-10 heteroatoms 1~20 Heteroaliphatic, C 6~20 Aryl, C 6~20 Arylaliphatic, C with 1-10 heteroatoms 6~20an optionally substituted group selected from 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 combine to form a covalent bond, or two or more R groups on the same atom optionally and independently combine with that atom to form an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic ring having, in addition to that atom, 0-10 heteroatoms; or two or more R groups on two or more atoms optionally and independently combine with their intervening atoms to form an optionally substituted 3- to 30-membered monocyclic, bicyclic, or polycyclic ring having from 0 to 10 heteroatoms in addition to the intervening atoms.

[0178] In some embodiments, ring BA (e.g., one of formula BA-I) has the structure of formula BA-Ia: [ka] It has.

[0179] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-Ia, etc.) has the structure of formula BA-Ib: [ka] It has.

[0180] In some embodiments, ring BA (e.g., one of formula BA-I) has the structure of formula BA-II: [ka] (In the formula, X 5 is -C(R B5 )2-, -N(R B5)-, -C(R B5 )=, -C(O)-, or -N=, and each R B5 are independently halogen or -L B5 -R B51 and R B51 is -R', -N(R')2, -OR', or -SR'; L B5 L B and each of the other variables independently as described herein).

[0181] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-Ia, BA-II, etc.) has the structure of formula BA-II-a: [ka] It has.

[0182] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-Ia, BA-Ib, BA-II, BA-II-a, etc.) has the structure of formula BA-II-b: [ka] It has.

[0183] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-II, etc.) has the structure of formula BA-III: [ka] (In the formula, X 6 is -C(R B6 )=, -C(OR B6 )=, -C(R B6 )2-, -C(O)-, or -N=(wherein each R B6 independently, -L B6 -R B61or two R on the same atom B6 Combined, =O, =C(-L B6 -R B61 )2, =NL B6 -R B61 or optionally substituted =CH or =NH, wherein each R B61 are independently R')); L B6 is LB; and each of the other variables independently as described herein).

[0184] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-Ia, BA-II, BA-II-a, BA-III, etc.) has the structure of formula BA-III-a: [ka] It has.

[0185] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-Ia, BA-Ib, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, etc.) has the structure of formula BA-III-b: [ka] It has.

[0186] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-II, etc.) has the structure of formula BA-IV: [ka] (In the formula, Ring BAA is an optionally substituted 5-14 membered monocyclic, bicyclic, or polycyclic ring having 0-5 heteroatoms; and each of the other variables independently as described herein).

[0187] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-Ia, BA-II, BA-II-a, etc.) has the structure of formula BA-IV-a: [ka] It has.

[0188] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-Ia, BA-II, BA-II-a, etc.) has the structure of formula BA-IV-b: [ka] It has.

[0189] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-II, BA-III, BA-IV, etc.) has the structure of formula BA-V: [ka] It has.

[0190] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-Ia, BA-II, BA-II-a, BA-III, BA-III-a, BA-IV, BA-IV-a, BA-V, etc.) has the structure of formula BA-Va: [ka] It has.

[0191] In some embodiments, ring BA (e.g., one of formulas BA-I, BA-Ia, BA-Ib, BA-II, BA-II-a, BA-II-b, BA-III, BA-III-a, BA-III-b, BA-IV, BA-IV-a, BA-IV-b, BA-V, BA-Va, etc.) has the structure of formula BA-Vb: [ka] It has.

[0192] In some embodiments, ring BA has the structure of formula BA-VI: [ka] (In the formula, X 1 ' is -N(-)- or -C(-)=; X 2 ' is -C(O)- or -C(R B2 ')=(In the formula, R B2 '-L B2 '-R') and; each [ka] are independently a single bond or a double bond; X 3 ' is -N(R B3 ')- or -N=(wherein R B3 '-L B3 '-R') and; X 4 ' is -C(R B4 ')=,-C(OR B4 ')=,-C(-N(R B4 ')2)=, -C(R B4 ')2-, -C(O)-, or -C(=NR B4 ')-(In the formula, each R B4 ' is independently -L B4 '-R B41' or two R on the same atom B4 ', =O, =C(-L B4 '-R B41 ')2, =NL B4 '-R B41 ', or optionally substituted =CH or =NH, where each R B41 ' is independently -R')) and; X 5 ' is -N(R B5 ')- or -N=(wherein R B5 '-L B5 '-R') and; X 6 ' is -C(R B6 ')=,-C(OR B6 ')=, -C(R B6 ')2-, -C(O)-, or -N=(wherein each R B6 ' is independently -L B6 '-R B61 ' or two R on the same atom B6 ', =O, =C(-L B6 '-R B61 ')2, =NL B6 '-R B61 ', or optionally substituted =CH or =NH, wherein each R B61 ' is independently R')) and; X 7 ' is -C(R B7 ')=,-C(OR B6 ')=, -C(R B7 ')2-, -C(O)-, -N(R B7 ')-, or -N=(wherein each R B7 ' is independently -L 7 '-R B71 ' or two R on the same atom B7 ', =O, =C(-L 7 '-R B71 ')2, =NL 7 '-R B71 ', or optionally substituted =CH or =NH, wherein each R B71 ' is independently R')) and; L B2 ', L B3 ', L B4 ', L B5 ' and L B6 each of ' is independently LB; and each of the other variables independently as described herein).

[0193] In some embodiments, [ka] is a single bond. In some embodiments, [ka] is a double bond.

[0194] In some embodiments, X 1 is -N(-)-. In some embodiments, X 1 is -C(-)=.

[0195] In some embodiments, X 2 is —C(O)—. In some embodiments, X 2 is -C(R B2 )=. In some embodiments, X 2 is -C(OR B2 )=. In some embodiments, X 2 is -CH=.

[0196] In some embodiments, L B2 is a covalent bond.

[0197] In some embodiments, R B2 is a protecting group, e.g., a hydroxyl protecting group suitable for oligonucleotide synthesis. In some embodiments, R B2 is R'. In some embodiments, R B2is -H.

[0198] In some embodiments, X 3 is -N(R B3 In some embodiments, X 3 is -NH-. In some embodiments, X 3 is -N=.

[0199] In some embodiments, L B3 is a covalent bond.

[0200] In some embodiments, R B3 is a protecting group, e.g., an amino acid protecting group suitable for oligonucleotide synthesis (e.g., Bz). In some embodiments, R B3 is R'. In some embodiments, R B3 is —C(O)R. In some embodiments, R B3 is R. In some embodiments, R B3 is -H.

[0201] In some embodiments, X 4 is -C(R B4 )=. In some embodiments, X 4 is -C(R)=. In some embodiments, X 4 is -CH=. In some embodiments, X 4 is -C(OR B4 )=. In some embodiments, X 4 is -C(-N(R B4 )2)=. In some embodiments, X 4 is -C(-NHR B4 )=. In some embodiments, X 4 is -C(=NHR')=. In some embodiments, X 4 is -C(=NHR')=. In some embodiments, X 4 is -C(=NH2)=. In some embodiments, X 4 is -C(-NHC(O)R)=. In some embodiments, X4 is -C(R B4 )2-. In some embodiments, X 4 is -CH-. In some embodiments, X 4 is —C(O)—. In some embodiments, X 4 is —C(O)—, where O forms an intramolecular hydrogen bond. In some embodiments, O is an X of the same BA. 5 In some embodiments, X 4 is -C(=NR B4 In some embodiments, X 4 is -C((=NR B4 )-(wherein N forms an intramolecular hydrogen bond). In some embodiments, N is an X of the same BA. 5 forms hydrogen bonds with the hydrogen bond donor.

[0202] In some embodiments, R B4 -L B4 -R B41 In some embodiments, two R on the same atom B4 Combined, =O, =C(-L B4 -R B41 )2, =NL B4 -R B41 or optionally substituted =CH2 or =NH.

[0203] In some embodiments, two R on the same atom B4 together to form =O. In some embodiments, two R B4 Combined, =C(-L B4 -R B41 )2. In some embodiments, ═C(-L B4 -R B41 )2 is =CH-L B4 -R B41 In some embodiments, ═C(-L B4 -R B41 )2 is =CHR'. In some embodiments, =C(-L B4 -RB41 )2 is =CHR. In some embodiments, two R on the same atom B4 Together, =NL B4 -R B41 In some embodiments, =NL B4 -R B41 is ═NR. In some embodiments, two R B4 together to form =CH2. In some embodiments, two R B4 together to form =NH. In some embodiments, the group formed is a suitable protecting group for oligonucleotide synthesis, for example, an amino protecting group.

[0204] In some embodiments, X 4 is -C(-N=C(-L B4 -R B41 )2)=. In some embodiments, X 4 is -C(-N=CH-L B4 -R B41 )=. In some embodiments, X 4 is -C(-N=CH-N(CH3)2)=

[0205] In some embodiments, X 4 (e.g., -C(=NR)-, =C(R)-, etc.) R can be, for example, X 5 optionally combined with another R to form a ring as described herein.

[0206] In some embodiments, R B4 is R'. In some embodiments, R B4 is R. In some embodiments, R B4 is -H. In some embodiments, R B4 is a protecting group, e.g., an amino or hydroxyl protecting group suitable for oligonucleotide synthesis. In some embodiments, R B4 is R'. In some embodiments, R B4is -CH2CH2-(4-nitrophenyl).

[0207] In some embodiments, L B4 is a covalent bond. In some embodiments, L B4 is not a covalent bond. In some embodiments, at least one methylene unit is replaced with -C(O)-. In some embodiments, at least one methylene unit is replaced with -C(O)N(R')-. In some embodiments, at least one methylene unit is replaced with -N(R')-. In some embodiments, at least one methylene unit is replaced with -NH-. In some embodiments, L B4 is or contains an optionally substituted -N=CH-.

[0208] In some embodiments, R B41 is R'. In some embodiments, R B41 is —H. In some embodiments, R B41 is R. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl.

[0209] In some embodiments, X 5 is -C(R B5 )2-. In some embodiments, X 5 -ChR B5 In some embodiments, X 5 is -CH-. In some embodiments, X 5 is -N(R B5 In some embodiments, X 5 is -NH-. In some embodiments, X 5 is -C(R B5 )=. In some embodiments, X 5 is -C(R)=. In some embodiments, X 5 is -CH=. In some embodiments, X 5In some embodiments, X 5 is -C(O)-.

[0210] In some embodiments, R B5 is halogen. In some embodiments, R B5 -L B5 -R B51 In some embodiments, R B5 -L B5 -R B 51 (In the formula, R B51 is R', -NHR', -OH, or -SH). In some embodiments, R B5 -L B5 -R B51 (In the formula, R B51 is -NHR', -OH, or -SH). In some embodiments, R B5 -L B5 -R B51 (In the formula, R B51 is —NH, —OH, or —SH). In some embodiments, R B5 is -C(O)-R B51 In some embodiments, R B5 is R'. In some embodiments, R B5 is R. In some embodiments, R B5 is —H. In some embodiments, R B5 is —OH. In some embodiments, R B5 is -CHOH.

[0211] In some embodiments, X 4 When is -C(O)-, X 5 is -C(R B5 )2-, -C(R B5 )=, or -N(R B5 )-(wherein, R B5 -L B5 -R B51 and R B51is —NHR′, —OH, or —SH). In some embodiments, X 4 is —C(O)— and R B51 is X 4 is or contains a hydrogen bond donor that forms a hydrogen bond with O of

[0212] In some embodiments, L B5 is a covalent bond. In some embodiments, L B5 is or includes -C(O)-. In some embodiments, L B5 In some embodiments, L B5 is or includes -OC(O)-. In some embodiments, L B5 is or contains —CH2OC(O)—.

[0213] In some embodiments, R 51 is -R'. In some embodiments, R 51 is -R. In some embodiments, R 51 is —H. In some embodiments, R 51 is -N(R'). In some embodiments, R 51 is -NHR'. In some embodiments, R 51 is -NHR. In some embodiments, R 51 is -NH. In some embodiments, R 51 is -OR'. In some embodiments, R 51 is -OR. In some embodiments, R 51 is —OH. In some embodiments, R 51 is -SR'. In some embodiments, R 51 is -SR. In some embodiments, R 51 is -SH. In some embodiments, R is benzyl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is methyl.

[0214] In some embodiments, R B5 is -C(O)-R B51 In some embodiments, R B5 is —C(O)NHCHPh. In some embodiments, R B5 is —C(O)NHPh. In some embodiments, R B5 is —C(O)NHCH. In some embodiments, R B5 is -OC(O)-R B51 In some embodiments, R B5 is -OC(O)-R. In some embodiments, R B5 is -OC(O)CH3.

[0215] In some embodiments, X 5 is X 1 and ring BA is five-membered.

[0216] In some embodiments, X 6 is -C(R B6 )=. In some embodiments, X 6 is -CH=. In some embodiments, X 6 is -C(OR B6 )=. In some embodiments, X 6 is -C(R B6 )2-. In some embodiments, X 6 is -CH-. In some embodiments, X 6 is —C(O)—. In some embodiments, X 6 is -N=.

[0217] In some embodiments, R B6 -L B6 -R B61 In some embodiments, two R on the same atom B6 Combined, =O, =C(-L B6 -R B61 )2, = NL B6-R B61 or optionally substituted =CH or =NH. In some embodiments, two R B6 and combine to form =O. In some embodiments, L B6 is a covalent bond. In some embodiments, R B6 is R. In some embodiments, R B6 is -H.

[0218] In some embodiments, R B6 is a protecting group, e.g., an amino or hydroxyl protecting group suitable for oligonucleotide synthesis. In some embodiments, R B6 is R. In some embodiments,

[0219] In some embodiments, L B6 is a covalent bond. In some embodiments, L B6 is an optionally substituted C 1~10 In some embodiments, L is alkylene. B6 is -CHCH-. In some embodiments, R B6 is -CH2CH2-(4-nitrophenyl).

[0220] In some embodiments, R B61 is R'. In some embodiments, R B61 is R. In some embodiments, R B61 is -H.

[0221] In some embodiments, ring BAA is 5-membered. In some embodiments, ring BAA is 5-membered. In some embodiments, ring BAA has 1 heteroatom. In some embodiments, ring BAA has 2 heteroatoms. In some embodiments, the heteroatom is nitrogen. In some embodiments, the heteroatom is oxygen.

[0222] In some embodiments, X 1In some embodiments, X' is -N(-)-. 1 ' is -C(-)=.

[0223] In some embodiments, X 2 In some embodiments, X′ is —C(O)—. 2 ' is -C(R B2 In some embodiments, X 2 ' is -CH=.

[0224] In some embodiments, L B2 ' is a covalent bond.

[0225] In some embodiments, R B2 In some embodiments, R B2 ' is R. In some embodiments, R B2 In some embodiments, X′ is —H. 2 ' is -CH=.

[0226] In some embodiments, X 3 ' is -N(R B3 In some embodiments, X 3 In some embodiments, X′ is —N(R′)—. 3 In some embodiments, X′ is —NH—. 3 ' is -N=.

[0227] In some embodiments, L B3 ' is a covalent bond.

[0228] In some embodiments, R B3 In some embodiments, R B3 ' is R. In some embodiments, R B3 ' is -H.

[0229] In some embodiments, X 4 ' is -C(R B4In some embodiments, X 4 ' is -C(OR B4 In some embodiments, X 4 ' is -C(-N(R B4 In some embodiments, X 4 ' is -C(-NHR B4 In some embodiments, X 4 In some embodiments, X′ is —C(—NH)═. 4 In some embodiments, So, X 4 In some embodiments, X′ is —C(—NHC(O)R)═. 4 ' is -C(R B4 In some embodiments, X 4 In some embodiments, X′ is —C(O)—. 4 ' is -C(=NR B4 ')-.

[0230] In some embodiments, R B4 '-L B4 '-R B41 In some embodiments, two R's on the same atom B4 ', =O, =C(-L B4 '-R B41 ')2, =NL B4 '-R B41 In some embodiments, two R B4 In some embodiments, two R's on the same atom form =O. B4 ' and =C(-L B4 '-R B41 In some embodiments, two R's on the same atom form B4 ' and =NL B4 '-R B41 In some embodiments, two R's on the same atom form a B4In some embodiments, two R's on the same atom form =CH2. B4 ' together to form =NH. In some embodiments, the group formed is a suitable protecting group for oligonucleotide synthesis, for example, an amino protecting group.

[0231] In some embodiments, X 4 ' is -C(-N=C(-L B4 '-R B41 In some embodiments, X 4 ' is -C(-N=CH-L B4 '-R B41 In some embodiments, X 4 ' is -C(-N=CH-N(CH3)2)=.

[0232] In some embodiments, R B4 In some embodiments, R B4 ' is R. In some embodiments, R B4 ' is -H.

[0233] In some embodiments, R B4 In some embodiments, R ' is a protecting group, e.g., an amino or hydroxyl protecting group suitable for oligonucleotide synthesis. B4 In some embodiments, R B4 ' is -CH2CH2-(4-nitrophenyl).

[0234] In some embodiments, L B4 In some embodiments, L′ is a covalent bond. B4 ' is an optionally substituted C 1~10 In some embodiments, L is alkylene. B4In some embodiments, R' is -CH2CH2-. In some embodiments, at least one methylene unit is replaced with -N(R')-. In some embodiments, R' is R. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is methyl. In some embodiments, R is -H.

[0235] In some embodiments, R B41 In some embodiments, R B41 ' is R. In some embodiments, R B41 ' is -H.

[0236] In some embodiments, X 5 ' is -N(R B5 In some embodiments, X 5 In some embodiments, X′ is —NH—. 5 ' is -N=.

[0237] In some embodiments, L B5 ' is a covalent bond.

[0238] In some embodiments, R B5 In some embodiments, R B5 ' is R. In some embodiments, R B5 ' is -H.

[0239] In some embodiments, X 6 ' is -C(R B6 In some embodiments, X 6 In some embodiments, X′ is —CH═. 6 ' is -C(OR B6 In some embodiments, X 6 ' is -C(R B6 In some embodiments, X 6In some embodiments, X′ is —C(O)—. 6 ' is -N=.

[0240] In some embodiments, R B6 '-L B6 '-R B61 In some embodiments, two R's on the same atom B6 ', =O, =C(-L B6 '-R B61 ')2, =NL B6 '-R B61 In some embodiments, two R B6 ' together to form =O.

[0241] In some embodiments, L B6 In some embodiments, L′ is a covalent bond. B6 ' is an optionally substituted C 1~10 In some embodiments, L is alkylene. B6 ' is -CH2CH2-.

[0242] In some embodiments, R B6 In some embodiments, R B6 ' is R. In some embodiments, R B6 In some embodiments, R B6 In some embodiments, R ' is a protecting group, e.g., an amino or hydroxyl protecting group suitable for oligonucleotide synthesis. B6 In some embodiments, R B6 ' is -CH2CH2-(4-nitrophenyl).

[0243] In some embodiments, R B61 In some embodiments, R B61 ' is R. In some embodiments, R B61 ' is -H.

[0244] In some embodiments, X 7 ' is -C(R B7 In some embodiments, X 7 In some embodiments, X′ is —CH═. 7 ' is -C(OR B7 In some embodiments, X 7 ' is -C(R B7 In some embodiments, X 7 In some embodiments, X′ is —C(O)—. 7 ' is -N(R B7 In some embodiments, X 7 In some embodiments, X′ is —NH—. 7 ' is -N=.

[0245] In some embodiments, R B7 '-L 7 '-R B71 In some embodiments, two R's on the same atom B7 ', =O, =C(-L 7 '-R B71 ')2, =NL 7 '-R B71 In some embodiments, two R B7 ' together to form =O. In some embodiments, L 7 In some embodiments, R B7 ' is R. In some embodiments, R B7 ' is -H.

[0246] In some embodiments, R B71 In some embodiments, R B71 ' is R. In some embodiments, R B71 ' is -H.

[0247] In some embodiments, L Bis a covalent bond. In some embodiments, L B is an optionally substituted divalent C 1~10 is a saturated or partially unsaturated aliphatic chain in which one or more methylene units are optionally and independently replaced with -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-. In some embodiments, L B is an optionally substituted divalent C having 1 to 6 heteroatoms 1~10 a saturated or partially unsaturated heteroaliphatic chain, wherein one or more methylene units are optionally and independently selected from -Cy-, -O-, -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R In some embodiments, at least one methylene unit is replaced. In some embodiments, L B is an optionally substituted C 1~10 In some embodiments, L is alkylene. B is -CH2CH2-. In some embodiments, at least one methylene unit is replaced with -C(O)-. In some embodiments, at least one methylene unit is replaced with -C(O)N(R')-. In some embodiments, at least one methylene unit is replaced with -N(R')-. In some embodiments, at least one methylene unit is replaced with -NH-. In some embodiments, at least one methylene unit is replaced with -Cy-. In some embodiments, L B is or includes an optionally substituted -N=CH-. In some embodiments, L B is or includes -C(O)-. In some embodiments, L BIn some embodiments, L B is or includes -OC(O)-. In some embodiments, L B is or contains —CH2OC(O)—.

[0248] In some embodiments, each -Cy- is independently an optionally substituted 3- to 20-membered monocyclic, bicyclic, or polycyclic saturated, partially saturated, or aromatic ring having 0-10 heteroatoms. Suitable monocyclic units of -Cy- are described herein. In some embodiments, -Cy- is monocyclic. In some embodiments, -Cy- is bicyclic. In some embodiments, -Cy- is polycyclic. In some embodiments, -Cy- is an optionally substituted divalent 3- to 10-membered monocyclic saturated or partially unsaturated ring having 0-5 heteroatoms. In some embodiments, -Cy- is an optionally substituted divalent 5- to 10-membered aromatic ring having 0-5 heteroatoms. In some embodiments, -Cy- is optionally substituted phenylene. In some embodiments, -Cy- is phenylene.

[0249] In some embodiments, R' is R. In some embodiments, R' is -C(O)R. In some embodiments, R' is -C(O)OR. In some embodiments, R' is -C(O)N(R). In some embodiments, R' is -S0R.

[0250] In some embodiments, R' in the various structures is a protecting group (e.g., amino, hydroxyl, etc.), e.g., one suitable for oligonucleotide synthesis. In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is 4-nitrophenyl. In some embodiments, R is -CHCH-(4-nitrophenyl). In some embodiments, R' is -C(O)NPh.

[0251] In some embodiments, each R is independently —H, or C 1~20 Aliphatic, C with 1-10 heteroatoms 1~20 Heteroaliphatic, C 6~30 Aryl, C 6~30 Arylaliphatic, C with 1-10 heteroatoms 6~30 An optionally substituted group selected from arylheteroaliphatic, 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms. In some embodiments, two R groups optionally and independently combine to form a covalent bond. In some embodiments, two or more R groups on the same atom optionally and independently combine 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. In some embodiments, two groups on the same atom optionally and independently combine 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. Forms a 20-membered monocyclic, bicyclic, or polycyclic ring. In some embodiments, two or more R groups on two or more atoms optionally and independently combine with their intervening atoms to form an optionally substituted 3- to 30-membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to the intervening atoms. In some embodiments, two groups on two or more atoms optionally and independently combine with their intervening atoms to form an optionally substituted 3- to 30-membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to the intervening atoms. In some embodiments, the ring formed is monocyclic. In some embodiments, the ring formed is bicyclic. In some embodiments, the ring formed is polycyclic. In some embodiments, each monocyclic ring unit is independently 3 to 10 (e.g., 3 to 8, 3 to 7, 3 to 6, 5 to 10, 5 to 8, 5 to 7, 5 to 6, 3, 4, 5, 6, 7, 8, 9, or 10) members, is independently saturated, partially saturated, or aromatic, and independently has 0 to 5 heteroatoms. In some embodiments, the ring is saturated. In some embodiments, the ring is partially saturated. In some embodiments, the ring is aromatic. In some embodiments, the ring formed has 1 to 5 heteroatoms. In some embodiments, the ring formed has 1 heteroatom. In some embodiments, the ring formed has 2 heteroatoms. In some embodiments, the heteroatom is nitrogen. In some embodiments, the heteroatom is oxygen.

[0252] In some embodiments, R is —H.

[0253] In some embodiments, R is optionally substituted C 1~20 , C 1~15 , C 1~10 , C 1~8 , C 1~6 , C 1~5 , C 1~4 , C 1~3 , or C 1~2In some embodiments, R is an optionally substituted alkyl. In some embodiments, R is an optionally substituted C 1~6 In some embodiments, R is an optionally substituted alkyl. In some embodiments, R is an optionally substituted methyl. In some embodiments, R is an optionally substituted alicyclic. In some embodiments, R is an optionally substituted cycloalkyl.

[0254] In some embodiments, R is an optionally substituted C 1~20 It is heteroaliphatic.

[0255] In some embodiments, R is optionally substituted C 6~20 In some embodiments, R is aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl.

[0256] In some embodiments, R is optionally substituted C 6~20 In some embodiments, R is an optionally substituted C 6~20 In some embodiments, R is arylalkyl. In some embodiments, R is benzyl. In some embodiments, R is an optionally substituted C alkyl group having 1-10 heteroatoms. 6~20 It is arylheteroaliphatic.

[0257] In some embodiments, R is an optionally substituted 5-20 membered heteroaryl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5 membered heteroaryl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 6 membered heteroaryl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 3-20 membered heterocyclyl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 3-10 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 3-10 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 3-10 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, the heterocyclyl is a 5- to 6-membered heterocyclyl. In some embodiments, the heterocyclyl is saturated. In some embodiments, the heterocyclyl is partially saturated.

[0258] In some embodiments, heteroatoms are selected from boron, nitrogen, oxygen, sulfur, silicon, and phosphorus. In some embodiments, heteroatoms are selected from nitrogen, oxygen, sulfur, and silicon. In some embodiments, heteroatoms are selected from nitrogen, oxygen, and sulfur. In some embodiments, heteroatoms are nitrogen. In some embodiments, heteroatoms are oxygen. In some embodiments, heteroatoms are sulfur.

[0259] As will be appreciated by those skilled in the art, the embodiments described with respect to variables can be readily combined to provide a variety of structures. The skilled artisan will also appreciate that the embodiments described with respect to variables can be easily combined to provide a variety of structures. B2 , R B3 , R B4 , R B5 , R B6 , R B2 ', R B3 ', R B4 ', R B5 ', R B6 ' etc.; L B2 , L B3 , L B4 , LB5 , L B6 , L B2 ', L B3 ', L B4 ', L B5 ', L B6 It is understood that other variables can be readily utilized for embodiments of LB, such as those relating to "." Exemplary embodiments and combinations thereof include, but are not limited to, the structures illustrated herein. Certain specific examples are described below.

[0260] For example, in some embodiments, ring BA is optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is: [ka] is.

[0261] In some embodiments, X 4 is -C(O)- and X 4 The O in -C(O)- is R 5 -H in, for example, -NHR', R 5 In some embodiments, X may form a hydrogen bond with the -OH or -SH of X'. 4 is -C(O)- and X 5 is -C(R 5 )=. In some embodiments, R 5 In some embodiments, R 5 -L B5 In some embodiments, L B5is an optionally substituted —CH—. In some embodiments, a methylene unit is replaced with —C(O)—. In some embodiments, L B5 is -C(O)-. In some embodiments, R' is an optionally substituted methyl. In some embodiments, R' is -CHPh. In some embodiments, R' is an optionally substituted phenyl. In some embodiments, R' is phenyl. In some embodiments, R' is an optionally substituted C 1~6 In some embodiments, R' is an optionally substituted C 1~6 In some embodiments, R' is an optionally substituted methyl. In some embodiments, R' is methyl. In some embodiments, ring BA is an optionally protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally protected [ka] In some embodiments, ring BA is: [ka] is.

[0262] In some embodiments, X 1 is -C(-)=X 4 is =C(-N(R B4 )2)-. In some embodiments, two R groups on the same atom, e.g., a nitrogen atom, combine to form an optionally substituted =CH2 or =NH. In some embodiments, two R groups on the same atom, e.g., a nitrogen atom, combine to form an optionally substituted =C(-L B4 -R)2, =NL B4 In some embodiments, the group formed is =CHN(R). In some embodiments, the group formed is =CHN(CH). In some embodiments, X 4 is =C(-N=CHN(CH3)2)-. In some embodiments, -N(R B4 )2 is -NR B4 In some embodiments, R B4 is -NHC(O)R. In some embodiments, ring BA is optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is: [ka] is.

[0263] In some embodiments, X1 is -N(-)- and X 2 is -C(O)- and X 3 is -N(R B3 In some embodiments, X 1 is -N(-)- and X 2 is -C(O)- and X 3 is -N(R B3 )- and X 4 is -C(R B4 )=. In some embodiments, X 1 is -N(-)- and X 2 is -C(O)- and X 3 is -N(R B3 )- and X 4 is -C(R B4 )= and X 5 is -C(R B5 In some embodiments, ring BA is optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] is.

[0264] In some embodiments, X 3 is -N(R')-. In some embodiments, R' is -C(O)R. In some embodiments, X 4 is -C(R B4 )2-. In some embodiments, R B4 is -R. In some embodiments, R B4 is —H. In some embodiments, X 4 is -CH-. In some embodiments, X 5 is -C(R B5 )2-. In some embodiments, RB5 is -R. In some embodiments, R B5 is —H. In some embodiments, X 5 In some embodiments, ring BA is an optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is: [ka] is.

[0265] In some embodiments, X 4 is -C(R B4 )=. In some embodiments, X 4 is -CH=. In some embodiments, X 5 is -C(R B5 )=. In some embodiments, X 5 is -CH=. In some embodiments, ring BA is optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] is.

[0266] In some embodiments, X 4 is -C(R B4 )2-. In some embodiments, X 4 is -CH-. In some embodiments, X 5 is -C(R B5 )=. In some embodiments, X 5 is -CH=. In some embodiments, ring BA is optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is: [ka] is.

[0267] In some embodiments, X 1 is -N(-)- and X 2 is -C(O)- and X 3 is -N(R B3 )- and X 4 is -C(R B4 )= and X 5 is -C(R B5 )= and X 6is —C(O)—. In some embodiments, R B3 , R B4 and R B5 Each of is independently R. In some embodiments, R B3 is —H. In some embodiments, R B4 is —H. In some embodiments, R B5 is -H. In some embodiments, BA is optionally substituted or protected [ka] In some embodiments, BA is: [ka] is.

[0268] In some embodiments, X 1 is -N(-)- and X 2 is -C(O)- and X 3 is -N(R B3 In some embodiments, X 4 is -C(R B4 )2-(wherein two R B4 together to form =O), or =C(-L B4 -R B41 )2, =NL B4 -R B41 In some embodiments, X 4 is -C(=NR B4 In some embodiments, X 5 is -C(R B5 )=. In some embodiments, R B41 or R B4 and R B5 is R and taken together with their intervening atoms form an optionally substituted ring as described herein. In some embodiments, ring BA is an optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, X 1 is -N(-)- and X 2 is -C(O)- and X 3 In some embodiments, X 4 is -C(-N(R B4 )2)=. In some embodiments, X 4 is -C(-NHR B4 )=. In some embodiments, X 5 is -C(R B5 )=. In some embodiments, one R B4 and R B5 are combined to form an optionally substituted ring as described herein. In some embodiments, the ring formed is an optionally substituted 5-membered ring having a nitrogen atom. In some embodiments, ring BA is an optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] In some embodiments, ring BA is optionally substituted or protected [ka] In some embodiments, ring BA is: [ka] is.

[0269] In some embodiments, ring BA has the structure of formula BA-IV or BA-V. 1 is -N(-)- and X 2 is -C(O)- and X 3 In some embodiments, X1 is -N(-)- and X 2 is -C(O)- and X 3 is -N = and X 6 is -C(R B6 )=. In some embodiments, ring BAA is 5-6 membered. In some embodiments, ring BAA is monocyclic. In some embodiments, ring BAA is partially unsaturated. In some embodiments, ring BAA is aromatic. In some embodiments, ring BAA has 0-2 heteroatoms. In some embodiments, ring BAA has 1-2 heteroatoms. In some embodiments, ring BAA has 1 heteroatom. In some embodiments, ring BAA has 2 heteroatoms. In some embodiments, the heteroatom is nitrogen. In some embodiments, the heteroatom is oxygen. In some embodiments, ring BA is optionally substituted or protected. [ka] In some embodiments, ring BA is: [ka] is.

[0270] In some embodiments, ring BA is an optionally substituted 5-membered ring. 1 is X 5 In some embodiments, X 4 and X 5 Each of X is independently -CH=. 1 is -N(-)- and X 2 is -C(O)- and X 3 is -NH- and X 4 is -CH= and X 5 is -CH=. In some embodiments, ring BA is optionally substituted or protected ...

Claims

【Request Item 1】 【Chemistry 1】 An oligonucleotide comprising a nucleic acid base having the structure, or a tautomer thereof, wherein the oligonucleotide has a nucleic acid base of length 10 to 200, and the oligonucleotide comprises one or more 2'-F modified sugars and 2'-OR modified sugars in which each R is independently and arbitrarily substituted with a C1-6 aliphatic group.

2. The oligonucleotide according to claim 1, wherein the oligonucleotide comprises one or more phosphorothioate bonds.

3. The oligonucleotide according to claim 1 or 2, wherein the oligonucleotide comprises one or more negatively charged bonds.

4. The oligonucleotide according to claim 1 or 2, wherein each oligonucleotide independently comprises one or more internucleotide bonds including a guanidine moiety.

5. The oligonucleotide is 【Chemistry 2】 The oligonucleotide according to claim 1 or 2, having one or more internucleotide bonds having the structure.

6. The oligonucleotide according to claim 2, wherein each chiral nucleotide bond is independently chiral-controlled.

7. The oligonucleotide according to claim 4, wherein each chiral nucleotide bond is independently chiral-controlled.

8. The oligonucleotide according to claim 5, wherein each chiral nucleotide bond is independently chiral-controlled.

9. The oligonucleotide according to claim 5, wherein each chiral nucleotide bond has a diastereopurity of at least about 90%.

10. The oligonucleotide according to claim 1, wherein the oligonucleotide comprises one or more natural DNA sugars.

11. The oligonucleotide according to claim 1, wherein the oligonucleotide comprises one or more 2'-OMe modified sugars.

12. The oligonucleotide according to claim 1, wherein the oligonucleotide comprises one or more 2'-OCH2CH2OCH3-modified sugars.

13. The oligonucleotide according to claim 1, wherein the oligonucleotide has nucleic acid bases of length 25 to 50.

14. The oligonucleotide according to any one of claims 1 to 13, wherein the oligonucleotide includes a targeting portion.

15. The oligonucleotide according to claim 14, wherein the portion is N-acetylgalactosamine (GalNAc) or a derivative thereof, or comprises the same.

16. A pharmaceutical composition comprising or for delivering an oligonucleotide according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

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