Double-stranded oligonucleotide compositions for RNA interference and methods relating thereto

Abstract

The present disclosure provides double-stranded oligonucleotides, compositions, and methods related thereto. The present disclosure embraces the recognition that structural elements of double-stranded oligonucleotides, such as base sequence, chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide linkages) or their patterns, and / or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotide linkages)) and / or their patterns, can significantly affect oligonucleotide properties and activity, such as RNA interference (RNAi) activity, Ago2 loading, thermal stability, in vivo stability, tissue delivery, and intracellular delivery. The present disclosure also provides methods for treating diseases, such as liver disease, central nervous system (CNS) disease, etc., using the provided double-stranded oligonucleotide compositions, for example, in RNA interference.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 487,823, filed March 1, 2023, U.S. Provisional Application No. 63 / 491,729, filed March 22, 2023, U.S. Provisional Application No. 63 / 581,614, filed September 8, 2023, and U.S. Provisional Application No. 63 / 585,133, filed September 25, 2023, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Gene targeting oligonucleotides are useful in various applications, such as therapeutics, diagnostics, research, and nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) in such applications can be limited, for example, by their susceptibility to endonucleases and exonucleases. Therefore, various synthetic counterparts have been developed to avoid these drawbacks. These include synthetic oligonucleotides containing chemical modifications, such as base modifications, sugar modifications, and backbone modifications. However, there remains a need in the art for double-stranded (ds) oligonucleotides with improved properties for use in the applications described above. Summary of the Invention

[0003] The present disclosure is directed, in part, to the recognition that controlling the structural elements of a double-stranded (ds) oligonucleotide can significantly affect the properties and / or activity of the ds oligonucleotide. In certain embodiments, such structural elements include one or more of: (1) chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide linkages) and their patterns; and (2) alterations in stereochemistry (e.g., the stereochemistry of backbone chiral internucleotide linkages) and their patterns. One or more of such structural elements can, in certain embodiments, be independently present in one or both oligonucleotides of a ds oligonucleotide. In certain embodiments, properties and / or activities influenced by such structural elements include, but are not limited to, contributions to or direction of reduction in the expression, activity, or levels of a gene or its gene product, e.g., mediated by RNA interference (RNAi interference), RNase H-mediated knockdown, steric hindrance of translation, etc. Furthermore, in certain embodiments, properties and / or activities influenced by such structural elements include, but are not limited to, contributions to Ago2 loading, thermal stability, in vivo stability, tissue delivery, and intracellular delivery, among others.

[0004] In certain embodiments, the present disclosure demonstrates that compositions comprising ds oligonucleotides with controlled structural elements (e.g., dsRNAi oligonucleotides, also referred to as dsRNAi agents) provide unexpected properties and / or activities.

[0005] In certain embodiments, the present disclosure encompasses the recognition that stereochemistry, for example, the stereochemistry of the main chain chiral center, can unexpectedly maintain or improve the properties of ds oligonucleotides.For example, but not limited to, the present disclosure relates in part to ds oligonucleotides that include a guide strand that includes a main chain phosphorylguanidine chiral center in the Sp configuration.

[0006] In certain embodiments, the guide strand comprises a backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars.

[0007] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide.

[0008] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modification at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the eighth (+8) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the ninth (+9) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the tenth (+10) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the twelfth (+12) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the sixteenth (+16) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 17th (+17) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 18th (+18) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 19th (+19) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 20th (+20) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 21st (+21) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 22nd (+22) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 23rd (+23) 3'-terminal nucleotide.

[0009] In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, such as a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, such as a 5'-(R)-methyl or a 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.

[0010] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the sixth (+6) nucleotide.

[0011] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a homo-DNA modification of the 5'-terminal (+1) nucleotide, the 6th (+6) nucleotide, the 9th (+9) nucleotide, the 10th (+10) nucleotide, the 12th (+12) nucleotide, the 16th (+16) nucleotide, the 17th (+17) nucleotide, the 18th (+18) nucleotide, the 19th (+19) nucleotide, the 20th (+20) nucleotide, the 21st (+21) nucleotide, the 22nd (+22), and / or the 23rd (+23) 3'-terminal nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a homo-DNA modification of the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the ninth (+9) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the tenth (+10) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the twelfth (+12) nucleotide.In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 16th (+16) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 17th (+17) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 18th (+18) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 19th (+19) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a homo-DNA modification of the 20th (+20) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a homo-DNA modification of the 21st (+21) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a homo-DNA modification of the 22nd (+22) nucleotide.In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains a homo-DNA modification of the 23rd (+23) 3'-terminal nucleotide.

[0012] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, that contain a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, which further comprise: (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0013] In certain embodiments, ds oligonucleotides comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise an LNA modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise an LNA modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise an LNA modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the sixth (+6) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises an LNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises an LNA modification of the eighth (+8) nucleotide. In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, which further (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral center internucleotide linkages in the Rp or Sp configuration occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0014] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain 2'-O-alkyl modifications of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain 2'-O-alkyl modifications of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains a 2'-O-alkyl modification of the fifth (+5) nucleotide.In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the seventh (+7) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the eighth (+8) nucleotide.

[0015] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, that contain a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, which further comprise: (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral center internucleotide linkages in the Rp or Sp configuration occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0016] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification at the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification at the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the sixth (+6) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a DNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a DNA modification of the eighth (+8) nucleotide.

[0017] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, that contain a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, which further comprise: (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0018] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the sixth (+6) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a GNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a GNA modification of the eighth (+8) nucleotide.

[0019] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, that contain a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, which further comprise: (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0020] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the sixth (+6) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the eighth (+8) nucleotide.

[0021] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, is (1) a guide strand containing backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand containing backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) A guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to a main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand containing a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, which contains one or more main chain chiral centers in the Rp or Sp configuration; (9) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide directly downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide directly downstream, i.e., in the 3' direction; and (11) further comprising one or more passenger strands in combination with one or more of the aforementioned guide strands, the passenger strands comprising main-chain phosphorothioate chiral centers in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by Rp, Sp, or sterically random non-negatively charged internucleotide bonds, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; and (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0022] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or steric random non-negatively charged internucleotide bond. In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide bonds incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide bond. In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide bond are Sp non-negatively charged internucleotide bond. In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide bond are steric random non-negatively charged internucleotide bond.

[0023] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the eighth (+8) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the ninth (+9) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the tenth (+10) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the twelfth (+12) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 16th (+16) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 17th (+17) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 18th (+18) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 19th (+19) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 20th (+20) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 21st (+21) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 22nd (+22) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 23rd (+23) 3'-terminal nucleotide.

[0024] In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, such as a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, such as a 5'-(R)-methyl or a 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.

[0025] In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain one or more 2'-F modifications. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain 2'-F modifications of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain 2'-F modifications of the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a 2'-F modification of the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a 2'-F modification of the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a 2'-F modification of the fifth (+5) nucleotide.In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains a 2'-F modification of the sixth (+6) nucleotide.

[0026] In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprise one or more homo-DNA modifications. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprise a homo-DNA modification of the 5'-terminal (+1) nucleotide, the 6th (+6) nucleotide, the 9th (+9) nucleotide, the 10th (+10) nucleotide, the 12th (+12) nucleotide, the 16th (+16) nucleotide, the 17th (+17) nucleotide, the 18th (+18) nucleotide, the 19th (+19) nucleotide, the 20th (+20) nucleotide, the 21st (+21) nucleotide, the 22nd (+22), and / or the 23rd (+23) 3'-terminal nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a homo-DNA modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a homo-DNA modification of the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a homo-DNA modification of the ninth (+9) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a homo-DNA modification of the tenth (+10) nucleotide.In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 12th (+12) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 16th (+16) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 17th (+17) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 18th (+18) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a homo-DNA modification of the 19th (+19) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a homo-DNA modification of the 20th (+20) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a homo-DNA modification of the 21st (+21) nucleotide.In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a homo-DNA modification of the 22nd (+22) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a homo-DNA modification of the 23rd (+23) 3'-terminal nucleotide.

[0027] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 5'-alkyl modification of the third (+3) nucleotide and / or the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 5'-methyl modification of the third (+3) nucleotide and / or the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 5'-alkyl modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a 5'-methyl modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a 5'-alkyl modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a 5'-methyl modification of the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains 5'-alkyl modifications of the third (+3) nucleotide and the fourth (+4) nucleotide.In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains 5'-methyl modifications of the third (+3) nucleotide and the fourth (+4) nucleotide.

[0028] In certain embodiments, ds oligonucleotides comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise one or more LNA modifications. In certain embodiments, ds oligonucleotides comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise one or more LNA modifications. In certain embodiments, ds oligonucleotides comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise an LNA modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the fourth (+4) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises an LNA modification of the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises an LNA modification of the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises an LNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises an LNA modification of the eighth (+8) nucleotide.

[0029] In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain one or more 2'-O-alkyl modifications. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain 2'-O-alkyl modifications of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain 2'-O-alkyl modifications of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the fourth (+4) nucleotide.In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains a 2'-O-alkyl modification of the eighth (+8) nucleotide.

[0030] In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain one or more DNA modifications. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain DNA modifications at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain DNA modifications at the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the fifth (+5) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a DNA modification of the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a DNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a DNA modification of the eighth (+8) nucleotide.

[0031] In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain one or more GNA modifications. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain GNA modifications at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain GNA modifications at the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the fifth (+5) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a GNA modification of the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a GNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a GNA modification of the eighth (+8) nucleotide.

[0032] In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain one or more (s)-GNA modifications. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain (s)-GNA modifications at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain (s)-GNA modifications at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains an (s)-GNA modification of the fifth (+5) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises an (s)-GNA modification of the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises an (s)-GNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises an (s)-GNA modification of the eighth (+8) nucleotide.

[0033] In certain embodiments, the present disclosure encompasses the recognition that the stereochemistry, for example, the stereochemistry of the chiral center in the 5'-end modification of the guide strand, can unexpectedly maintain or improve the properties of the ds oligonucleotides described herein. For example, but not limited to, the present disclosure relates in part to ds oligonucleotides comprising a guide strand comprising one or more of the following: (1) a backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, (2) a phosphorothioate chiral center in the Rp or Sp configuration, (3) an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, wherein the 3' nucleotide of the nucleotide pair joined by the Rp, Sp, or sterically random non-negatively charged internucleotide linkage comprises a 2' modification, e.g., a 2'F or a 2'-OMe, (4) a modified sugar selected from a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, e.g., a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, e.g., a 5'-(R)-methyl or a 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA, and (5) a 5'-terminal modification selected from: (a) 5'PO modifications, including but not limited to: [ka] (b) 5'VP modifications, including but not limited to: [ka] (c) 5'MeP modifications, including but not limited to: [ka] (d) 5'PN and 5'triazole-P modifications, including but not limited to: [ka] [ka] wherein the bases are selected from A, C, G, T, U, abasic, and modified nucleobases; R 1 is selected from alkyl, methyl, ethyl, isopropyl, propyl, cyclohexyl, benzyl, phenyl, tolyl, xylyl, aryl, or arene groups. R 2’ represents H, OH, O-alkyl, O-Me, F, MOE, locked nucleic acid (LNA) bridges, and [ka] The nucleotides may be selected from bridged nucleic acid (BNA) bridges to the 4'C, such as, but not limited to, Rp, Sp, or steric random non-negatively charged internucleotide linkages incorporated into the guide strand. In certain embodiments, the one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages are Rp non-negatively charged internucleotide linkages. In certain embodiments, the one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, the one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages are steric random non-negatively charged internucleotide linkages.

[0034] In certain embodiments, the guide strand comprises a main-chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and a main-chain phosphorothioate chiral center in Rp configuration between the +2 nucleotide and the (+3) nucleotide immediately downstream. In certain embodiments, the 5'-terminal modification is [ka] In some embodiments, the 5' end modification is [ka] In certain embodiments, the 5' end modification is [ka] In certain embodiments, the guide strand comprises a 5'-end modification selected from, but not limited to, a 5'MeP modification and a 5'triazole-P modification. In certain embodiments, the guide strand comprises a 5'MeP modification [ka] and a main-chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and a main-chain phosphorothioate chiral center in Rp configuration between the +2 nucleotide and the immediately downstream (+3) nucleotide.

[0035] In certain other embodiments, the present disclosure encompasses the recognition that the stereochemistry, e.g., of the chiral center at the 5'-terminal nucleotide of the guide strand, can unexpectedly maintain or improve the properties of a ds oligonucleotide, wherein the guide strand of the ds oligonucleotide also comprises a phosphorothioate chiral center in the Rp or Sp configuration. For example, but not by way of limitation, the present disclosure relates in part to a ds oligonucleotide comprising a guide strand comprising: (1) a phosphorothioate chiral center in the Rp or Sp configuration; (2) an Rp, Sp, or a sterically random non-negatively charged internucleotide linkage, wherein the 3' nucleotide of the nucleotide pair joined by the Rp, Sp, or a sterically random non-negatively charged internucleotide linkage comprises a 2' modification, e.g., 2'F or 2'-OMe; and (3) a 5'-terminal modification selected from the following: (a) 5'PO nucleotides, such as, but not limited to: [ka] , (b) 5'VP nucleotides, such as, but not limited to: [ka] (c) 5'MeP nucleotides, such as, but not limited to: [ka] (d) 5'PN and 5'triazole-P nucleotides, including but not limited to: [ka] [ka] (e) 5' abasic VP and 5' abasic MeP nucleotides, including but not limited to: [ka] In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds incorporated into the guide strand are Rp non-negatively charged internucleotide bonds. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds are Sp non-negatively charged internucleotide bonds. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds are sterically random non-negatively charged internucleotide bonds.

[0036] In certain other embodiments, the present disclosure encompasses the recognition that the stereochemistry, for example, of the chiral center at the 5'-terminal nucleotide of the guide strand, can unexpectedly maintain or improve the properties of ds oligonucleotides, where the guide strand of the ds oligonucleotide also contains a phosphorothioate chiral center in the Rp or Sp configuration. For example, but not by way of limitation, the present disclosure provides a backbone phosphorylguanidine chiral center in the Rp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, (2) a backbone phosphorylguanidine chiral center in the Rp configuration between the +7 nucleotide and the immediately downstream (+8) nucleotide, (3) a phosphorothioate chiral center in the Rp or Sp configuration, and (4) [ka] The present invention relates in part to a ds oligonucleotide comprising a guide strand comprising a protected phosphonate selected from: In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are sterically random non-negatively charged internucleotide linkages.

[0037] In certain embodiments, the present disclosure encompasses the recognition that non-naturally occurring internucleotide linkages, such as neutral internucleotide linkages, can be used to attach one or more molecules to the double-stranded oligonucleotides described herein. In certain embodiments, such attached molecules can facilitate the targeting and / or delivery of double-stranded oligonucleotides. For example, but not limited to, such attached molecules include lipophilic molecules. In certain embodiments, the attached molecules are molecules comprising one or more GalNAc moieties. In certain embodiments, the attached molecules are receptors. In certain embodiments, the attached molecules are receptor ligands.

[0038] In certain embodiments, the present disclosure provides techniques for incorporating various additional chemical moieties into ds oligonucleotides. In certain embodiments, the present disclosure provides reagents and methods for, for example, introducing additional chemical moieties through the nucleobase (e.g., by covalent bonding, optionally via a linker, at a site on the nucleobase).

[0039] In certain embodiments, the present disclosure provides techniques, e.g., ds oligonucleotide compositions and methods, for achieving allele-specific suppression, wherein the transcript from one allele of a particular target gene is selectively knocked down relative to at least one other allele of the same gene.

[0040] Among other things, the present disclosure provides structural elements, techniques, and / or features that can be incorporated into ds oligonucleotides to impart or tailor one or more properties thereof (e.g., relative to an otherwise identical ds oligonucleotide lacking the relevant technique or feature). In certain embodiments, the present disclosure documents that one or more of the provided techniques and / or features can be usefully incorporated into ds oligonucleotides of various sequences.

[0041] In certain embodiments, the present disclosure demonstrates that certain provided structural elements, techniques, and / or features are particularly useful for ds oligonucleotides that participate in and / or induce RNAi mechanisms (e.g., RNAi agents). Nevertheless, the teachings of the present disclosure are not limited to ds oligonucleotides that participate in or operate via any particular mechanism. In certain embodiments, the present disclosure relates to any ds oligonucleotides that operate through any mechanism and are useful for any purpose, including any sequence, structure, or format (or portion thereof) described herein. In certain embodiments, the present disclosure relates to any ds oligonucleotides that operate through any mechanism and are useful for any purpose, including any sequence, structure, or format (or portion thereof) described herein, including a guide strand that includes a backbone phosphorylguanidine chiral center in the Sp configuration.

[0042] In certain embodiments, the guide strand comprises a main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, ie, in the 3' direction.

[0043] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, further comprises a phosphoryl guanidine chiral center in the Sp configuration between the +7 and the immediately downstream (+8) nucleotide, between the +10 nucleotide and the immediately downstream (+11) nucleotide, between the +18 nucleotide and the immediately downstream (+19) nucleotide, or combinations thereof.

[0044] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modification at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the eighth (+8) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the ninth (+9) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the tenth (+10) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the twelfth (+12) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the sixteenth (+16) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 17th (+17) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 18th (+18) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 19th (+19) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 20th (+20) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 21st (+21) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 22nd (+22) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 23rd (+23) 3'-terminal nucleotide.In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, such as a 2'-O-methyl (OMe) modification, a 2'-O-C16 lipid modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, such as a 5'-(R)-methyl or a 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.

[0045] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0046] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, that contain a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, which further comprise: (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0047] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a homo-DNA modification of the 5'-terminal (+1) nucleotide, the 6th (+6) nucleotide, the 9th (+9) nucleotide, the 10th (+10) nucleotide, the 12th (+12) nucleotide, the 16th (+16) nucleotide, the 17th (+17) nucleotide, the 18th (+18) nucleotide, the 19th (+19) nucleotide, the 20th (+20) nucleotide, the 21st (+21) nucleotide, the 22nd (+22), and / or the 23rd (+23) 3'-terminal nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contain a homo-DNA modification of the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the ninth (+9) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the tenth (+10) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the twelfth (+12) nucleotide.In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 16th (+16) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 17th (+17) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 18th (+18) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contains a homo-DNA modification of the 19th (+19) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a homo-DNA modification of the 20th (+20) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a homo-DNA modification of the 21st (+21) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a homo-DNA modification of the 22nd (+22) nucleotide.In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains a homo-DNA modification of the 23rd (+23) 3'-terminal nucleotide.

[0048] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, that contain a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, which further comprise: (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0049] In certain embodiments, ds oligonucleotides comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise an LNA modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise an LNA modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprise an LNA modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the sixth (+6) nucleotide.In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the seventh (+7) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain an LNA modification of the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the third (+3) nucleotide.In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-O-alkyl modification of the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-O-alkyl modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-O-alkyl modification of the eighth (+8) nucleotide.

[0050] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, that contain a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, which further comprise: (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0051] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification at the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification at the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a DNA modification of the sixth (+6) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a DNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a DNA modification of the eighth (+8) nucleotide.

[0052] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, that contain a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, which further comprise: (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0053] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a GNA modification of the sixth (+6) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a GNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a GNA modification of the eighth (+8) nucleotide.

[0054] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, that contain a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, which further comprise: (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0055] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, and / or the eighth (+8) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the sixth (+6) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the seventh (+7) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises an (s)-GNA modification of the eighth (+8) nucleotide.

[0056] In certain embodiments, the present disclosure provides ds oligonucleotides useful for any purpose, operating through any mechanism, that contain a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, which further comprise: (1) a guide strand comprising main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the (N-2) nucleotide immediately upstream, i.e., in the 5' direction; (2) a guide strand comprising backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the +2 nucleotide and the (+3) nucleotide immediately downstream; (3) a guide strand comprising one or more main-chain phosphorothioate chiral centers upstream, i.e., in the 5' direction, relative to the main-chain phosphorothioate chiral center in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream main-chain phosphorothioate chiral center is in the Rp or Sp configuration; (4) a guide strand comprising one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (6) a guide strand containing one or more main chain phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction; (7) a guide strand comprising a 5'-end modification; (8) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main chain chiral centers in the Rp or Sp configuration; (9) a passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; (10) A passenger strand in combination with one or more of the aforementioned guide strands, comprising one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction; (11) a passenger strand in combination with one or more of the aforementioned guide strands, comprising a main chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; ds oligonucleotides are (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) Further comprising one or more passenger strands comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0057] In certain embodiments, a ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or steric random non-negatively charged internucleotide linkage. In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages are steric random non-negatively charged internucleotide linkages. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide directly downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains a modified sugar at the 5'-terminal (+1) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains a modified sugar at the sixth (+6) nucleotide.

[0058] In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, such as a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, such as a 5'-(R)-methyl or a 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.

[0059] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0060] In certain embodiments, provided ds oligonucleotides can participate in (e.g., induce) the RNAi mechanism. In certain embodiments, provided ds oligonucleotides can participate in the RNase H (ribonuclease H) mechanism. In certain embodiments, provided ds oligonucleotides can act as translation inhibitors (e.g., provide a steric block to translation).

[0061] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, and the guide strand comprises backbone phosphorothioate chiral centers in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) One or more passenger strands further comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0062] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises 0-n Rp, Sp, or sterically random non-negatively charged internucleotide linkages, where n is from about 1 to 49.

[0063] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide.

[0064] In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, such as a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, such as a 5'-(R)-methyl or a 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the sixth (+6) nucleotide.

[0065] In certain embodiments, the passenger strand comprises an Sp main chain phosphorothioate chiral center between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and an Sp main chain phosphorothioate chiral center between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0066] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, and the guide strand comprises backbone phosphorothioate chiral centers in the Rp, Sp, or alternating configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) nucleotide and the 11th (+11) nucleotide relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 7th (+7) nucleotide and the 8th (+8) nucleotide relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) One or more passenger strands further comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0067] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises 0-n Rp, Sp, or sterically random non-negatively charged internucleotide linkages, where n is from about 1 to 49.

[0068] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises a modified sugar. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide.

[0069] In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, such as a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, such as a 5'-(R)-methyl or a 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.

[0070] In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0071] In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are sterically random non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises an Sp main-chain phosphorothioate chiral center between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and an Sp main-chain phosphorothioate chiral center between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0072] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, and the guide strand comprises one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration upstream of a backbone phosphorothioate chiral center in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) One or more passenger strands further comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0073] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises 0-n Rp, Sp, or sterically random non-negatively charged internucleotide linkages, where n is from about 1 to 49.

[0074] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide.

[0075] In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, such as a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, such as a 5'-(R)-methyl or a 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the sixth (+6) nucleotide.

[0076] In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain further embodiments, the passenger strand comprises an Sp main-chain phosphorothioate chiral center between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and an Sp main-chain phosphorothioate chiral center between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0077] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, and the guide strand comprises one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occurring between the second (+2) nucleotide and the third (+3) nucleotide relative to the 5'-terminal nucleotide of the guide strand, and an internucleotide linkage to the penultimate 3'(N-1) nucleotide; (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) One or more passenger strands further comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0078] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises 0-n Rp, Sp, or sterically random non-negatively charged internucleotide linkages, where n is from about 1 to 49.

[0079] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, such as a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, such as a 5'-(R)-methyl or a 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the second (+2) nucleotide.In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0080] In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are sterically random non-negatively charged internucleotide linkages. In certain embodiments, the passenger strand comprises an Sp main-chain phosphorothioate chiral center between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and an Sp main-chain phosphorothioate chiral center between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0081] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, and the guide strand comprises backbone phosphorothioate chiral centers in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) One or more passenger strands further comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0082] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are sterically random non-negatively charged internucleotide linkages. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, e.g., a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, e.g., a 5'-(R)-methyl or 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0083] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, and the guide strand comprises backbone phosphorothioate chiral centers in the Rp, Sp, or alternating configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) One or more passenger strands containing one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide. The ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are sterically random non-negatively charged internucleotide linkages. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, e.g., a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, e.g., a 5'-(R)-methyl or 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0084] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, and the guide strand comprises one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration upstream of the backbone chiral center in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) One or more passenger strands further comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0085] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are sterically random non-negatively charged internucleotide linkages. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, e.g., a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, e.g., a 5'-(R)-methyl or 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0086] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, and the guide strand comprises one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) One or more passenger strands further comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0087] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are sterically random non-negatively charged internucleotide linkages. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, e.g., a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, e.g., a 5'-(R)-methyl or 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0088] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, and the guide strand comprises a backbone phosphoryl guanidine chiral center in the Sp configuration between the +7 and the immediately downstream (+8) nucleotide, and (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) One or more passenger strands further comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0089] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are sterically random non-negatively charged internucleotide linkages. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, e.g., a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, e.g., a 5'-(R)-methyl or 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0090] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, and the guide strand comprises one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the (+2) nucleotide and the immediately downstream (+3) nucleotide, and between the (+5) nucleotide and the (+6) nucleotide; (1) A guide strand in which one or both of the 5'- and 3'-terminal dinucleotides are not linked by a non-negatively charged internucleotide bond, i.e., the guide strand includes one or more non-negatively charged internucleotide bonds downstream, i.e., in the 3' direction, relative to the bond between the 5'-terminal dinucleotides, and / or upstream, i.e., in the 5' direction, relative to the bond between the 3'-terminal dinucleotides; (2) a guide strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; (3) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the 10th (+10) and 11th (+11) nucleotides relative to the 5'-terminal nucleotide; (4) a guide strand in which an Rp, Sp, or sterically random non-negatively charged internucleotide bond occurs between the seventh (+7) and eighth (+8) nucleotides relative to the 5'-terminal nucleotide; (5) a guide strand containing one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, where N is the 3'-terminal nucleotide, and where the bond occurs between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide; (6) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (7) a passenger strand in which one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; (8) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; (9) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp-configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; and (10) One or more passenger strands further comprising one or more modified sugars, e.g., homoDNA, between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide.

[0091] In certain embodiments, the ds oligonucleotide further comprises a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages incorporated into the guide strand or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages are sterically random non-negatively charged internucleotide linkages. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, e.g., a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, e.g., a 5'-(R)-methyl or 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA.In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0092] In certain embodiments, the ds oligonucleotide comprises a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide directly downstream, i.e., in the 3' direction, and the guide strand further comprises one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occurring between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide, and a 2' modification, such as a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises one or more main chain chiral centers in the Rp or Sp configuration. In certain embodiments, the one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages are steric random non-negatively charged internucleotide linkages. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in an Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, e.g., a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, e.g., a 5'-(R)-methyl or 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide containing a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, contains a 2'-F modification of the third (+3) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a 2'-F modification of the sixth (+6) nucleotide.

[0093] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction; the guide strand further comprises backbone phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; and a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide linkage; and the passenger strand comprises an 0-n Rp, Sp, or sterically random non-negatively charged internucleotide linkage and one or more backbone chiral centers in the Rp or Sp configuration, where n is from about 1 to 49.

[0094] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the twenty-third (+23) 3'-terminal nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the second (+2) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the third (+3) nucleotide.In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fourth (+4) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the fifth (+5) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, comprises a modified sugar at the sixth (+6) nucleotide. In certain embodiments, the modified sugar comprises a 2'-F modification, a 2'-H modification, a 2'-OH modification, a 2'-O-alkyl modification, such as a 2'-O-methyl (OMe) modification, a 2'-methoxyethyl (MOE) modification, a 5'-alkyl modification, such as a 5'-(R)-methyl or a 5'-(S)-methyl, DNA, a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), or a homoDNA. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, and / or the sixth (+6) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the 5'-terminal (+1) nucleotide. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a 2'-F modification of the second (+2) nucleotide.In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the third (+3) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fourth (+4) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the fifth (+5) nucleotide. In certain embodiments, ds oligonucleotides containing a guide strand main chain phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction, contain a 2'-F modification of the sixth (+6) nucleotide.

[0095] In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages incorporated into the guide strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain further embodiments, one or more Rp, Sp, or steric random non-negatively charged internucleotide linkages are steric random non-negatively charged internucleotide linkages. In certain further embodiments, the passenger strand comprises an Sp main-chain phosphorothioate chiral center between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and an Sp main-chain phosphorothioate chiral center between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

[0096] In certain embodiments, the ds oligonucleotide comprises a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide, i.e., in the 3' direction; the guide strand further comprises backbone phosphorothioate chiral centers in the Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide; and a 2' modification, e.g., a 2'F modification, of the 3' nucleotide of the nucleotide pair linked by the Rp, Sp, or sterically random non-negatively charged internucleotide linkage; and the passenger strand comprises an 0-n Rp, Sp, or sterically random non-negatively charged internucleotide linkage and one or more backbone chiral centers in the Rp or Sp configuration, where n is from about 1 to 49.

[0097] In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, further comprises one or more modified sugars. In certain embodiments, a ds oligonucleotide comprising a guide strand backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction, comprises a modified sugar at the 5'-terminal (+1) nucleotide, the second (+2) nucleotide, the third (+3) nucleotide, the fourth (+4) nucleotide, the fifth (+5) nucleotide, the sixth (+6) nucleotide, the seventh (+7) nucleotide, the eighth (+8) nucleotide, the ninth (+9) nucleotide, the tenth (+10) nucleotide, the twelfth (+12) nucleotide, the sixteenth (+16) nucleotide, the seventeenth (+17) nucleotide, the eighteenth (+18) nucleotide, the nineteenth (+19) nucleotide, the twentieth (+20) nucleotide, the twenty-first (+21) nucleotide, the twenty-second (+22), and / or the ...

Claims

1. A double-stranded RNAi (dsRNAi) agent comprising a guide strand and a passenger strand, a) the guide strand is complementary or substantially complementary to a target RNA sequence, the guide strand comprising a backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction; and i. backbone phosphorothioate chiral centers in Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; ii. backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; iii. one or more main-chain phosphorothioate chiral centers in Rp or Sp configuration upstream of a main-chain phosphorothioate chiral center in Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; iv. one or more backbone phosphorothioate chiral centers in an Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and / or between the (+2) nucleotide and the immediately downstream (+3) nucleotide; v. one or more backbone phosphorothioate chiral centers in the Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' (N-1) nucleotide and the upstream N-10 nucleotide of the guide strand, where N is the 3' terminal nucleotide; vi. one or more backbone phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction, and / or vii. comprising a 5' end modification; b) the guide strand comprises one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds that occur between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3' (N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; c) the guide strand comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide bond; d) the guide strand comprises an Rp, Sp, or sterically random non-negatively charged internucleotide bond between the 10th (+10) nucleotide and the 11th (+11) nucleotide relative to the 5′-terminal nucleotide; e) the guide strand comprises an Rp, Sp, or sterically random non-negatively charged internucleotide bond between the seventh (+7) nucleotide and the eighth (+8) nucleotide relative to the 5'-terminal nucleotide; f) the guide strand comprises one or more backbone phosphorothioate chiral centers in an Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide and the upstream N-10 nucleotide of the guide strand, where N is the 3' terminal nucleotide; g) the passenger strand comprises one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occurring upstream, i.e., in the 5' direction, relative to the central nucleotide of said passenger strand; h) the passenger strand comprises one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occurring downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; i) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; j) a passenger strand, wherein one or more main chain phosphorothioate chiral centers at Rp or Sp configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; k) a passenger strand in combination with one or more of the aforementioned guide strands, the passenger strand comprising one or more modified sugars between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide; l) the passenger strand is i. 0-n Rp, Sp, or sterically random non-negatively charged internucleotide linkages, where n is from about 1 to 49; ii. one or more main chain chiral centers in the Rp or Sp configuration; iii. one or more backbone phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; iv. one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction, and / or v. comprising one or both of the main-chain phosphorothioate chiral centers in the Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; m) each strand of the dsRNAi agent independently has a length of from about 15 to about 49 nucleotides; and / or n) A dsRNAi agent, wherein said dsRNAi is capable of inducing target-specific RNA interference.

2. 1. A chiral control oligonucleotide composition comprising a double-stranded oligonucleotide, wherein the guide strand and passenger strand of the double-stranded oligonucleotide are independently: a) consensus sequence and length; b) common patterns of main-chain bonding, and c) characterized by a common pattern of main-chain chiral centers; the composition is chiral-controlled in that it is enriched for oligonucleotides having a common pattern of chiral centers relative to a substantially racemic preparation of guide strands having the same common base sequence and length; a) the guide strand is complementary or substantially complementary to a target RNA sequence, the guide strand comprising a backbone phosphorylguanidine chiral center in the Sp configuration between the +3 nucleotide and the (+4) nucleotide immediately downstream, i.e., in the 3' direction; and i. backbone phosphorothioate chiral centers in Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; ii. backbone phosphorothioate chiral centers in Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide; iii. one or more main-chain phosphorothioate chiral centers in Rp or Sp configuration upstream of a main-chain phosphorothioate chiral center in Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; iv. one or more main-chain phosphorothioate chiral centers in Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and / or between the (+2) nucleotide and the immediately downstream (+3) nucleotide, and / or main-chain phosphorothioate chiral centers in Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; v. one or more backbone phosphorylguanidine chiral centers in the Sp configuration between +7 and (+8) directly downstream, i.e., in the 3' direction, and / or vi. comprising a 5' end modification; b) the guide strand comprises one or more Rp, Sp, or sterically random non-negatively charged internucleotide bonds occurring between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3' (N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; c) the guide strand comprises a 2' modification of the 3' nucleotide of a nucleotide pair linked by an Rp, Sp, or sterically random non-negatively charged internucleotide bond; d) the guide strand comprises an Rp, Sp, or sterically random non-negatively charged internucleotide bond between the 10th (+10) nucleotide and the 11th (+11) nucleotide relative to the 5′-terminal nucleotide; e) the guide strand comprises an Rp, Sp, or sterically random non-negatively charged internucleotide bond between the seventh (+7) nucleotide and the eighth (+8) nucleotide relative to the 5'-terminal nucleotide; f) the guide strand comprises one or more backbone phosphorothioate chiral centers in an Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide; g) the passenger strand comprises one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occurring upstream, i.e., in the 5' direction, relative to the central nucleotide of said passenger strand; h) the passenger strand comprises one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occurring downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; i) a passenger strand in which one or more main-chain phosphorothioate chiral centers at Rp or Sp configured internucleotide linkages occur upstream, i.e., in the 5' direction, relative to the central nucleotide of the passenger strand; j) a passenger strand, wherein one or more main chain phosphorothioate chiral centers at Rp or Sp configured internucleotide linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand; k) a passenger strand in combination with one or more of the aforementioned guide strands, the passenger strand comprising one or more modified sugars between the 5'-terminal (+1) nucleotide and the penultimate (N-1) nucleotide; l) the passenger strand is i. 0-n Rp, Sp, or sterically random non-negatively charged internucleotide linkages, where n is from about 1 to 49; ii. one or more main chain chiral centers in the Rp or Sp configuration; iii. one or more backbone phosphorylguanidine chiral centers in the Rp configuration between the +7 nucleotide and the (+8) nucleotide immediately downstream, i.e., in the 3' direction; iv. one or more main-chain phosphorylguanidine chiral centers in the Rp configuration between the +15 nucleotide and the (+16) nucleotide immediately downstream, i.e., in the 3' direction, and / or v. comprising one or both of the main-chain phosphorothioate chiral centers in the Sp configuration between the 5'-terminal (+1) nucleotide and the (+2) nucleotide immediately downstream, i.e., in the 3' direction, and between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide; m) the guide strand and passenger strand have a length of about 15 to about 49 nucleotides; and / or n) A composition wherein the guide strand and passenger strand are capable of inducing target-specific RNA interference.

3. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises backbone phosphorothioate chiral centers in Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotide linkages, where n is from about 1 to 49.

4. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises backbone phosphorothioate chiral centers in Rp, Sp, or alternating configurations between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and the passenger strand comprises 0-n Rp, Sp, or stereo-random non-negatively charged internucleotide linkages, where n is from about 1 to 49.

5. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers in an Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotide linkages, where n is from about 1 to 49.

6. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises one or more main-chain phosphorothioate chiral centers in the Rp or Sp configuration upstream of main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotide linkages, where n is from about 1 to 49.

7. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises one or more Rp, Sp, or sterically random non-negatively charged internucleotide linkages occurring between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; and the passenger strand comprises 0-n Rp, Sp, or sterically random non-negatively charged internucleotide linkages, where n is from about 1 to 49.

8. 3. The double-stranded oligonucleotide of claim 1, wherein the guide strand comprises main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises one or more main-chain chiral centers in the Rp or Sp configuration.

9. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises a main-chain phosphorothioate chiral center in an Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and the passenger strand comprises one or more main-chain chiral centers in an Rp or Sp configuration.

10. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises one or more main-chain phosphorothioate chiral centers in an Rp or Sp configuration, with bonds occurring between any two adjacent nucleotides between the penultimate 3' nucleotide of the guide strand and the upstream N-10 nucleotide, where N is the 3' terminal nucleotide, and the passenger strand comprises one or more main-chain chiral centers in an Rp or Sp configuration.

11. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises a backbone phosphorylguanidine chiral center in the Sp configuration between the +7 and the immediately downstream (+8), i.e., in the 3' direction, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotide linkages, where n is from about 1 to 49.

12. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises a main-chain phosphorylguanidine chiral center in the Sp configuration between the +7 and the directly downstream, i.e., (+8) in the 3' direction, and the passenger strand comprises one or more main-chain chiral centers in the Rp or Sp configuration.

13. 3. The double-stranded oligonucleotide of claim 1, wherein the guide strand comprises one or more main-chain phosphorothioate chiral centers in the Rp or Sp configuration upstream of main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises one or more main-chain chiral centers in the Rp or Sp configuration.

14. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises one or more main-chain phosphorothioate chiral centers in an Rp or Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and / or between the (+2) nucleotide and the immediately downstream (+3) nucleotide.

15. the guide strand comprises a 5' end modification selected from: 【Chemistry 1-1】 【Chemistry 1-2】 Bases: A, C, G, T, U, abasic and modified nucleobases; R １ : H, OH, O-alkyl, O-Me, F, MOE, LNA bridge at 4' position, BNA bridge at 4' position, R ２ 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein: is an alkyl, methyl, ethyl, isopropyl, propyl, cyclohexyl, benzyl, phenyl, tolyl, xylyl, aryl, or arene group.

16. 14. The double-stranded oligonucleotide or composition of claim 13, wherein the guide strand comprises a 5'-end modification selected from a 5' MeP modification and a 5' Trizol-P modification.

17. The 5'MeP modification is 【Chemistry 2】 15. The double-stranded oligonucleotide or composition of claim 14, wherein:

18. 16. The double-stranded oligonucleotide or composition of claim 15, comprising a main-chain phosphorothioate chiral center in Sp configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and a main-chain phosphorothioate chiral center in Rp configuration between the +2 nucleotide and the immediately downstream (+3) nucleotide.

19. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises one or more Rp, Sp, or stereo-random non-negatively charged internucleotide linkages occurring between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide, and the passenger strand comprises one or more main-chain chiral centers in the Rp or Sp configuration.

20. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide, and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotide linkages, where n is from about 1 to 49, and one or more main-chain chiral centers in the Rp or Sp configuration.

21. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises backbone phosphorothioate chiral centers in the Rp, Sp, or alternating configuration between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotide linkages and one or more backbone chiral centers in the Rp or Sp configuration, where n is from about 1 to 49.

22. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises one or more main-chain phosphorothioate chiral centers in the Rp or Sp configuration upstream of main-chain phosphorothioate chiral centers in the Sp configuration between the 3'-terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0-n Rp, Sp, or stereorandom non-negatively charged internucleotide linkages and one or more main-chain chiral centers in the Rp or Sp configuration, where n is from about 1 to 49.

23. 3. The double-stranded oligonucleotide of claim 1, or the composition of claim 2, wherein the guide strand comprises one or more Rp, Sp, or stereo-random non-negatively charged internucleotide linkages occurring between any two adjacent nucleotides between the second (+2) nucleotide relative to the 5'-terminal nucleotide of the guide strand and the penultimate 3'(N-1) nucleotide of the guide strand, where N is the 3'-terminal nucleotide; and the passenger strand comprises 0-n non-negatively charged internucleotide linkages, where n is from about 1 to 49, and one or more main-chain chiral centers in the Rp or Sp configuration.

24. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the Rp, Sp, or sterically random non-negatively charged backbone internucleotide linkages have a neutral charge.

25. Neutral backbone internucleotide linkages are 【Transformation 3】 25. The double-stranded oligonucleotide or composition of claim 24, wherein:

26. The guide strand has the following structure between the third (+3) nucleotide and the fourth (+4) nucleotide of the guide strand, between the tenth (+10) nucleotide and the eleventh (+11) nucleotide of the guide strand, or both: 【Chemistry 4】 26. The double-stranded oligonucleotide or composition of claim 25, comprising a linkage having the formula:

27. The guide strand has the following structure between the third (+3) nucleotide and the fourth (+4) nucleotide of the guide strand, between the seventh (+7) nucleotide and the eighth (+8) nucleotide of the guide strand, between the tenth (+10) nucleotide and the eleventh (+11) nucleotide of the guide strand, between the eighteenth (+18) nucleotide and the nineteenth (+19) nucleotide of the guide strand, or a combination thereof: 【Transformation 5】 26. The double-stranded oligonucleotide or composition of claim 25, comprising a linkage having the formula:

28. the passenger strand has the following structure 5' to the central nucleotide of the passenger strand, 3' to the central nucleotide of the passenger strand, or both: 【Transformation 6】 26. The double-stranded oligonucleotide or composition of claim 25, comprising a linkage having the formula:

29. The composition of claim 2, wherein the guide strands and passenger strands in the composition that independently share a common base sequence, a common pattern of base modifications, a common pattern of sugar modifications, and / or a common pattern of internucleotide linkages are at least 90% of all the guide strands and passenger strands in the composition.

30. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein said double-stranded oligonucleotide comprises a carbohydrate moiety connected by an internucleoside or internucleotide bond, optionally through a linker.

31. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the double-stranded oligonucleotide comprises a lipid moiety connected to the double-stranded oligonucleotide at an internucleoside or internucleotide bond, optionally through a linker.

32. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein one or both strands of the double-stranded oligonucleotide comprise targeting moieties connected by nucleobases, optionally through a linker.

33. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the internucleotide linkages of the double-stranded oligonucleotide are independently chiral internucleotide linkages.

34. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% of the nucleotide units of said double-stranded oligonucleotide independently comprise a 2'-substitution.

35. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the modified sugar of said oligonucleotide comprises a 2'-F modification, a 2'-OH modification, a 2'-OMe modification, a 2'-O-C16 lipid modification, a 5'-alkyl modification, a 2'-MOE modification, DNA, LNA, UNA, GNA, or homoDNA.

36. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the modified sugar of the oligonucleotide is at one position or at multiple positions.

37. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the modified sugar of the oligonucleotide is at one or more of the following positions: (a) +1, (b) +2, (c) +3, (d) +4, (e) +5, and (f) +6.

38. The double-stranded oligonucleotide or composition of claims 35-37, wherein the modified sugar of the oligonucleotide is at the +4 position and the modified sugar of the oligonucleotide is a 2'-F modification.

39. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the 2'-substitution of said oligonucleotide is -L-, wherein L connects C2 and C4 of the sugar unit.

40. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% of the nucleotide units of the double-stranded oligonucleotide do not comprise a 2'-substitution.

41. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the guide strand comprises a target binding sequence that is perfectly complementary to a target sequence, the target binding sequence having a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases, each base being an optionally substituted adenine, cytosine, guanosine, thymine, or uracil, and the target sequence comprises one or more allelic sites, wherein the allelic site is a SNP or mutation.

42. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the target sequence comprises two SNPs.

43. 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the target sequence comprises an allelic site and the target binding sequence is perfectly complementary to the target sequence of a disease-associated allele but not to the target sequence of an allele less frequently associated with the disease.

44. the double-stranded oligonucleotide comprises a guide strand that binds to a transcription product of a target nucleic acid sequence of which multiple alleles exist within a population, each of the multiple alleles comprising a specific nucleotide signature sequence element that defines the allele relative to other alleles of the same target nucleic acid sequence; The base sequence of the guide strand is or comprises a sequence that is complementary to the characteristic sequence element that defines a particular allele; A double-stranded oligonucleotide or composition according to any one of the preceding claims, characterized in that the guide strand, when contacted with a cell containing a transcription product of the target nucleic acid sequence, exhibits suppression of the transcription product of the particular allele, or the protein encoded thereby, at a level that exceeds the level of suppression observed for another allele of the same nucleic acid sequence.

45. the passenger strand is an Sp backbone phosphorothioate chiral center between the 5'-terminal (+1) nucleotide and the immediately downstream (+2) nucleotide; 10. The double-stranded oligonucleotide or composition of any one of the preceding claims, comprising an Sp backbone phosphorothioate chiral center between the penultimate (N-1) nucleotide and the 3'-terminal (N) nucleotide.

46. 10. A method for reducing the level and / or activity of a transcript or the protein encoded thereby, comprising administering to a cell expressing said transcript a double-stranded oligonucleotide or composition according to any one of the preceding claims, wherein the guide strand of the double-stranded oligonucleotide or composition comprises a target binding sequence that is perfectly complementary to a target sequence in said transcript.

47. 42. The method of claim 41, wherein the cell is an immune cell, a blood cell, a heart cell, a lung cell, a photoreceptor cell, a muscle cell, a liver cell, a kidney cell, a brain cell, a cell of the central nervous system, or a cell of the peripheral nervous system.

48. 1. A method for allele-specific suppression of a transcript from a nucleic acid sequence of which multiple alleles exist in a population, each of said multiple alleles comprising a specific nucleotide signature sequence element that defines said allele relative to other alleles of the same target nucleic acid sequence, said method comprising: contacting a sample containing a transcription product of the target nucleic acid sequence with a double-stranded oligonucleotide or composition according to any one of the preceding claims, the guide strand of the double-stranded oligonucleotide or composition comprises a target binding sequence that is identical to or completely complementary to a target sequence in the nucleic acid sequence, the target sequence comprising a characteristic sequence element that defines a particular allele; When the guide strand of the double-stranded oligonucleotide or composition is contacted with a cell containing transcripts of both a target allele and another allele of the same nucleic acid sequence, the transcript of the specific allele is suppressed at a level that exceeds the level of suppression observed for the other allele of the same nucleic acid sequence.

49. 1. A method for allele-specific suppression of a transcript from a nucleic acid sequence of which multiple alleles exist in a population, each of said multiple alleles comprising a specific nucleotide signature sequence element that defines said allele relative to other alleles of the same target nucleic acid sequence, said method comprising: Administering to a subject a double-stranded oligonucleotide or composition according to any one of the preceding claims, together with a transcription product of the target nucleic acid sequence, the guide strand of the double-stranded oligonucleotide or composition comprises a target binding sequence that is identical to or completely complementary to a target sequence in the nucleic acid sequence, the target sequence comprising a characteristic sequence element that defines a particular allele; When the guide strand of the double-stranded oligonucleotide or composition is contacted with a cell containing transcripts of both a target allele and another allele of the same nucleic acid sequence, the transcript of the specific allele is suppressed at a level that exceeds the level of suppression observed for the other allele of the same nucleic acid sequence.

50. When the oligonucleotide or the oligonucleotide of the composition is contacted with a cell containing transcripts of both the target allele and another allele of the same nucleic acid sequence, a) a level greater than that in the absence of said composition; b) a level of suppression that exceeds the level of suppression observed for another allele of the same nucleic acid sequence, or c) exhibiting repression of the transcript of said particular allele at a level that exceeds that observed in the absence of said composition and that exceeds the level of repression observed for another allele of the same nucleic acid sequence.

52. 51. The method of claim 50, wherein the cell is an immune cell, a blood cell, a heart cell, a lung cell, a photoreceptor cell, a muscle cell, a liver cell, a kidney cell, a brain cell, a cell of the central nervous system, or a cell of the peripheral nervous system.

53. 53. The method of any one of claims 46-52, wherein the repression of the transcript of the particular allele is greater than in the absence of the composition and at a level that exceeds the level of repression observed for another allele of the same nucleic acid sequence.

Images