Double stranded oligonucleotide compositions for RNA interference and methods relating thereto

Abstract

The present disclosure provides double stranded oligonucleotides, compositions, and methods relating thereto wherein the oligonucleotides comprise modifications of internucleotidic linkages. The present disclosure encompasses the recognition that structural elements of double stranded oligonucleotides, such as base modifications, as well as base sequence, chemical modifications (e.g., modifications of sugar and / or internucleotidic linkages) or patterns thereof, and / or stereochemistry, e.g., stereochemistry of chiral internucleotidic linkages, and / or patterns thereof, can have significant impact on oligonucleotide properties, e.g., RNA interference activity. The present disclosure also provides methods for treatment of diseases, e.g., hepatic diseases, central nervous system diseases, etc., using the provided double stranded oligonucleotide compositions.

Description

[0001] DOUBLE STRANDED OLIGONUCLEOTIDE COMPOSITIONS FORRNA INTERFERENCE AND METHODS RELATING THERETO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to 63 / 744,330, filed January 12, 2025; 63 / 750,252, filed January 27, 2025; and International Patent Application No. PCT / US2025 / 040766, filed August 5. 2025, the contents of which are incorporated by reference herein in their entireties.

[0003] This application incorporates herein by reference United States Provisional Application No.

[0004] 63 / 620,768, filed January 12. 2024; United States Provisional Application No. 63 / 625,263, filed January 25, 2024; United States Provisional Application No. 63 / 679,508, filed August 5, 2024; United States Provisional Application No. 63 / 691,245. filed September 5, 2024; International Patent Application No. PCT / US2025 / 011458, filed January 13, 2025; International Patent Application No, PCT / US2025 / 013253, filed January 27, 2025; International Patent Application No. PCT / IB2021 / 000351, filed May 24, 2021; International Patent Application No. PCT / US2022 / 044296, filed September 21. 2022; International Patent Application No. PCT / US2024 / 0181 9, filed March 1, 2024: and International Patent Application No. PCT / US2025 / 018204. filed March 3. 2025.

[0005] BACKGROUND

[0006] Gene-targeting oligonucleotides are useful in various applications, e.g., therapeutic, diagnostic, research and nanomaterials applications. The use of naturally -occurring nucleic acids (e.g.. unmodified DNA or RNA) in such applications can be limited by, for example, their susceptibility to endo- and exo-nucleases. As such, various synthetic counterparts have been developed to circumvent these shortcomings. These include synthetic oligonucleotides that contain chemical modifications, e.g., base modifications, sugar modifications, backbone modifications. There remains, however, a need in the art for double-stranded (ds) oligonucleotides with improved properties for use in connection with the above-described applications.

[0007] SUMMARY

[0008] The present disclosure is directed, in part, to the recognition that controlling structural elements of the oligonucleotides of a double -stranded (ds) oligonucleotide can have a significant impact on the ds oligonucleotide’s properties and / or activity. In certain embodiments, such structural elements include one or more of: (1) chemical modifications (e.g., modifications of a sugar, base and / or internucleotidic linkage) and patterns thereof; and (2) alterations in stereochemistry (e.g.. stereochemistry of a backbone chiral internucleotidic linkage) and patterns thereof. 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, theproperties and / or activities impacted by such structural elements include, but are not limited to, participation in, direction of a decrease in expression, activity or level of a gene or a gene product thereof, mediated, for example, by RNA interference (RNAi interference), RNase H-mediated knockdown, steric hindrance of translation, etc. Moreover, in certain embodiments, the properties and / or activities impacted by such structural elements include, but are not limited to, participation in, Ago2 loading, thermal stability, in vivo stability, delivery to tissues and into cells, among others.

[0009] in certain embodiments, the present disclosure demonstrates that compositions comprising ds oligonucleotides (e.g., dsRNAi oligonucleotides, also referred to as dsRNAi agents) with controlled structural elements provide unexpected properties and / or activities.

[0010] In certain embodiments, the present disclosure encompasses the recognition that stereochemistry, e.g., stereochemistry of backbone chiral centers, can unexpectedly maintain or improve properties of ds oligonucleotides. For example, but not by 'ay of limitation, the instant disclosure relates, in part, to ds oligonucleotides comprising a guide strand comprising backbone phosphor l guanidine chiral centers in the Sp configuration.

[0011] In a first aspect, the invention relates to a double -stranded RNAi (dsRN Ai) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: the guide strand is complementary or substantially complementary to a target RNA sequence; the guide strand comprises a seed region at its 5’ end region that is capable of mediating the initial recognition of the target RNA sequence, e.g., nucleotides at positions 2-6, at positions 2-7, or at positions 2-8, relative to the 5 ’ -end of the guide strand: the guide strand comprises

[0012] — p— o ''N 6 a backbone phosphoryl guanidine chiral center comprising the structure of, e.g.,

[0013]

[0014] ! 5(nOOl), e.g, in the Sp configuration, in the seed region, e.g., between the third (+3) nucleotide and the immediately downstream (+4) nucleotide, relative to its 5’ terminal nucleotide; and the guide strand comprises a contiguous or consecutive stretch of, e.g., five backbone phosphorothioate (PS) chiral centers, e.g., between the +13 nucleotide and the +18 nucleotide, relative to the 5’ terminal nucleotide, and / or a contiguous or consecutive stretch of, e.g., four backbone PS chiral centers, e.g., between the +19 nucleotide and the 3‘ terminal (N) nucleotide, each independently in, e.g, the Sp configuration or the / / p configuration. In an exemplary embodiment, the contiguous stretch of five backbone PS chiral centers between the +13 nucleotide and the +18 nucleotide, relative to the 5’ terminal nucleotide, and the contiguous stretch of four backbone PS chiral centers between the +19 nucleotide and the 3’ terminal (N) nucleotide, of the guide strand, are in the Sp configmation. In an exemplary embodiment, the passenger strand comprises a backbone PS chiral center, e.g., between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and / or a backbone PS chiral center, e.g.. between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide, each independently in. e.g. the Sp configuration or the Rp configuration. In an exemplaryembodiment, the backbone PS chiral center between the 5 termmal (+1) nucleotide and the +2 nucleotide, and the backbone PS chiral center between the 3" terminal (N) nucleotide and the penultimate (N-l), of the passenger strand, are in the Sp configuration. In an exemplary embodiment, the guide strand further comprises a backbone phosphoryl guanidine chiral centerr-'EN;

[0015] ■ V-N — P— O

[0016] "■■N p

[0017] comprising the structure of, e.g.,

[0018]

[0019] ' " (n(K)l). e.g., between the +7 nucleotide and the +8 nucleotide, and / or a backbone phosphoryl guanidine chiral center comprising the structure of, e.g., i — i

[0020] [ > N P O

[0021] '"hf O

[0022]

[0023] *r(nOOl), e.g.. between the +18 nucleotide and the +19 nucleotide thereof In an exemplary embodiment, the backbone phosphoryl guanidine chiral center comprising the structure1o

[0024] f P-O

[0025] ~" N 6

[0026] of?(nOOl) between the +7 nucleotide and the +8 nucleotide, and the backbone -N 9

[0027] [ \=N— p-0

[0028] "''N O

[0029]

[0030] phosphoiyl guanidine chiral center comprising the structure of 'f(nOOl) between the +18 nucleotide and the +19 nucleotide, of the guide strand, are in the S configuration. In an exemplary' embodiment, the passenger strand comprises or further comprises a backbone phosphory I r--N?

[0031] I )=N -P=O

[0032] "~N 6

[0033] guanidine chiral center comprising the structure of, e.g., 'f(nOOl), e.g.. between the +7 nucleotide and the + 8 nucleotide, and / or a backbone phosphoryl guanidine chiral center comprising r-'S’d T

[0034] I ^=N—p=0

[0035] 6

[0036]

[0037] the structure of, e g.,1 s(nOOl), e.g., between the +15 nucleotide and the +16 nucleotide, each independently in, e.g., the Sp configuration or the i?p configuration. In an exemplary embodiment, the backbone phosphoryl guanidine chiral center comprising the structure of

[0038]

[0039] (nOOl) between the +7 nucleotide and the +8 nucleotide, and the backbone

[0040] f'N- '

[0041]

[0042] phosphoryl guanidine chiral center comprising the structure of! s(nOOl) between the +15 nucleotide and the +16 nucleotide, of the passenger strand, are in the A’p configuration.

[0043] In an exemplary' embodiment of the first aspect, neither the guide strand nor the passengerstrand oi the siRNAi agent comprises a lipid ligand or a non-hpid ligand.

[0044] In a second aspect, the invention relates to a double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein the guide strand is complementary or substantially complementary' to a target RNA sequence, and wherein the guide strand comprises one or more N-3-uridine base

[0045]

[0046] modifications (N3U) having the structure '

[0047] In certain embodiments, the guide strand comprises an N3U base modification at the 5" terminal (+1) nucleotide. In particular embodiments, the guide strand comprises an N3U base modification at the 3’ terminal nucleotide, the penultimate (N-l) nucleotide, or at both the 3’ terminal nucleotide and the penultimate (N-l) nucleotide. In certain embodiments, the guide strand comprises an N3U base modification at the penultimate (N-l) nucleotide.

[0048] In a third aspect, the invention relates to a double-stranded RNAi (dsRNAi) agent capable of directing target-specific RN A interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: the guide strand is complementary or substantially complementary to a target RNA sequence; the guide strand comprises a seed region at its 5’ end region that is capable of mediating the initial recognition of the target RNA sequence, e.g., nucleotides at positions 2-6, at positions 2-7. or at positions 2-8, relative to the 5 ’-end of the guide strand; the guide strand comprises r —! 0 I b=N — P=O

[0049]

[0050] o a backbone phosphoryl guanidine chiral center comprising the structure of, e.g.,!(nOOl). e.g., in the. Sp configuration, in the seed region, e.g., between the third (+3) nucleotide and the immediately downstream (+4) nucleotide, relative to its 5 ’ terminal nucleotide; and the passenger strand comprises a backbone phosphoryl guanidine chiral center comprising the structure of, e.g.,rN?

[0051] | P O

[0052] ~''N 6

[0053]

[0054] ! ’ (nOOl), between the 5’ terminal ( h nucleotide and the immediately downstream (+2) nucleotide in, e.g., the Rp configuration. In an exemplary1embodiment, the passenger strand further comprises a backbone phosphoryl guanidine chiral center comprising the structure of, e.g.,

[0055] f )=N— P-O

[0056] ''N

[0057]

[0058] s(nOOl), between the 3’ terminal (N) nucleotide and the penultimate ( -l) nucleotide in, e.g., the Rp configuration. In an exemplary' embodiment, the nucleoside 3' to the backbone phosphoryl guanidine chiral center in the seed region comprises a 2"-F ribose modification. In an exemplary embodiment, the guide strand further comprises a backbone phosphory l guanidine chiral.?

[0059] I }=N-P=O

[0060] '' N 6

[0061] center comprising the structure of, e.g., 'f(nOOl), between the +10 nucleotide and the immediately downstream (+11) nucleotide. relative to the 5‘ terminal nucleotide of the guide strand. In an exemplary embodiment, the nucleoside 3’ to the backbone phosphoryl guanidine chiral center between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5' terminal nucleotide of the guide strand, comprises a 2‘-F ribose modification. In an exemplary embodiment, the backbone phosphoryl guanidine chiral center between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5' terminal nucleotide of the guide strand, is in, e.g., the Rp configuration. In an exemplary embodiment, the guide strand comprises a 5’ phosphate modification, e.g., a 5’ phosphate mimic modification. In an exemplary embodiment, the

[0062] 5’ phosphate mimic modification is, e

[0063]

[0064] .g., ’, wherein: the base is, e.g., N3U, or is selected from, e.g., A, C, G. T, U, abasic, and modified nucleobases other than N3U; and R1is selected from, e.g., H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2’- 0,4 'C-methylene -bridged or locked nucleic acid (2’,4’-BNA or LNA). In an exemplary embodiment. R1is, e.g., O-methyl (O-Me). In an exemplary’ embodiment, the guide strand further comprises a backbone phosphoryl guanidine chiral center comprising the structure of. e.g.,1o

[0065] f ) N — P“O

[0066] ''N 6

[0067]

[0068] *f(nOOl), between, e.g., the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide in, e.g., the / <p configuration, and / or a backbone phosphoryl guanidine chiral center r-N 9

[0069] L =N— P-0

[0070] ' N Q

[0071] comprising the structure of. e.g,.?(nOOl), between, e.g., the penultimate (N-l) nucleotide and the immediately’ upstream (N-2) nucleotide in. e.g., the p configuration. In an exemplary’ embodiment, the guide strand does not comprise a backbone phosphoryl guanidine chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide. In an exemplary’ embodiment, the guide strand does not comprise a backbone phosphory l guanidine chiral center between the penultimate (N-l) nucleotide and the immediately' upstream (N-2) nucleotide. In an exemplary embodiment, the guide strand comprises a phosphorothioate (PS) chiral center between, e.g., the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide in, e.g.. the Sp configuration. In an exemplary embodiment, the guide strand comprises, e.g., a phosphoryl guanidine cap at, e.g., its 5 ’-end (5 ’-end PN cap). In an exemplary embodiment, the 5 ’-end PN cap is selected from, e.g.,

[0072]

[0073] wherein: the base is N3U, or is selected from, e.g., A, C, G. T, U, abasic, and modified nucleobases other than N3U; and R1is selected from H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2’-O,4’C-methylene-bridged or locked nucleic acid (2’,4’-BNA or LNA). In an exemplary embodiment, R1is, e.g., O-methyl (O- Me). In an exemplary' embodiment, the guide strand comprises, e.g., a natural phosphate linkage (PO) between, e.g., the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and / or, e.g., a natural phosphate linkage (PO) between, e.g., the +2 nucleotide and the +3 nucleotide, relative to the 5’ terminal nucleotide.

[0074] hi a fourth aspect, the invention relates to a double-stranded RNAi (dsRNAi) agent capable of directing target-specific RN interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: the guide strand is complementary or substantially complementary to a target RNA sequence; the guide strand comprises a seed region at its 5’ end region that is capable of mediating the initial recognition of the target RNA sequence, e.g., nucleotides at positions 2-6, at positions 2-7, or at positions 2-8. relative to the 5 ’ -end of the guide strand: the guide strand comprises 9 =N— p=o 6 a backbone phosphoryl guanidine chiral center comprising the structure of. e.g., ' (nOOl), e.g., in the > Sp configuration, in the seed region, e.g., between the third (+3) nucleotide and the immediately downstream (+4) nucleotide, relative to its 5’ terminal nucleotide; and the guide strand comprises a phosphoryl guanidine cap at its 5 ’-end (5 ’-end PN cap) In an exemplary

[0075] embodiment, the 5 ’-end PN cap is selected from, e.g.,

[0076]

[0077] and

[0078]

[0079] .wherein: the base is, e.g., N3U, or is selected from, e.g., A, C, G, T, U,abasic, and modified nucleobases other than N3U; and R is selected from, e.g., H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2’-(?.4'C-methylene-bridged or locked nucleic acid (2',4’-BNA or LNA). In an exemplar}' embodiment, R!is, e.g., O-methyl (O-Me). In an exemplar}' embodiment, the guide strand further comprises a backbone phosphoryl guanidine chiral center i I

[0080] Ri M —? P-- o

[0081] ~'N 6

[0082] comprising the structure of, e.g.,

[0083]

[0084] *5(nOOl), between, e.g., the 3 ' terminal (N) nucleotide and the penultimate (N-l) nucleotide in. e.g., the A'p configuration, and / or a backbone phosphoryl U 9

[0085] i >=N— P'" O

[0086] 'N 6

[0087] guanidine chiral center comprising the structure of, e.g..

[0088]

[0089] *11(tiOOI). between, e.g.. the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide in, e.g.. the Rp configuration. In an exemplar ' embodiment, the guide strand does not comprise a backbone phosphoryl guanidine chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-1) nucleotide. In an exemplar ' embodiment, the guide strand does not comprise a backbone phosphoiy'I guanidine chiral center between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide. In an exemplary embodiment, the guide strand comprises a phosphorothioate (PS) chiral center between, e.g., the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide in. e.g., the. Sp configuration.

[0090] In a fifth aspect, the invention relates to a double -stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: the guide strand is complementary or substantially complementary to a target RNA sequence; the guide strand comprises a seed region at its 5’ end region that is capable of mediating the initial recognition of the target RNA sequence, e.g., nucleotides at positions 2-6, at positions 2-7. or at positions 2-8, relative to the 5 ’ -end of the guide strand; the guide strand comprises U, y=N— 9 p~o ~ N p a backbone phosphoryl guanidine chiral center comprising the structure of. e.g.,!' (nOOl), e.g., in the Sp configuration, in the seed region, e.g., between the third (+3) nucleotide and the immediately downstream (+4) nucleotide, relative to its 5’ terminal nucleotide; and the guide strand comprises a natural phosphate linkage (PO) between, e.g., the 5‘ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and / or a natural phosphate linkage (PO) between, e.g., the +2 nucleotide and the +3 nucleotide, relative to the 5’ terminal nucleotide. In an exemplary embodiment, the guide strand comprises a 5’ phosphate modification, e.g., a 5’ phosphate mimic modification. In an exemplary' embodiment, the 5’ phosphate mimic modification is. e.g..

[0091]

[0092] , wherein: the base is, e.g., N3U. or is selected from, e.g.. A, C, G. T, U, abasic, and modified nucleobases other than N3U; and R1is selected from, e.g., H. OH, O-alkyl, O-methyl (O-Me), F. O-methoxyethyl (MOE), and 2’-O,4’C-methylene-bridged or locked nucleic acid (2’,4’-BNA or LNA). Tn an exemplary embodiment, R1is, e.g.. O-methyl (O-Me). Tn an exemplaty embodiment, the guide strand further comprises a backbone phosphory l guanidine chiral i 1

[0093] -N?

[0094] I =N — P— O

[0095] '"''N 6

[0096] center comprising the structure of, e.g,, * (nOOl), between, e.g,, the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide in, e.g., the Ap configuration, and / or a backbone1o

[0097] ! >=N-P=O

[0098] '■'N O

[0099] phosphory l guanidine chiral center comprising the structure of, e.g.,! J(nOOl), between, e.g., the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide in, e.g., the Rp configuration, hi an exemplary embodiment, the guide strand does not comprise a backbone phosphoiyd guanidine chiral center between the 3’ terminal (N) nucleotide and the penultimate (N- 1) nucleotide. In an exemplary embodiment, the guide strand does not comprise a backbone phosphoryl guanidine chiral center between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide. In an exemplary embodiment, the guide strand comprises a phosphorothioate (PS) chiral center between, e.g.. the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide in, e.g. the 5’p configuration.

[0100] In a sixth aspect, the invention relates to a double -stranded RNAi (dsRNAi) agent capable of directing target-specific RN interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: the guide strand is complementary’ or substantially complementary’ to a target RNA sequence; the guide strand comprises a seed region at its 5‘ end region that is capable of mediating the initial recognition of the target RNA sequence, e.g., nucleotides at positions 2-6, at positions 2-7, or at positions 2-8, relative to the 5 ’-end of the guide strand: the guide strand comprises J?

[0101]

[0102] a backbone phosphoryl guanidine chiral center comprising the structure of, e.g.,! J(nOOl). e.g., in the Sp confi uration, in the seed region, e.g., between the third (+3) nucleotide and the immediately downstream (+4) nucleotide, relative to its 5’ terminal nucleotide; wherein the nucleoside 3‘ to the backbone phosphoryl guanidine chiral center in the seed region comprises a 2’-F ribose modification. In an exemplary embodiment, the guide strand further comprises a backbone,-N?

[0103] | )=N—P=O

[0104] '''N 6

[0105] phosphoryl guanidine chiral center comprising the structure of, e.g., 'f(nOOl), between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5’ terminal nucleotide of the guide strand. In an exemplary' embodiment, the guide strand further comprises a backbone phosphoryl guanidine chiral center between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5‘ terminal nucleotide of the guide strand. In an exemplary embodiment, the nucleoside 3’ to the backbone phosphoryl guanidine chiral center between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5‘ terminal nucleotide of the guide strand, comprises a 2’-F ribose modification. In an exemplary' embodiment, the backbone phosphoryl guanidine chiral center between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5’ terminal nucleotide of the guide strand, is in. e.g., the Rp configuration. In an exemplary embodiment, the guide strand further comprises a I

[0106] r-N? y=N— p=o

[0107]

[0108] 6 backbone phosphoryl guanidine chiral center comprising the structure of, e.g.,!(nOOl). between, e.g., the 3’ terminal (N) nucleotide and the penultimate ( N-l ) nucleotide in. e.g., the Rp configuration, and / or a backbone phosphoryl guanidine chiral center comprising the structure.?

[0109] r-N 9

[0110] ; )=N— P=O

[0111] ~'N 6

[0112] of, e.g., ‘ (nOOl). between, e.g., the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide in, e.g., the Rp configuration. In an exemplary' embodiment, the guide strand does not comprise a backbone phosphory l guanidine chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide. In an exemplary embodiment, the guide strand does not comprise a backbone phosphoryl guanidine chiral center between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide. In an exemplary' embodiment, the guide strand comprises a phosphorothioate (PS) chiral center between, e g, the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide in, e.g., the Sp configuration. In an exemplary embodiment, the guide strand comprises a 5’ phosphate modification, e.g., a 5’ phosphate mimic modification. In an exemplary embodiment, the 5’ phosphate mimic modification is, e.g.,

[0113] 6 R1

[0114]

[0115] , wherein: the base is, e.g., N3U. or is selected from, e.g.. A, C, G, T, U, abasic, and modified nucleobases other than N3U; and R1is selected from, e.g., H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2’-O,4’C-methylene-bridged or locked nucleicacid (2’,4’-BNA or LNA). In an exemplary embodiment, R1is, e.g., O-nietliyl (O-Me).

[0116] hi particular embodiments of the first, second, third, fourth, fifth, and sixth aspects of the present disclosure, the dsRNAi agent comprises a 5' phosphate modification. In certain embodiments, the 5’ phosphate modification is a 5’ phosphate mimic modification, hi particular embodiments, the 5’ phosphate mimic modification is selected from

[0117]

[0118] 

[0119]

[0120]

[0121]

[0122]

[0123] 10

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] N3U, or is selected from, e.g.. A, C. G, T, U. abasic, and modified nucleobases other than N3U; R1is selected from, e.g., H. OH, O-alkyl, O-methyl (O-Me), F. O-methoxy ethyl (MOE), and 2’-O,4’C-methylene-bridged or locked nucleic acid (2',4'-BNA or LNA); R2is selected from alkyl, methyl, ethyl, isopropyl, propyl, cyclohexyl, benzyl, phenyl, tolyl, xylyl, aryl, and arene group; and R6is selected from H, methyl, ethyl, isopropyl, propyl, cyclohexyl, benzyl, phenyl, tolyl, xylyl, aryl, and arene group. In certain embodiments, R1is O-Me. In particular embodiments, the 5’ phosphate

[0131] mimic modification i

[0132]

[0133] s In particular embodiments. R2in

[0134] 0

[0135]

[0136] I is methyl. In particular embodiments, the guide strand further comprises a backbone phosphorothioate (PS) chiral center in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and a backbonePS chiral center in the Sp configuration between the +2 nucleotide and immediately downstream (+3) nucleotide.

[0137] In particular embodiments, the guide strand further comprises a backbone phosphorothioate (PS) chiral center in the Sp configuration between the 5’ terminal ( b nucleotide and the immediately downstream (+2) nucleotide, and wherein the backbone linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide of the guide strand is an unmodified phosphodiester (PO) linkage.

[0138] In accordance with another aspect of the disclosed subject matter, the present disclosure provides a double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein the guide strand is complementary' or substantially complementary to a target RNA sequence, wherein the guide strand comprises a 5’ phosphate modification, and wherein the guide strand further comprises a backbone phosphorothioate (PS) chiral center in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and a backbone PS chiral center in the Sp or Rp configuration between the +2 nucleotide and immediately downstream (+3) nucleotide.

[0139] In particular embodiments, the backbone PS chiral center between the +2 nucleotide and immediately downstream (+3) nucleotide is in the Sp configuration. In alternative embodiments, the backbone PS chiral center between the +2 nucleotide and immediately downstream (+3) nucleotide is in the Rp configuration.

[0140] In accordance with another aspect of the disclosed subject matter, the present disclosure provides a double -stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsR Ai agent comprising a guide strand and a passenger strand, wherein the guide strand is complementary or substantially complementary to a target RNA sequence; wherein the guide strand comprises a 5" phosphate modification; wherein the guide strand further comprises a backbone phosphorothioate (PS) chiral center in the Sp configuration between the 5‘ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide; and wherein the backbone linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide of the guide strand is an unmodified phosphodiester (PO) linkage.

[0141] In particular embodiments, the 5' phosphate modification is a 5’ phosphate mimic modification. In certain embodiments, the 5‘ phosphate mimic modification is selected from:

[0142]

[0143]

[0144]

[0145] the base is selected from A. C, G, T. U, abasic. and modified nucleobases other than N3U;

[0146] R1is selected from H. OH, O-alkyi, O-methyl (O-Me). F, O-methoxy ethyl (MOE), and 2’- 0.4’C-methylene-bridged or locked nucleic acid (2’, 4’ -BN A or LNA); and

[0147] R2is selected from alkyl, methyl, ethyl, isopropyl, propyl, cyclohexyl, benzyl, phenyl, tolyl, xylyl, aryl, and arene group.

[0148] In certain embodiments. the 5’ phosphate mimic modification is

[0149]

[0150] ’, In particular embodiments, R’ is LNA bridge to the --T position. In certain embodiments. R1is MOE. In particular embodiments, R1is F. In certain embodiments, R2in

[0151]

[0152] o- O- P::=O

[0153] R2Base

[0154] I

[0155] 0 R1

[0156]

[0157] * is methyl. In certain embodiments, the 5’

[0158] phosphate mimic modification i

[0159]

[0160] s *, where the base is U or abasic, R’ is H or O-alkyl. In particular embodiments, R’ is O-Cie alkyl. In particular embodiments. R6is H.Base oj.orf OR8O R1hi particular embodiments, the 5’ phosphate mimic modification i

[0161]

[0162] s

[0163] where R1is O-Me and Rcis H. In particular embodiments,

[0164]

[0165] is

[0166]

[0167] In certain embodiments, the 5’ phosphate mimic modification

[0168]

[0169] is where the base is abasic and R‘ is H.

[0170] In particular embodiments, the 5’ phosphate mimic modification is

[0171]

[0172] where the R1is O-Me and R6is H.

[0173] In particular embodiments, the guide strand further comprises a backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide. In certain embodiments, the guide strand further comprises backbone PS chiral centers in Sp configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide. In particular embodiments, the guide strand comprises an Rp, Sp, or stereorandom non- negatively charged internucleotidic linkage between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5 ' terminal nucleotide. In particular embodiments, the guide strand further comprises a backbone phosphoryl guanidine chiral center between the +10 nucleotide and the +11 nucleotide is in the Rp configuration, hr certain embodiments, the guide strand comprises one or more Rp, Sp, or stereorandom non-oegatively charged internucleotidic linkage occurs 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-l) nucleotide of the guide strand, where N is the 3" terminalnucleotide. In particular embodiments, the guide strand comprises a 2 modification, of the 3 nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage. In certain embodiments, the guide strand comprises an Rp, Sp. or stereorandom non-negatively charged internucleotidic linkage between the seventh (+7) and eighth (+8) nucleotides, relative to the 5' terminal nucleotide. In particular embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkage occurs between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide.

[0174] In certain embodiments, the passenger strand comprises one or more Rp. Sp. or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand. In particular embodiments, the passenger strand comprises one or more Rp, Sp. or stereorandom non-negatively charged internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand. In certain embodiments, the passenger strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage occurs downstream, i.e.. in the 3’ direction, relative to the central nucleotide of the passenger strand. In particular embodiments, the passenger strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage occurs upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand. In certain embodiments, the passenger strand in combination with one or more of the aforementioned guide strands, comprises one or more modified sugars between the 5’ terminal (+1) nucleotide and the penultimate (N-l) nucleotide.

[0175] In particular embodiments, the passenger strand comprises one or more of 0-n Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49, one or more backbone chiral centers in Rp or Sp configuration, one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +7 nucleotide and the immediately downstream (+8) nucleotide, i.e., in the 3’ direction, one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +15 nucleotide and the immediately downstream (+16) nucleotide, i.e., in the 3' direction, and / or backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+ 1) nucleotide and the immediately downstream, i.e., in the 3’ direction, (+2) nucleotide and between the 3‘ terminal nucleotide and the penultimate (N-l) nucleotide.

[0176] In particular embodiments, each strand of the dsRNAi agent independently has a length of about 15 to about 49 nucleotides.

[0177] In particular embodiments, the dsRNAi agent of any of the preceding claims, wherein the Rp. Sp. or stereorandom non-negatively charged backbone internucleotidic linkages have neutralcharge. In particular embodiments, the neutral backbone internucleotidic linkage is [CH2]nCH3

[0178] r"\, OA

[0179] V=--N— P<

[0180] % O o, /

[0181]

[0182] [CHzlmCH3, wherein n is about 0 to 49 and m is about 0 to 49.

[0183] In particular embodiments, the guide strand comprises a linkage having the following structure between the third ( +-3) and fourth (+4) nucleotides of the guide strand, between the tenth (+10) and eleventh (+11) nucleotides of the guide strand, or both.

[0184] In particular embodiments, guide strand comprises a linkage having the following structure /

[0185] r >N-pr°

[0186]

[0187] between the third (+3) and fourth (+4) nucleotides of the guide strand, between the seventh (+7) and eighth (+8) nucleotides of the guide strand, between the tenth (+10) and eleventh (+11) nucleotides of the guide strand, between the eighteenth (+18) and nineteenth (+19) nucleotides of the guide strand, or combinations thereof.

[0188] In particular embodiments, the passenger strand comprises a linkage having the following [CH2]nCH3

[0189] r y=N-- p<°

[0190] 6

[0191] structure

[0192]

[0193] [CH2]mCH3, where n is about 0 to 49 and m is about 0 to 49, at 5' to the central nucleotide of the passenger strand. 3’ to the central nucleotide of the passenger strand, or both.

[0194] In particular embodiments, the passenger strand comprises a linkage having the following [CH2]nCH3

[0195] ~'N o

[0196]

[0197] structure (1CH21JmCH3. wherein n is about 11 to 49 and m is 0, or n is 11 or 15.

[0198] In particular embodiments, the passenger strand comprises a non-negatively charged backbone internucleotidic linkage in the Rp configuration between the +7 nucleotide and the +8 nucleotide, a non-negatively charged backbone internucleotidic linkage in the Rp configuration between the +15 nucleotide and the +16 nucleotide and / or a backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+1) nucleotide and the +2 nucleotide and between the 3" terminal nucleotide and the penultimate (N-l) nucleotide.

[0199] In particular embodiments, the passenger strand comprises:p-o' "

[0200] /

[0201] N

[0202] 'N N

[0203]

[0204] in the Rp configuration between the +7 nucleotide and the +8 nucleotide.

[0205] P~O

[0206] /

[0207] N N N

[0208]

[0209] in the Rp configuration between the +15 nucleotide and the +16 nucleotide, and a backbone phosphorothioate chiral centers m the Sp configuration between the +1 nucleotide and the +2 nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

[0210] In particular embodiments, the passenger strand comprises:

[0211] " K / ? X

[0212] / p-o

[0213] N

[0214] / ftN

[0215]

[0216] '' — / in the Rp configuration between the +7 nucleotide and the +8 nucleotide and backbone phosphorothioate chiral centers in the Sp configuration between the +1 nucleotide and the -;-2 nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

[0217] In accordance with another aspect of the disclosed subject matter, the present disclosure provides a method for reducing level and / or activity of a transcript or a protein encoded thereby, comprising administering to a ceil expressing the transcript a double-stranded RNAi (dsRNAi) agent of any of the above aspects or embodiments.

[0218] In certain embodiments, the cell is an immune cell, a blood cell, a cardiac cell, a lung cell, an optic cell, a muscle cell, a Us er ceil, a kidney cell, a cell of the central nervous system, or a cell of the peripheral nervous system. In particular embodiments, the cell of the central nervous system is a brain cell. In certain embodiments, when the double-stranded RNAi (dsRNAi) agent is contacted with a cell comprising transcripts of both the target allele and another allele of the same nucleic acid sequence, it shows suppression of transcripts of the particular allele at a level that is greater than when the double-stranded RNAi (dsRNAi) agent is absent, greater than a level of suppression observed for another allele of the same nucleic acid sequence, both greater than when the double¬ stranded RNAi (dsRNAi) agent is absent, and greater than a level of suppression observed for another allele of the same nucleic acid sequence.It is to be understood that both the foregoing general description and the following detailed description are examples and are provided for purpose of illustration and not intended to limit the scope of the disclosed subject matter in any manner.

[0219] I. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Technologies of the present disclosure may be understood more readily by reference to the following detailed description of certain embodiments.

[0220] Definitions

[0221] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the elements are identified in accordance with the Periodic 'Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in " Organic Chemistry ", Thomas Sorrell. University Science Books, Sausalito: 1999, and " March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M. B. and March. J.. John Wiley & Sons, New York: 2001.

[0222] As used herein, the terms “aliphatic”, “alkenyl”, “alkyl”, “alkynyl”, “analog”, “animal”, “aryl”, “chiral control”, “chirally controlled oligonucleotide composition”, “cycloaliphatic,” “heteroaliphatic”, “heteroalkyl”, “heteroaryl”, “heteroatom”, “heterocycle”, “identity”, “internucleotidic linkage”, “in vitro”, “in vivo”, “linkage phosphorus”, “modified nucleobase”, “modified nucleoside”, “modified nucleotide”, “modified sugar”, “nucleic acid”, “nucleobase”, “nucleoside”, “nucleotide”, “oligonucleotide”, “oligonucleotide ty pe”, “substituted”, “optionally' substituted”, “stable”, “P-modification”, “partially unsaturated”, “pharmaceutical composition”, “pharmaceutically' acceptable”, “pharmaceutically acceptable carrier”, “pharmaceutically acceptable salt”, “predetermined”, “5' phosphate mimic modification”, “protecting group”, “subject” or “test subject”, “substantially”, “sugar”, “susceptible to”, “therapeutic agent”, “therapeutically' effective amount”, “treat,” “treatment,” or “treating”, “unsaturated”, “wild-type”, and the like have their art-understood meaning as would be appreciated by those of ordinary skill in the art, and as specifically defined in WO 2024 / 182749, at pages 147-179, the contents of which are incorporated herein by reference in their entirety.

[0223] L P^riptiQn yf C rtain Emfrodintyitys

[0224] As examples, certain dsRNAi oligonucleotides comprising certain example base sequences, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, internucleotidic linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and / or additional chemical moieties are presented in Table 1 (e.g.. Tables IB. 1C, ID, and IE), below. Among other things, ds oligonucleotides, e g.. those in Table 1, may be utilized to target a transcript, e.g.. to reduce the level of a transcript and / or a product thereof.kd S N HELMaeequence

[0225] GG SS UUAUAACAAR- 006266 GG1AACACUUUU

[0226] U GG SS UUAUAACAAR- GG 00626AACACUUUU17

[0227] U

[0228] £ s < GG SS UUAUAACAAR3- <3 £ <3 2,

[0229] E E E GG 006268AACACUUUU1 E E E E E

[0230] jd d d i d d d d d

[0231] <£ q <c A <" A U

[0232] r— ’ rN " rsi i i " <' A < A < A < A < A

[0233] x; rj

[0234] rsi > Csi v> rsi < / > i GG SS < UUAUAACAARN- v> fXj ” >

[0235] v> r\i v> rxi

[0236] v M-, w “ l / V>; ■-!- r - GG 006269iAACACUUUU1zv M-, V> M-, w M-, V>

[0237] r-A X5- <z> x vi- X) vF di <z> J2; | <z> Ai <z> 82; UF UF U

[0238] o 2 CD •— % O ZD o b~ o b O 23 O Z) O ZDGG SS UUAUAACAAR- O 7T O 7T o ~ o 'cr O 'X o ^d c £ c: E I c E c e: c E X E jS GG 00620AACACUUUU17

[0239] <£ CL; <t CL <* lx <“ lx < C £X < C ex < F lx -S lx > — ■.y) > — > ( / j - — < - — '.yj ■— - - co: ( / i - — t / i U E Kl i E A. E E ¥1 E 82, E X E 82, E 82,

[0240] d — GG SS UUAUAACAAR- d 23 Ji Z) d 23 9- =)! d 23 d 23

[0241] O ~ 2 'g s’ 88 T X T GG 0062AACACUUUU171 ii ~E ii 'E

[0242] E X E X E £ E.--- E i E E -A O- „ O- U < S °- d d S’ 1 ^> L I / ) d S’ d S’ un X « «— * < / )

[0243] <3 £ <3GG SS UUAUAACAAR- <g. “ <3. “ S' - S ~

[0244] E 5' E Z E 2 E 2 E 2, a E S E d, GG 00622AACACUUUU17

[0245] d d E _d E d E _d E _d E d p _d E U < d <£ d! < Q. < d < d d,9; 3i. <77 2f i IT 'jz 23 'g’ 22! g T GG SS UUAUAACAAR- E S E o

[0246] d E d E d E i d E d E d E.a? GG 00623AACACUUUU17

[0247] <3 _d <3 d. (3 _d <3 _d <3 X! <3 -S fx S X U 13 T? <3r“ ’ <3 T7 <3 TT <3 T As S £c o {()()()()()()()()())()[][fl][][][fl][fl]()[][fl]([GGS200S22GG00S2G SS UUAUAACAA RNAlUURA1UAACA1ARmsprspmnrpmprpmpmpmpmnrp! Ti-........... S E S £ S E S E E. X S £ XL SC F, E

[0248] d J2: d d d d Ji i d Ji d X d 2 $$$()()()()()()()()()()()}$][][fl][j[fl][][] GG00S00S200S2GSS200062AACACUUUU1A1A1CACUUUUUV174 nmnrnmprpmpmpmpmpmspmspm,...........

[0249] <■ O <f 2- < " 2- <" 2. < ZD < 2. < o XL O U '7C' o £ E E i E x SC c. X, E E E JE 1 i, {()()()()()()()()()()()()[][fl][][][fl][fl][][fl]GGS200S22GG00S2G SS UUAUAACAA RNAlUURA1UAACA1ARmsprspmnrpmprpmpmpmpmnrpm-...,....... d.2: ■ d.ii d „A d ^d d jx jx JJ O

[0250] S X ZD — - ZD ° ZD 7) o GG 0062AACACUUUU175$$$$()(()(()()(()())()}[][fl])[][][fl]))[]([]00S200S00S2GSS20A1AC1ACUUUUUV1 pmnrnmnrpmpmpmpmpmspmspm............2. id o o E H i H 1= TT c E d U rxi CM CM -it £4 d ™ Ji s H= Z) s A: 9= 2. ±L < ■ ±X f“l ±L < X <- o _x -X i _X O _X, < <z> E < Z) AX 1 < Z) o < Z) UF E <7F T2’ x s <z> 2 H CN H c: rH rsj Q ■.; Q o S s s O cjr o UF o O " O r“! o o o u O <3 o -r i o <r O *2 c o SC DC' SC c ", S=. 9- o JE Ji Del E “ X ice E — E

[0251] <£ Ji, <t u < X O; ™ d <, d < d

[0252] E <_ E E E <, E E E c E

[0253] ci i ci ZC£ Zx Q. Ji TT S' ex cj i ex r-i CN 2 rN S" Aj w £Z? -JC-! W f—> tr SL ne >- t£ x-j £3C ne 52ce, c i ££ O * § — ’: — c S < N S. S S Ji ZD Ji ZD Ji ZD Cl 2- 12- — IJ <, H 2, id r-J A § i A 'j= E 5 E 5 E <5 E £ I' ±L £N x, EL X d d d d _A O

[0254] CX O lx X < X < lx < X < lx <_ lx uo sc uo uo w rcr i <2 yj i / ) 77 7T LD T7 <zi TF <zi 7Z7 <z) T7 szs!ct CN! fN r\i CM fN -. X?.< z? o 2, E, 2 F, 2, F, 2 ^.2 ° — E

[0255] E E.2 i E.2 E J2, E E J: cn dr < ijd < *P1 < 'Pl < ■pi < Pl < Pl <

[0256] < C c I c < L Q sc C sc sc *£ sc 2» x; "? x z: E z: E H E E E O £t SC Ct CX tx: ex c£ ex ex: ex ex c£ d i cc d

[0257] Q

[0258]

[0259]

[0260]

[0261] {()()()()()()()()()()()()[][fl][][][fl][fl][][fl]GGS200S22GG00S2G SS UUAUAACAA RNAlUURA1UAACA1ARmsprspmnrpmprpmpmpmpmnrpm-...........

[0262] $$$$()(()(()()(()()))}[fl])[][][fl])[])[]([]( GG200S00S200SGSS2000626AACACUUUUAAC1ACUUUUUV1711 pmprnmnrnmpmpmpmpmspiTispm............

[0263] U GG UUAUAACAA GGAACACUUUU U GG UUAUAACAA GGAACACUUUU U GG UUAUAACAA GGAACACUUUU U H GG UUAUAACAA GGAACACUUUU U GG UUAUAACAA GAACACUGUUU

[0264] 1 i I I

[0265] 1 U

[0266] GG UUAUAACAA 1 GGAACACUUUU X U X GG UUAUAACAA GGAACACUUUU U T GG UUAUAACAA GGAACACUUUU

[0267] 1

[0268] U

[0269] x X x GG UUAUAACAA GGAACACUUUU

[0270] | X U

[0271] GG UUAUAACAA H H GGAACACUUUU U

[0272] T

[0273] ~ZL,

[0274]

[0275]

[0276] {()()()()()()()()()()()()[][fl][][][fl][fl][][fl]GGS200S22GG00S2G SS UUAUAACAA RNAlUURA1UAACA1ARmsprspmnrpmprpmpmpmpmnrpm-...........

[0277] $$$$()(()(()()(()()))}[fl])[fl])[])[j([]( GG 0062982200SGSS20AACACUUUU1AACAC1UUUUUV pmprpmprpmnmpmpmpmspmspm............

[0278] U GG SS UUAUAACAAR- GG 006299AACACUUUU1

[0279] U GG SS UUAUAACAAR- GG 006300AACACUUUU1

[0280] U

[0281] s. <5 s

[0282] £ E GG UUAUAACAA. £ s £ £ 3 S 3 SSR E- E E E E E E E _d jd. jd GG 00630AACACUUUU11 jd jd jd jd jd _d Ji < < <_ U < £ £ < < < Ai Ai o CD

[0283] 5GG SS UUAUAACAAR- A CM CM (N CM o

[0284] CM " fc. q i±. q X OM~, r-j / J, £N

[0285] > c s > s >!> CM GT CJ un GG 006302AACACUUUU1 -A < / > X GT S un < / > un < / > un < / > CQ v> <z> v> CH v>

[0286] > o >

[0287] o O o V7 U S

[0288] o JJ- § s § gv>ri o v> s gv>

[0289] § § s £ c v> cr GG SS UUAUAACAAR- IE. z> IE, z> X23-1X

[0290] £ GAACACUGUUU 0106303 < E < E < E < E < E < E < f p X c X C r-^, E E E U E Q. E Q.. Q_ r> CJ- ft tn ft tn ft tn i l 1 1 11 & JiGG SS UUAUAACAAR- s o S3- O O o (J o' X o' X Si S’ 2 s- E 3 r 3 £ 2, GG 00630AACACUUUU14 E 3 ~E 3 'E 3 E 3 'E X E “ E “

[0291] d d. E d E d E jd E d E jd E jd ZD d z) E U o b cZ o cZ o £ b aS « aS £ 1- £ E S 'E TT V) " X <s> x x xGG SS UUAUAACAAR- p X P £ X £ E *£>, E E E dL E dL " CL " CL " CL E X Q Q CL ‘-A n Q Cl Cl n Q CL GG 00630AACACUUUU15 X GO x on

[0292] < X < < 3 s ~ £ '•d £ U -S- ~£ 'E -2- 'E E E £ E 3 E 3 ~E £ E d E d E d E d E d E Q. GG SS UUAUAACAAR- CL E CL E gL £L £L d E d ji.2: Q° X*

[0293] o — o — o X o' 3 a x (D X O 3 a x GG 006306AACACUUUU1 £ s E E E X E X X •“3E T7 d TZ1*N \T o U CM J2L CM,d CM jd CM jd- CM o CM o g E g E S £ JE X X ±L ID ±L < D ±L ^D ±L (D X d GG SS UUAUAACAAR- d jx 55 d d? ji 55 Sk 3 £ E 2z £ 3 O - E" < P GG o 00AACACUUUU14474 <? o < E < d -d o •X o S E < V £ E " S " ji ji

[0294] E 3 E:□ E b E' 77 U E 5 E d E JE E d E 2. X § jd d. ~— ji ^d 73 ji o o ji O Ji 3 -i E £ E SSR-GG UUAUAACAA —; £ O ZD CD ZD ZD ZD ZD 3 q~ O — ■ o 3 ji £ s " d GG 00630AACACUUUU17 TT c TD c TT o TD O Ai (J CM ji CM Ji CM X S’ ji S’ CM CM X U s X ID M=, Z> X ID X X, XL fc. E E= £ S CM ■—r o d. ^d. fd E GT E GT E on ±L ts? E IZI O GT E GT S i / > -s? — i <

[0295] S S S s S Ji S Ji o “ o o S Ji o SSL o u o u o u O " o o u o u o u o o c " c: o r: X c X £Z X c: ’Tt c: x c: x £, CM o E X E X E “ E X E X E X E i s

[0296] < ■±? £ d <. _d < d <, ji <_ Ji S sd £ &- i ^d £

[0297] E <_ E E <_ E E ~ E <j E E < E <_ E < Z • U

[0298] S TD s S' S’ £ E S" < N 5" S" < N un CM S’ w Ct Ct Ct ct qz: Ct CE S= CE Ct X Ct X

[0299] S. H S Ji 3 Ji S S. H S. H S Ji S.2: 2. S5-;2 oT x u X u x £ x £ ' 3 id “.y.

[0300] tN CM £ § d E d E d E d E 5 E E 2 E 2 E ±L i±. t. d d d d d X d d. " EZ “EZ “d < “d < " CL ’al < “EL < a < £ s- < tn tn tn < Zi; —; < Z) d? on 7? <z> T7 < Z) T7 <zi T7 on, T7 <z> T7 <z> T7 < Z) T7

[0301] X fM X £M x < N X CM X £M di di d CM S' ££ £ 32- 3

[0302] E” E E E' 'E 'E E- O; E 3 'E E- " S' sz> 77 < 77 < 77 < 77 < 77 < 77 < 77 < 77 < 77 < cM < c c cz c < ~a < c *1 O E 2 E H E E 2 E E z E z E H E o Ct CL Ct CL Ct CL ai c Ct CL Ct CL Ct CL Ct CL Ct CL CC CL

[0303]

[0304] SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001S].[fl2r](A)p.m(G) UUAUAGAGCAA 0104475 p.m(A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 GAACACUGUUU U SSR- RNAl{m(U)[sp].[fi2r](U)[sp].m(A)p.m(U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)p.m(A)p.m(G)p.m(A)p.[fl2r](A)p.m( UUAUAGAGCAA 0104720 C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[sp].m(U)[sp].m(U)}$$$$V2.0 GAACACUGUUU U SSR- RNAl{p.m(A)[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2rj(G)p.m(U)p.[fl2rj(U)p.[fl2r](C)p.[fl2r](U)p.m(U)p.m(G)p.m(C)p. AACAGUGUUCU 0101599 m(U)p.m(C)p.m(U)p.m(A)p.m(U)p.m(A)[Ssp].m(A)}|CHEMl{[GalNAc3C12oyl]}|CHEM2{[nC6o]}$CHEM2, RNAl,l: Rl- UGCUCUAUAA 1: R1 |CHEM2, CHEMI, 1: R2-1: R1$$$V2.

[0305] SSR- RNAl{p.m(A)[sp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fl2r](U)p.[fl2r](C)p.[fl2r](U)p.m(U)p.m(G)p.m(C)p. AACAGUGUUCU 0101596 m(U)p.m(C)p.m(U)p.m(A)p.m(U)p.m(A)[sp].m(A)}|CHEMl{[GaiNAc3C12oyi]}|CHEM2{[nC6o]}$CHEM2, RNAl,l: Rl- UGCUCUAUAA 1: R1|CHEM2, CHEMI, 1: R2-1: R1$$$V2. O

[0306] SSR- RNAl{p.m(U)[sp].m(G)p.m(G)p.m(U)p.m(A)p.nn(U)p.[fl2r](C)p.m(A)p.[fl2r](A)p.[fl2r](A)p.[fl2r](A)p.m(C)p.m(C)p.m(U)p. UGGUAUCAAAAC 0101630 m(C)p.m(A)p.m(U)p.m(G)p.m(U)p.m(C)[sp].m(A)}|CHEMl{[GalNAc3C12oyl]}|CHEM2{[nC6o]}$CHEM2, RNAl,l: Rl- CUCAUGUCA 1: R1|CHEM2, CHEMI, l: R2-l: Rl$$$V2.0

[0307] SSR- RNAl{p.m(U)[Ssp].m(G)p.m(G)p.m(U)p.m(A)p.m(U)p.[fl2r](C)p.m(A)p.[fl2r](A)p.[fl2r](A)p.[fl2r](A)p.m(C)p.m(C)p.m(U)p. UGGUAUCAAAAC 0101695 m(C)p.m(A)p.m(U)p.m(G)p.m(U)p.m(C)[Ssp].m(A)}|CHEMl{[GalNAc3C12oyl]}|CHEM2{[nC6o]}$CHEM2, RNAl,l: Rl- CUCAUGUCA 1: R1|CHEM2, CHEMI, 1: R2-1: R1$$$V2. O

[0308] SSR- RNAl{m(U)[sp].[fi2r](G)[sp].m(A)p.[fl2r](C)p.m(A)p.[fl2r](U)p.m(G)p.m(A)p.m(G)p.m(G)p.[fl2r](U)p.m(U)p.m(U)p.[fl2r](U UGACAUGAGGU 0105101 )p.m(G)p.[fl2r](A)p.m(U)p.m(A)p.m(C)p.m(C)p.m(A)[sp].m(U)[sp].m(U)}$$$$V2.0 UUUGAUACCAU U SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)p.[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)p.m(A)p.m(G)p.m(A)p.[fl2r](A) UUAUAGAGCAA 0106582 p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 GAACACUGUUU

[0309]

[0310] U

[0311] Compoun

[0312]

[0313] d ID HELM Naked SequenceSSR- RNAl{p.m(A)[sp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fl2r](U)p.[fl2r](C)p.[fl2r](U)p.m(U)p.m(G)p.m(C)p. AACAGUGUUCUU 0101596 m(U)p.m(C)p.m(U)p.m(A)p.m(U)p.m(A)[sp].m(A)}|CHEMl{[GalNAc3C12oyl]}|CHEM2{[nC6o]}$CHEM2, RNAl,l: Rl- GCUCUAUAA 1: R1|CHEM2, CHEMI, 1: R2-1: R1$$$V2. O

[0314] SSR- RNAl{p.iTn(A)[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fi2r](U)p.[fl2r](C)p.[fl2r](U)p.m(U)p.m(G)p.m(C)p. AACAGUGUUCUU 0101599 m(U)p.m(C)p.m(U)p.m(A)p.m(U)p.m(A)[Ssp].m(A)}|CHEMl{[GalNAc3C12oyl]}|CHEM2{[nC6o]}$CHEM2, RNAl,l: Rl- GCUCUAUAA 1: R1|CHEM2, CHEMI, 1: R2-1: R1$$$V2. O

[0315] SSR- RNAl{m(A)[Ssp].m(G)[Ssp].[fl2r](A)[Ssp].m(U)[Ssp].[fl2rHG)[Ssp].m(C)[Ssp].[fl2r](C)[n001R].m(U)p.[fl2r](G)p.[fl2r](A)p.[fl AGAUGCCUGAAC 0104021 2r](A)p.m(C)p.[fl2r](U)p.m(U)p.[fl2r](G)[n001R].m(A)[Ssp].[fl2r](A)[Ssp].m(U)[Ssp].[fi2r](U)[Ssp].m(A)[Ssp].m(A)}$$$$V2. UUGAAUUAA 0

[0316] SSR- RNAl{m(A)[Ssp].m(G)[5sp].m(A)[Ssp].m(U)[Ssp].m(G)[Ssp].m(C)[Ssp].[fl2r](C)[n001R].m(U)p.[fl2r](G)p.[fl2r](A)p.[fl2r](A) AGAUGCCUGAAC 0104022 p.m(C)p.m(U)p.m(U)p.m(G)[n001R].m(A)[Ssp].m(A)[Ssp].m(U)[Ssp].m(U)[Ssp].m(A)[Ssp].m(A)}$$$$V2.0 UUGAAUUAA SSR- RNAl{m(A)[sp].m(G)p.[fl2r](A)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](C)p.m(U)p.[fl2r](G)p.[fl2r](A)p.[fl2r](A)p.m(C)p.[fl2r](U)p. AGAUGCCUGAAC 0104024 m(U)p.[fl2r](G)p.m(A)p.[fl2r](A)p.m(U)p.[fl2r](U)p.m(A)[sp].[fl2r](A)}$$$$V2.0 UUGAAUUAA SSR- RNAl{m(A)[sp].m(G)p.m(A)p.m(U)p.m(G)p.m(C)p.[fl2r](C)p.m(U)p.[fl2r](G)p.[fl2r](A)p.[fl2r](A)p.m(C)p.m(U)p.m(U)p.m( AGAUGCCUGAAC 0104025 G)p.m(A)p.m(A)p.m(U)p.m(U)p.m(A)[sp].m(A)}$$$$V2.0 UUGAAUUAA SSR- RNAl{m(C)[Ssp].m(U)[Ssp].[fi2r](G)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[f!2r](U)[n001R].m(G)p.[fl2r](U)p.[f!2r](A)p.[f CUGUGCUGUAAC 0104026 l2r](A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)[n001R].m(A)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].m(A)}$$$$V2. ACAAGUAGA 0

[0317] SSR- RNAl{m(C)[Ssp].m(U)[Ssp].m(G)[Ssp].m(U)[Ssp].m(G)[Ssp].m(C)[Ssp].[fl2r](U)[n001R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A CUGUGCUGUAAC 0104027 )p.m(C)p.m(A)p.m(C)p.m(A)[n001R].m(A)[Ssp].m(G)[Ssp].m(U)[Ssp].m(A)[Ssp].m(G)[Ssp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[sp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](U)p.m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A)p.m(C)p.[fl2r](A)p. CUGUGCUGUAAC 0104028 m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[sp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[sp].m(U)p.m(G)p.m(U)p.m(G)p.m(C)p.[fl2r](U)p.nn(G)p.[fl2r](U)p.[fl2r](A}p.[fl2r](A)p.m(C)p.m(A)p.m(C)p.m( CUGUGCUGUAAC 0104029 A)p.m(A)p.m(G)p.m(U)p.m(A)p.m(G)[sp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(G)[Ssp].m(G)[Ssp]4fl2r](A)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(A)[Ssp].[fl2r](U)[n001R].m(U)p.[fl2r](G)p.[fl2r](U)p.[f GGAUGAUUGUAC 0104030 l2r](A)p.m(C)p.[fl2r](A)p.m(G)p.[fl2r](A)[n001R].m(A)[Sspj.[fl2r](U)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].m(U)[Ssp].m(A)}$$$$V2 AGAAUCAUA.0

[0318] SSR- RNAl{m(U)[Rsp]4fl2r](A)[Ssp].m(A)[n001Si.[fl2r](A)p.m(U)p.[f'l2r](G)p.m(C)[n001S].[fl2r](A)p.m(U)p.[fl2rHA)p.m(G)p.m( UAAAUGCAUAGU 0104031 U)p.m(G)[Ssp]4fl2r](A)[Ssp].m(U)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp]4fl2r](G)[n001S].m(G)[Ssp].[^2r](A)[Ssp].m(A)[Ssp].m(U)[S GAUCAGGAAUU sp].m(U)}$$$$V2.0

[0319] SSR- RNAl{m(U)[Rsp]4fl2r](A)[Ssp].m(U)[nOOlS].[fl2r](G)p.m(A)p.[fl2r](U)p.m(U5[nOOlS].[fl2r](C)p.m(U)p.[fl2r](G)p.m(U)p.m( UAUGAUUCUGUA 0104035 A)p.m(C)[Ssp].[fl2r](A)[Ssp].m(A)[Ssp].[fl2r](U)[Ssp].nn(C)[Ssp].[fl2r](A)[n001S].nn(U)[Ssp].[fl2r](C)[Ssp].m(C)[Ssp].m(U)[S CAAUCAUCCUU

[0320]

[0321] sp].m(U)}$$$$V2.0SSR- RNAl{m(U)[Rsp].[fl2r](C)[Ssp].m(A)[n001S].[fl2r](U)p.m(G)p.[fl2r]{U)p.m(U)[n001S].[fl2r](G)p.m(A)p.[fl2r](G)p.m(U)p.m( UCAUGUUGAGUU 0104036 U)p.m(U)[Ssp].[fl2r](A)[Ssp].m(C)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](C)[n001S].m(A)[Ssp].[fl2r](G)[Ssp].m(A)[Ssp].m(U)[S UACCACAGAUU sp].m(U)}$$$$V2.0

[0322] SSR- RNAl{m(U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r](G)p.m(U)p.m( UCUACUUGUGUU 0104037 U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[f!2r](A)[n001S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].m(U)[S ACAGCACAGUU sp].m(U)}$$$$V2.0

[0323] SSR- RNAl{m(U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.m(G)p.m(U)p.nn(G)[n001R].[fl2r](U)p.m(U) UCUACUUGUGUU 0104038 p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].nn(C)[Ssp].m(A)[Ssp].m(G)[Ssp].m(U)[Ssp].m(U)}$ ACAGCACAGUU $$$V2.0

[0324] SSR- RNAl{m(U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.nn(C)p.[fl2r](U)p.nn(U)p.m(G)p.m(U)p.nn(G)[n001S].[fl2r](U)p.m(U) UCUACUUGUGUU 0104039 p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Sspj.[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].nn(C)[Ssp].m(A)[Ssp].m(G)[Ssp].m(U)[Ssp].m(U)}$ ACAGCACAGUU $$$V2.0

[0325] SSR- RNAl{m(U)[Rsp].[fl2r](G)[Ssp].m(A)[n001S].[fl2r](G)p.m(A)p.[fl2r](A)p.m(U)[n001S].[fl2r](C)p.m(U)p.[fl2r](A)p.m(U)p.m( UGAGAAUCUAUU 0104043 U)p.m(C)[Ssp].[fl2r](A)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(C)[Ssp].[fl2r](U)[Ssp].m(A)[Ssp].m(U)[S CAUGCACUAUU sp].m(U)}$$$$V2.0

[0326] SSR- RNAl{m(U)[Rsp].[fl2r](G)[Ssp].m(A)[n001S].[fl2r](G)p.m(A)p.[fl2r](A)p.m(U)p.m(C)p.m(U)p.m(A)[n001R].[fl2r](U)p.m(U) UGAGAAUCUAUU 0104044 p.m(C)[Ssp].[fl2r](A)[Ssp].m(U)[Ssp].[fi2rj(G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(C)[Ssp].m(U)[Ssp].m(A)[Ssp].m(U)[Ssp].m(U)}$ CAUGCACUAUU $$$V2.0

[0327] SSR- RNAl{m(U)[Rsp].[fl2r](G)[Ssp].m(A)[n001S].[fl2r](G)p.m(A)p.[fl2r](A)p.in(U)p.m(C)p.m(U)p.m(A)[n001S].[fl2r](U)p.m(U) UGAGAAUCUAUU 0104045 p.m(C)[Ssp].[fl2r](A)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(C)[Ssp].m(U)[Ssp].m(A)[Ssp].m(U)[Ssp].m(U)}$ CAUGCACUAUU $$$V2.0

[0328] SSR- RNAl{m(U)[Rsp].[fl2r](U)[Ssp].m(A)[n001S].[fl2r](A)p.m(U)p.[fl2r](U)p.m(C)[n001S].[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.m( UUAAUUCAAGUU 0104052 U)p.m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].[fl2rHG)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].iTi(U)[Ssp].[fl2rKC)[Ssp].m(U)[Ssp].m(U)[S CAGGCAUCUUU sp].m(U)}$$$$V2.0

[0329] SSR- RNAl{m(U)[Rsp].[fl2r](U)[Ssp].m(Aj[n001S].[fl2r](A)p.m(U)p.[fl2r3(U)p.m(Cjp.m(A)p.m(A)p.iTi(G)[n001R].[fl2r](U)p.m(U) UUAAUUCAAGUU 0104053 p.m(C)[Ssp].[fl2r](AHSsp].m(G)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(U)[Ssp].m(C)[Ssp].m(U)[Ssp].m(U)[Ssp].m(U)}$ CAGGCAUCUUU $$$V2.0

[0330] SSR- RNAl{m(U)[Rsp]4fl2r](U)[Ssp].m(A)[n001S].[fl2r](A)p.m(U)p.[fl2rHU)p.m(C)p.m(A)p.m(A)p.m(G)[n001S].[fl2r](U)p.m(U) UUAAUUCAAGUU 0104054 p.m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].[fl2rj(G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(U)[Ssp].m(C)[Ssp].m(U)[Ssp].m(U)[Ssp].m(U)}$ CAGGCAUCUUU $$$V2.0

[0331] SSR- RNAl[m(U)[Ssp].[i:!2r](A)[Rsp].m(A)[n001Si.[fi2rHA)p.m(U)p.[fl2r](G)p.m(C)[n001S]4fl2r](A)p.m(U)p.[fl2r](A)p.m(G)p.m( UAAAUGCAUAGU 0104061 U)p.m(G)[Ssp].[fl2r](A)[Ssp].m(U)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[n001S].m(G)[Ssp].[fi2rj(A)[Ssp].m(A)[Ssp].m(U)[S GAUCAGGAAUU

[0332]

[0333] sp].m(U)}$$$$V2.0SSR- RNAl{m(U)[Ssp].[fl2r](A)[Rsp].m(U)[n001S].[fl2r](G)p.m(A)p.[fl2r](U)p.m(U)[n001S].[fl2r](C)p.m(U)p.[fl2r](G)p.m(U)p.m( UAUGAUUCUGUA 0104065 A)p.m(C)[Ssp].[fl2r](A)[Ssp].m(A)[Ssp].[fl2r](U)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(U)[Ssp].[fl2r](C)[Ssp].m(C)[Ssp].m(U)[S CAAUCAUCCUU sp].m(U)}$$$$V2.0

[0334] SSR- RNAl{m(LJ)[Ssp].[fl2r](C)[Rsp].m(A)[n001S].[fl2r](U)p.nn(G)p.[fl2r](U)p.nn(U)[n001S].[fl2r](G)p.m(A)p.[fl2r](G)p.m(U)p.m( UCAUGUUGAGUU 0104066 U)p.m(U)[Ssp].[fl2r](A)[Ssp].m(C)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](C)[n001S].m(A)[Ssp3.[fl2r](G)[Ssp].m(A)[Ssp].m(U)[S UACCACAGAUU sp].m(U)}$$$$V2.0

[0335] SSR- RNAl{m(U)[Ssp].[fl2r](C)[Rsp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r](G)p.m(U)p.m( UCUACUUGUGUU 0104067 U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[f!2r](A)[n001S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].m(ll)[S ACAGCACAGUU sp].m(U)}$$$$V2.0

[0336] SSR- RNAl{m(U)[Ssp].[fl2r](C)[Rsp].m(U)[n001Sj.[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.m(G)p.m(U)p.m(G)[n001R].[fl2r](U)p.m(U) UCUACUUGUGUU 0104068 p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Sspj.[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].nn(C)[Ssp].m(A)[Ssp].m(G)[Ssp].m(U)[Ssp].m(U)}$ ACAGCACAGUU $$$V2.0

[0337] SSR- RNAl{m(U)[Ssp].[fl2r](C)[Rsp].m(U)[n001S].[fl2r](A)p.nn(C)p.[fl2r](U)p.m(U)p.m(G)p-m(U)p.nn(G)[n001S].[fl2r](U)p.m(U) UCUACUUGUGUU 0104069 p.m(A)[Ssp].[fl2r](C3[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(C)[Ssp].m(A)[Ssp].m(G)[Ssp].m(U)[Ssp].m(U)}$ ACAGCACAGUU $$$V2.0

[0338] SSR- RNAl[m(U)[SspJ.[fl2r](G)[Rsp].m(A)[n001S].[fl2r](G)p.m(A)p.[fl2r](A)p.m(U)[n001SJ.[fl2r](C)p.m(U)p.[fl2r](A)p.m(U)p.m( UGAGAAUCUAUU 0104073 U)p.m(C)[Ssp].[fl2r](A)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(C)[Ssp].[fl2r](U)[Ssp].m(A)[Ssp].m(U)[S CAUGCACUAUU sp].m(U)}$$$$V2.0

[0339] SSR- RNAl{m(U)[SspJ.[fl2r](G)[RspJ.m(A)[n001S].[fl2rJ(G)p.m(A)p.[fl2r](A)p.in(U)p.m(C)p.m(U)p.m(A)[n001R].[fl2r](U)p.m(U) UGAGAAUCUAUU 0104074 p.m(C)[Ssp].[fl2r](A3[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(C)[Ssp].m(U)[Ssp].m(A)[Ssp].m(U)[Ssp].m(U)}$ CAUGCACUAUU $$$V2.0

[0340] SSR- RNAl{m(U)[SspJ.[fl2r](G)[Rsp].m(A)[n001S].[fl2r](G)p.m(A)p.[fl2r](A)p.m(U)p.m(C)p.m(U)p.m(A)[n001Sj.[fl2r](U)p.m(U) UGAGAAUCUAUU 0104075 p.m(C)[Ssp].[fl2r](A)[Ssp].m(U)[Ssp].[fi2rj(G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(C)[Ssp].m(U)[Ssp].m(A)[Ssp].m(U)[Ssp].m(U)}$ CAUGCACUAUU $$$V2.0

[0341] SSR- RNAl{m(U)[SspJ.[fl2r](U)[Rsp].m(A)[nOOlSJ.[fl2r](A)p.m(U)p.[fl2r3(U)p.m(Cj[nOOlS].[fl2rJ(A)p.m(A)p.[fl2r](G)p.m(U)p.m( UUAAUUCAAGUU 0104082 U)p.m(C)[Ssp].[f!2r](A)[Ssp].m(G)[Ssp].[f!2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].nn(U)[Ssp].[fl2r](C)[Ssp].m(U)[Ssp].m(U)[S CAGGCAUCUUU sp].m(U)}$$$$V2.0

[0342] SSR- RNAl{m(U)[SspJ.[fl2r](U)[RspJ.m(A)[n001SJ.[fl2rJ(A)p.m(U)p.[fl2r](U)p.m(C)p.m(A)p.m(A)p.m(G)[n001R].[fl2r](U)p.m(U) UUAAUUCAAGUU 0104083 p.m(C)[Ssp].[fl2r](A3[Ssp].m(G)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(U)[Ssp].m(C)[Ssp].m(U)[Ssp].m(U)[Ssp].m(U)}$ CAGGCAUCUUU $$$V2.0

[0343] SSR- RNAl[m(U)[SspJ.[fl2r](U)[Rsp].m(A)[n001SJ.[fl2r](A)p.m(U)p.[fl2r3(U)p.m(C)p.m(A)p.m(A)p.m(G)[n001S].[fl2r](U)p.m(U) UUAAUUCAAGUU 0104084 p.m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].[fl2rj(G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(U)[Ssp].m(C)[Ssp].m(U)[Ssp].nn(U)[Ssp].nn(U)}$ CAGGCAUCUUU

[0344]

[0345] $$$V2.0SSR- RNAl{m(U)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001R].m(U)p.[fl2r](G)p.[fl2r](A)p.[f UAGUGCAUGAAU 0104092!2r](A)p.m(U) p.[fl2r] (A)p.m(G)p.[fl2r] (A)[n001R].m( U)[Ssp].[fl2r] ( U ) [Ssp].m (C)[Ssp],[f!2r] (U )[Ssp].m (C)[Ssp].m(A)}$$$$V2 AGAUUCUCA.0

[0346] SSR- RNAl{m(LJ)[Ssp].m(A)[Sspj.m(G)[Ssp].m(U)[Ssp].nn(G)[Ssp].m(C)[Ssp].[fi2r](A)[n001R].m(U)p.[fl2rj(G)p.[fl2r](A)p.[fi2rj(A UAGUGCAUGAAU 0104094 )p.m(U)p.m(A)p.m(G)p.m(A)[n001R].m(U)[Ssp].m(U)[Ssp].m(C)[Ssp].m(U)[Ssp].m(C)[Ssp3.m(A)}$$$$V2.0 AGAUUCUCA SSR- RNAl{m(U)[Ssp].m(C)[Ssp].[fl2r](U)[Ssp].m(G)[Ssp].[fl2r](U)[Ssp].m(G)[Ssp].[fl2r](G)[n001R].m(U)p.[fl2r](A)p.[fl2r](A)p.[f UCUGUGGUAAAC 0104097 l2r](A)p.m(C)p.[fl2r](U)p.m(C)p.[fl2r](A)[n001R].m(A)[Sspj.[fi2r](C)[Ssp].m(A)[Ssp].[fl2r](U)[Ssp].m(G)[Ssp].m(A)}$$$$V2. UCAACAUGA 0

[0347] SSR- R N Al{m ( U )[Ssp].m (U) [Ssp]. [fl2r](C) [Ssp].m(C) [Ssp].[f I2r] ( U ) [Ssp].m (G)[Ssp]. ]fl2r](A) [nOOl R].m( U)p. [f!2r] (C)p. [fl2r] (A)p. [f! UUCCUGAUCACU 0104099 2r](C)p.m(U)p.[fl2r](A)p.nn(U)p.[fl2r](G)[n001R].m(C)[Ssp].[fl2r](A)[Ssp].m(U)[Ssp].[fl2r](U)[Ssp].m(U)[Ssp].m(A)}$$$$V2. AUGCAUUUA 0

[0348] SSR- RNAl{m(U)[sp].[fl2r](C)!sp].m(U)p.[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(U)p.m(U)p.m(A)p.[fl2r UCUACUUGUGUU 0104100 ](C)p.m(A)p.[fl2r](G)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(G)[sp].m(U)[sp].m(U)}$$$$V2.0 ACAGCACAGUU SSR- RNAl{m(U)[sp].[fl2r](C)[sp].m(U)p.m(A)p.m(C)p.[f!2r](U)p.m(U)p.m(G)p.m(U)p.m(G)p.m(U)p.m(U)p.m(A)p.[fl2r](C)p.m( UCUACUUGUGUU 0104101 A)p.[fl2r](G)p.m(C)p.m(A)p.m(C)p.m(A)p.m(G)[sp].m(U)[sp].m(U)}$$$$V2.0 ACAGCACAGUU SSR- RNAl{m(U)[sp].[fi2r](G)[sp].m(A)p.[fl2r](G)p.m(A)p.[fl2r](A)p.m(U)p.[fl2r](C)p.m(U)p.[fl2r](A)p.m(U)p.m(U)p.m(C)p.[fl2r UGAGAAUCUAUU 0104102 ] (A)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](A)p.nn (C)p. [fl2r]( U)p.m(A) [sp].m (U)[sp].m(U)}$$$$V2.0 CAUGCACUAUU SSR- RNAl{m(U)[sp].[fl2r](G)[sp].m(A)p.m(G)p.m(A)p.[fl2r](A)p.m(U)p.m(C)p.m(U)p.m(A)p.m(U)p.m(U)p,m(C)p.[fl2r](A)p.m( UGAGAAUCUAUU 0104103 U)p.[fl2r](G)p.m(C)p.m(A)p.m(C)p.m(U)p.m(A)[sp].m(U)[sp].m(U)}$$$$V2.0 CAUGCACUAUU SSR- RNAl{m(U)[sp].[fi2r](U)[sp].m(A)p.[fl2r](A)p.nn(U)p.[fl2r](U)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.m(U)p.m(C)p.[fl2r UUAAUUCAAGUU 0104104 ](A)p.m(G)p.[fl2r](G)p.m(C)p.[fl2r](A)p.m(U)p.[fl2r](C]p.m(U)[sp].m(U)[sp].m(U)}$$$$V2,0 CAGGCAUCUUU SSR- RNAl{m(U)[sp].[fl2r](U)[sp].m(A)p.m(A)p.m(U)p.[fl2r](U)p.m(C)p.m(A)p.m(A)p.m(G)p.m(U)p.m(U)p.m(C)p.[fl2r](A)p.m( UUAAUUCAAGUU 0104105 G)p.[fl2r](G)p.m(C)p.m(A)p.m(U)p.m(C)p.m(U)[sp].m(U)[sp].m(lJ)}$$$$V2.0 CAGGCAUCUUU SSR- RNAl{m(U)[sp].m(A)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](A)p.m(U)p.[fl2r]{G)p.[fi2r](A)p.[fl2r](A)p.m(U)p.[fl2r](A)p. UAGUGCAUGAAU 0104106 m(G)p.[fl2r](A)p.m(U)p.[fl2r](USp.m(C)p.[fl2r](U)p.m(Cj[sp].[fl2r](A)}$$$$V2.0 AGAUUCUCA SSR- RNAl{m(U)[sp].m(A)p.m(G)p.m(U)p.m(G)p.m(C)p.[fl2r](A)p.m(U)p.[fl2r](G)p.[fl2r](A)p.[fl2r](A)p.m(U)p.m(A)p.m(G)p.m( UAGUGCAUGAAU 0104107 A)p.m(U)p.m(U)p.m(C)p.m(U)p.m(C)[sp].m(A)}$$$$V2.0 AGAUUCUCA SSR- RNAl{m(U)[Ssp].[fi2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[f!2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001Sj.[fl2r](A)p.m(G) UUAUAGAGCAAG 0104474 p.m(A)pJfl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)[n001S]Jfl2r](U)p.m(U)[Sspj.m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[fi2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001S].[fl2r](A)p.m(G) UUAUAGAGCAAG 0104475 p.m(A)p.[fl2r](A)p,m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G5p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2. O AACACUGUUUU SSR- RNAl{m(U)[sp].[fl2rHU)[sp].m(A)p.m(U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)p.m(A)p.m(G)p.m(A)p.[fl2r](A)p.m( UUAUAGAGCAAG

[0349]

[0350] 0104720 C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[sp].m(U)[sp].m(U)}$$$$V2.0 AACACUGUUUU

[0351]

[0352] SSR- RNAl{m(U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.[fl2r](G)p.m(U)p.m(G)[n001S].[fl2r](U)p.m( UCUACUUGUGUU 0106181 U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[n001S].[fl2r](G)[Ssp].m(C)[Ssp].[fi2r](A)[Ssp].m(C)[n001S].[f!2r](A)[Ssp].m(G)[Ssp].m(U ACAGCACAGUU )[Ssp].m(U)}$$$$V2.0

[0353] SSR- RNAl{m(U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.m(G)p.m(U)p.m(G)[n001S].[fl2r](U)p.m(U) UCUACUUGUGUU 0106182 p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[n001S].[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Sspi.m(C)[n001S].[fi2r](A)[Ssp].m(G)[Ssp].m(U)[Ssp] ACAGCACAGUU.m(U)}$$$$V2.0

[0354] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fi2r](G)p.m(U)p.[fl2r](G) UCUACUUGUGUU 0106183 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(C)[Sspi.[fl2r](A)[Ssp].m(G)[ ACAGCACAGUU Ssp].m(U)[Ssp].m(U)}$$$$\ / 2.0

[0355] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.nn(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r](G) UCUACUUGUGUU 0106184 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].nn(C)[n001S].[fl2r](A)[Ssp].m(G)[ ACAGCACAGUU Ssp].m(U)[Ssp].m(U)}$$$$V2.0

[0356] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)[n001S UCUACUUGUGUU 0106185 ].m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].m(C)[n001S].[fl2r](A)[Ssp].m(G)[S ACAGCACAGUU sp].m(U)[Ssp].m(U)}$$$$V2.0

[0357] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.[fl2r](G)p.m(U)p.m(G)[n001S].[fl UCUACUUGUGUU 0106186 2r](U)p.m(U)p.m(A)[Sspj.[^2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp]4fl2r](A)[Ssp].m(C)[n001S]4fl2r](A)[Sspj.m(G)[S ACAGCACAGUU sp].m(U)[Ssp].m(U)}$$$$V2.0

[0358] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.m(G)p.m(U)p.m(G)[n001S].[fl2r] UCUACUUGUGUU 0106187 (U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(C)[n001S].[fl2r](A)[Ssp].m(G)[Ssp].m( ACAGCACAGUU U)[Ssp].m(U)}$$$$V2.0

[0359] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r](G) UCUACUUGUGUU 0106188 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].m(C)[Ssp].[f!2r](A)[Sspj.m(G)[Ssp ACAGCACAGUU ].m(U)[Ssp].m(U)}$$$$V2.0

[0360] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001Sj.[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)[n001S UCUACUUGUGUU 0106189 ].m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[f!2r](A)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp ACAGCACAGUU ].m(U)[Ssp].m(U)}$$$$V2.0

[0361] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)lSsp].m(U)[n001Sj.[fl2r](A)p.m(C)p.[f'l2r](U)p.m(U)p.[fl2r](G)p.m(U)p.m(G)ln001S].[fl UCUACUUGUGUU 0106190 2r](U)p.m(U)p.m(A)[Sspj.[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp] ACAGCACAGUU.m(U)[Ssp].m(U)}$$$$V2.0

[0362] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.m(G)p.m(U)p.m(G)[n001S].[fl2r] UCUACUUGUGUU 0106191 (U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Sspj.[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(C)[Ssp].[fl2rj(A)[Ssp].m(G)[Ssp].m(U) ACAGCACAGUU

[0363]

[0364] [Ssp].m(U)}$$$$V2.0SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.m(G)p.m(U)p.m(G)[n001S].[fl2r] UCUACUUGUGUU 0106192 (U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Sspj.[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(C)[Ssp].m(A)[Ssp].m(G)[Ssp].m(U)[Ss ACAGCACAGUU p].m(U)}$$$$V2.0

[0365] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fi2r](G)p.m(U)p.[fl2r](G) UCUACUUGUGUU 0106193 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[n001S].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S3.m(C)[Ssp].[fl2r](A)[Ssp].m( ACAGCACAGUU G)[Ssp].m(U)[Ssp].m(U)}$$$$V2,0

[0366] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fi2r](G)p.m(U)p.[fl2r](G) UCUACUUGUGUU 0106194 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[n001S].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].m(C)[ri001S].[fl2r](A)[Ssp],m( ACAGCACAGUU G)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0

[0367] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.nn(C)p.[fl2r](U)p.m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)[n001S UCUACUUGUGUU 0106195 ].m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[n001S].[fi2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].m(C)[n001S].[fi2r](A)[Ssp].m(G ACAGCACAGUU )[Ssp].m(U)[Ssp].m(U)}$$$$V2.0

[0368] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.[fl2r](G)p.m(U)p.m(G)[n001S].[fl UCUACUUGUGUU 0106196 2r](U)p.m(U)p.m(A)[Sspj.[fl2r](C)[Ssp].m(A)[n001S].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].m(C)[n001S].[fl2r](A)[Ssp].m(G ACAGCACAGUU )[Ssp].m(U)[Sspj.m(U)}$$$$V2.0

[0369] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.m(G)p.m(U)p.rr!(G)[n001S].[fl2r] UCUACUUGUGUU 0106197 (U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[n001S].[fl2r](G)[Ssp].m(C)[Ssp].m(A)[Ssp].m(C)[n001S].[fl2r](A)[Ssp].m(G)[Ssp]. ACAGCACAGUU m(U)[Ssp].m(U)}$$$$V2.0

[0370] SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2rHG)p.m(C)p.[fl2r](U)[n001R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A)p.m(C)p.[fl2 CUGUGCUGUAAC 0106206 r](A)p.m(C)p.[fl2r](A)[n001R].m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](U)[n001R].m(G)p.[fl2rHU)p.[fl2r](A)p.[fl2r](A)p.m(C)p.[fl2 CUGUGCUGUAAC 0106207 r] (A)p.m (C)p. [f!2r] (A) [nOOIR].m (A)[SspJ,[^l2r](G) [Ssp].m( U ) [Ssp]. [fl2rj ( A)[Ssp].m(G)[Ssp].m (A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](U)[n001R].m(G)p.[fl2r](U)p.[fl2r](A)p.[f CUGUGCUGUAAC 0106208 l2r](A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)[n001R].m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Sspj.m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fi2r](G)p.m(C)p.[fl2r](U)[n001R],m(G)p.[n2rHU)p.[fl2r](A)p.[fl2r](A)p.m(C)[Ssp] CUGUGCUGUAAC 0106209.[fl2r](A)[Ssp].m(C)[Ssp].[fl2r](A)[n001R].m(A)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp],m(U)[Ssp],[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].iri(C)[Ssp].[fl2r](U)[n001R],m(G)[Ssp],[fl2r](U)[Ssp].[fi2r CUGUGCUGUAAC 0106210 ](A)[Ssp].[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)[n001R].m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](U)[n001R].m(G)[Ssp].[fl2r](U)[Ssp].[fl2r CUGUGCUGUAAC 0106211 ](A)[Ssp].[fi2r](A)p.m(C)[Ssp].[fl2r](A)[Ssp].m(C)[Ssp].[fl2r](A)[n001R].m(A)[Ssp].[fi2r](G)[Ssp].m(U)[Ssp].[fl2r](A)[Ssp].m( ACAAGUAGA

[0371]

[0372] G)[Ssp].m(A)}$$$$V2.0SSR- RNAl{m(C)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](U)[n001R].m(G)[Ssp].[fl2r](U)[Ssp].[fl2r CUGUGCUGUAAC 0106212 ](A)[Ssp].[fi2r](A)[Ssp].m(C)[Ssp].[f!2r](A)[Ssp].m(C)[Ssp].[fl2r](A)[n001R].m(A)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](A)[Ssp ACAAGUAGA ],m(G)[Ssp].m(A)}$$$$V2.0

[0373] SSR- RNAl{m(C)[Ssp].m(U)p.m(G)p.m(U)p.m(G)p.m(C)p.[fl2r](U)[n001R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A)p.m(C)p.m(A)p.m CUGUGCUGUAAC 0106213 (C)p.m(A)[n001R].m(A)p.m(G)p.m(U)p.m(A)p.m(G)[Ssp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.m(G)p.m(U)p.m(G)p.m(C)p.[fl2r](U)[n001R].m(G)p.[fl2rHU)p.[fl2r](A)p.[fl2r](A)p.m(C)p.m(A)p.m CUGUGCUGUAAC 0106214 (C)p.m(A)[n001R].m(A)[Ssp].m(G)[Ssp].m(U)[Ssp].m(A)[Ssp].m(G)[Ssp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)[Ssp].m(G)[Ssp].m(U)[Ssp].m(G)[Ssp].m(C)[Ssp].[fl2r](U)[n001R].m(G)p.[fl2r](U)p.ffl2r](A)p.[fl2r](A CUGUGCUGUAAC 0106215 )p.m(C)p.m(A)p.m(C)p.m(A)[n001R].m(A)p.iTi(G)p.m(U)p.m(A)p.m(G)[Ssp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.m(G)p.m(U)p.m(G)p.m(C)p.[fl2r](U)[n001R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A)p.m(C)[Ssp].m(A)f CUGUGCUGUAAC 0106216 Ssp].m(C)[Ssp].m(A)[n001R].m(A)[Ssp].m(G)[Ssp].m(U)[Ssp].m(A)[Ssp].m(G)[Ssp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp],m(U)[Ssp],m(G)[Ssp].m(U)[Ssp].m(G)[Ssp].m(C)[Ssp].[fl2r](U)[n001R],m(G)[Ssp],[fl2r](U)[Ssp].[fl2r](A)[S CUGUGCUGUAAC 0106217 sp].[fl2r](A)p.m(Cjp.m(A)p.m(C)p.m(A)[n001R].m(A)p.m(G)p.m(U)p.m(A)p.m(G)[Ssp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)[Ssp].m(G)[Ssp].m(U)[Ssp].m(G)[Ssp].m(C)[Ssp].[fl2r](U)[n001R].m(G)[Ssp].[fl2r](U)[Ssp].[fl2r](A)[S CUGUGCUGUAAC 0106218 sp].[fl2r](A)p.m(C)[Ssp].m(A)[Ssp].m(C)[Ssp].m(A)[n001R].m(A)[Ssp].m(G)[Ssp].m(U)[Ssp].m(A)[Ssp].m(G)[Ssp].m(A)}$$$ ACAAGUAGA $V2.0

[0374] SSR- RNAl{m(C)[Ssp].m(U)[Ssp].m(G)[Ssp].m(U)[Ssp].m(G)[Ssp].m(C)[Ssp].[f!2r](U)[n001R].m(G)[Ssp].[f!2r](U)[Ssp].[fl2r](A)[S CUGUGCUGUAAC 0106219 sp].[fl2r](A)[Sspj.m(C)[Ssp^.m(A)[Ssp],m(C)[Ssp].m(A)[n001R].m(A)[Ssp].m(G)[Ssp].m(U)[Ssp].m(A)[Ssp].m(G)[Ssp].m(A)} ACAAGUAGA $$$$V2.0

[0375] SSR- RNAl{p.[Rm5d5m](U)[Ssp].[fl2r](C)[Rsp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r](G) UCUACUUGUGUU 0106220 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m! C)[Ssp].[fl2r](A)[n001S].m(C)[Sspj.[fl2r](A)[Ssp].m(G)[ ACAGCACAGUU Ssp].m(U)[Ssp].m(U)}$$$$V2.0

[0376] SSR- RNAl{p.[Rm5d5m](U)[Ssp].[fl2r](C)[Rsp].m(U)[n001Sj.[fl2r](A)p.nn(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r](G) UCUACUUGUGUU 0106221 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[Ssp].m(C)Psp].[fl2r](A)[Ssp].m(G)[Ssp ACAGCACAGUU ].m(U)[Ssp].m(U)}$$$$V2.0

[0377] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Rsp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2rj(G) UCUACUUGUGUU 0106222 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(C)[Sspj.[fl2r](A)[Ssp].m(G)[ ACAGCACAGUU Ssp].m(ll)[Ssp].m(U)}$$$$V2.0

[0378] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Rsp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2rj(G) UCUACUUGUGUU 0106223 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m{C)[Ssp].[fl2r](A)[Ssp].m(C)[Ssp].[fl2rHA)[Ssp].m(G![Ssp ACAGCACAGUU

[0379]

[0380] ].m(U)[Ssp].m(U)}$$$$V2.0£ £ E E

[0381] Q. ji <

[0382] Tn o o c: S

[0383] E

[0384] jj LJ

[0385] E

[0386] ji £ E

[0387] d. <_ E

[0388] Q- £ ci E

[0389] jci -S

[0390] E

[0391] ££ S

[0392] Aj

[0393] < or

[0394]

[0395]

[0396] {()()()()()()()()()()()()[][fl][][][fl][fl][][fl]GGGS200S22GG00S2G SS UUAUAACAA RNAlUURA1UAACA1ARmsprspmnrpmprpmpmpmpmnrpm-...........

[0397] ()(()(()())()())[][fl])[][fl])[][fl]([][fl][]([](G 00629000S200S200S2G00S2SS AACACUUUUU1A1AC1AC1U1UUUU pmnrpmnrpmnrpmnrpmspmspm...........

[0398] $$$$ )}20V.

[0399] GGG SS UUAUAACAAR- G 00629 AACACUUUUU11

[0400] GGG SS UUAUAACAAR- G 006292 AACACUUUUU1

[0401] v>

[0402] <£ 3 Si v> £ S £ 2 3

[0403] U 2 2 3 2 GGG SSUAUAACAAR E- £ ~ £ ~ E E E E E £ E E E E 1 3 jd G 006293 AACACUUUUU1 ji _d d _d ji _d 2 2 _d 2 S E <_ GGG SS < UUAUAACAAR- <_ <£ <_ 2 2 < 2 CX < N Ai ” m o Z7 o ~ uo 5 g- < N G 00629 AACACUUUUU14 ni < N Aj (N O nz < N CM CM 00 on lie o de p O ic. o O o ±L > ±L >

[0404] 2^ Q

[0405] 2 S 2 5 tn r-j U7 g; eGGG SS UUAUAACAAR-r gj, —. <n- S > s Q > X 5 er d er g P V> on V> o -tn- S o v> G 00629 AACACUUUUU15 o <n- o <n- o -tn O £ o v> o 2 O 't / >o v> o - Q V> s g § o • O v> O iQ: ° ° v> °? £ X £ C c 2”{()()()()()()()()()()()()[][fl][][][fl][fl][][fl]GGGS200S22GG00S2G SS UUAUAACAA RNAlUURA1UAACA1ARmsprspmnrpmprpmpmpmpmnrpm-........... £, a 2 de Ln -E,::::■ ix. v> 2E::: J—J v> “ v> Z> - co < on^ <_ Lie < < P' 026 AACACUGUUUU1069$$$()[fl]()()[][fl]()()(){)()()[]()[]()}$20S2GS20AAC01ACUUUSUUV pmprpmnrpmpmpmpmpmspmspm............ < £ < E < E 2 2 < 5 < s < f < s

[0406] E 3 E r-X E S E E z> E £* £ " EGGG UUAUAACAA SSR- E s' tn Q- ex ^n d E ex « ex ~ ex 1 1 n I S 2: >■' i l 2 s' — on —. on O AACACUGUUUU 0106297 ^x o o. (J °- o Q- O °- U Q” O O 2” 2 in £ S’. LL. in •ZL. < Z5 CL. yj 3 d Si d 3

[0407] E 2 E S ~£ 3 3 E S E <n GGG SS UUAUAACAAR- 23 £ S £ S £ S £ “ £ S £ S

[0408] d jd p d:□ ex c d E _d £ ji. rD d 7--' d z> d z> d z) d z) 2 2 G 006298 AACACUUUUU1 « s « " S £ E 2 Ef 2 E 323 ~E ex v £ s X XT’ yi 3 J p.-A GGG SS UUAUAACAAR- G yi E on £ jn, £ o £ d ~ ex £ 'd lx E on

[0409] ri ° CL d CL ‘S' d d1 / 1ex CL d §• G 006299 AACACUUUUU12 c 2 O on < Z) 2: X on 2 J=t on <£ H < “ < < 3 < “ < “ < < ~ < < X < 2 < TGGG SS UUAUAACAAR- -£ E E 2 E d c — ■ 2 E jS X 3 E B I - £ d £ d CL E ex G 006300 AACACUUUUU1 E ji E Q_ E CL E d g: CL " d £ d £ d.£ L §.£■■ E 2 2 (J r-X < D — < D _S S' Ji S' Ji S' Ji S' Ji. S O “ 3 3GG SS UUAUAAGCAAR- S 3 S jj 2 on ^t, L Z> z> T7 E ’U’ Z’ -1zr o Ai _d < N O CM d rxi o S' 3 G 00630 AACACUUUUU11 S | g £ S £ S £ S 2 S£S £ £ E 2 2 ±L (£ “ d “ _d “ d d “ d “ 3GGG SS UUAUAACAAR- ex -~- d CL £; er 2 2 2 S E 2 E £ ■<■ o < PG 006302 AACACUUUUU1 < 2.< Q. SL £ I f ZX o 2 jx o 2 E E X E 7=* E f £ E £ c “ " E E E 2 E " E 5 E 5 E d E 2. GGG SS UUAUAACAAR- £?. Q,. Q.. Q. E d d X £L JX jx 2 JX JS ex c? CT _d er Ji 2 2 ST 2 o 2 £ J: E — - Z3 — Z) G 006303 AACACUUUUU1 Z) — Z) Z) S £ Z> '— 2 -Z S _ o 2 E 2 ji 2 _d H "g ex | -g X £ “ E E z? c ZGGG SS UUAUAACAAR-7 u* z? O rxl — CN JX r--! jX CN rN Aj 2 S 2 CM d CM d CM 5 2 2 Ji s s G 00630 AACACUUUUU14 O ±L U U 2 2 2 ±L ±L E <_ 2 o d <7? E <7F E er t / F E er £ er £ 2 " IT on on 2 S’ 2 < ri CM

[0410] S Ji S 2 S o O o <£: o jj o 2 O v? o o SL o on o d

[0411] O " o -7 o O <“! o U o o o o o o o a 77? s C i — > e: x o c 'Zt c: “x e: c e2 o P. OJ £. o o E E E E< Z)i s <■ CM <" £4 < 3 < < uS rN 2 d 2 2 2 o 2 2; 2 2 2 ji E E ~ E? E E <_ E E o E" £ E 2 E 2 E 2 E 2 £ X ■ o - Q.r~nd —.£ a 2 zz ex 2 u o' o' W CM « JN S' ™ 6 E tn <xj m CS- 1 / 12 ££ nc ££ DC ce nr & L ct qz 2 d £ § 2 E “ £ “ E

[0412] S ji ji S Ji S Ji 5 2: S. Si jj £ er 2, S. 2 2 2 2 2 o*l “ o_ o, £. 2 2 2 T? < T? <. <• 2 O tN r— fN Aj cj CM s E <5 E 5 o O 5 E E 5 E <2 E S? H~, er ±L £sl ±L ££ ±L [£. ±L2=2d 2 d 2* d ^2 d r-X d

[0413] £ < lx <? "d < < 2 < ex 2 <? 2 < lx 2 £X Si* "p- 3 e'j < E y> "^t <zi T? $ jq on Z7 on ZT on T7 on TZ on ZT on Z? on Z7 on Z? 2 < N izi CM fN £=? fN o 2 3 E. 2 2 2 2 2 2 2 2 2 2 2. ° E

[0414] 'E 2 E 2 E Ji E 2 E E Jx E 2 E 2 E “ ~E 1= E J: £ T7 < 2* < 7^ < TT* < T^7 < < < rt" <

[0415] < C C3 < <t o *£ o 'S fc t: c c <£ p, Z 5 z £ z ~ H E CM H oi~ E z £ 2 £ 2Z ~ CM 2 o rr r> ct Cl Ct c ct ex oe d ct ex cc ex ct d ct d ct ex ct ex > ct ex > ct c

[0416]

[0417] SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001S].[fl2r](A)p.m(G) UUAUAGAGCAAG 0106305 p.m(A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)[n001S].[fl2r](U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[f!2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001S].[fl2r](A)p.m(G) UUAUAGAGCAAG 0106306 p.nn(A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)[n001S].[fl2r](G)p.m(U)p.m(U) [Ssp]. m(U)[Ssp].m(U )}$$$$ V2.0 AACACUGUUUU SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001S].[fl2r](A)p.m(G) UUAUAGAGCAAG 0106307 p.m(A)p.[fl2r[(A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)[n001S].[f!2r](U)[Ssp].m(U)[Ssp[.m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(C)[sp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](U)[n009R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A)p.m(C)p.[fl2r CUGUGCUGUAAC 0106445 ] (A)p.m(C)p.[fi2r] ( A)p.m(A)p.[f!2r] (G )p.m( U)p. [fl2r] (A)p.m(G) [sp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[sp].m(U)p.[fl2r](G)p.m(U)p.[fi2r](G)p.m(C)p.[fl2r](U)[n033R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A)p.m(C)p.[fl2r CUGUGCUGUAAC 0106446 ](A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2rKG)p.m(U)p.[fl2r](A)p.m(G)[sp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r[(G)p.m(C)p.[fl2r](U)[n009R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A)p.m(C)p.[fl2 CUGUGCUGUAAC 0106447 rj(A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r[(G)p.m(C)p.[fl2r](U)[n033R].m(G)p.[fl2r[(U)p.[fl2r](A)p.[fl2r](A)p.! Ti(C)p.[fl2 CUGUGCUGUAAC 0106448 r[(A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C) [Ssp].m ( U )p.[fl2r] (G )p.m(U)p.[fl2r](G)p.m [C)p.[fl2r] ( U ) [n009R].m(G)p.[fl2r] (U )p.[fl2r] (A)p.[f!2r](A)p.m(C)p. [f!2 CUGUGCUGUAAC 0106449 r] (A)p.m (C)p. [f!2r] (A) [nOOIR].m (A)p.[f!2r] (G)p.m (U )p.[f I2r] (A)p.m (G) [Ssp]. [fl2r] ( A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](U)[n033R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A)p.m(C)p.[fl2 CUGUGCUGUAAC 0106450 r](A)p.m(C)p.[fl2r](A)[n001R].m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](U)[n009R].m(G)p.[fl2r](U)p.[fl2r](A)p.[f CUGUGCUGUAAC 0106451 l2r](A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)[n001R].m(A)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].m(A)}$$$$V2. ACAAGUAGA 0

[0418] SSR- RNAl{m(C)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](U)[n033R].m(G)p.[fl2r](U)p.[fl2r](A)p.[f CUGUGCUGUAAC 0106452 l2r](A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)[n001R].m(A)[Ssp].[fl2r](G)[Ssp].m(U)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].m(A)}$$$$V2. ACAAGUAGA 0

[0419] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n065S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n065S].[fl2r](G)p.m(U)p.[fl2r](G) UCUACUUGUGUU 0106479 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n065S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[ ACAGCACAGUU Ssp].m(U)[Ssp].m(U)}$$$$V2.0

[0420] SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n070S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n070S].[fl2r](G)p.m(U)p.[fl2r](G) UCUACUUGUGUU 0106480 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n070S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[ ACAGCACAGUU Ssp].m(U)[Ssp].m(U)}$$$$V2.0

[0421] SSR- RNAl{p.[Rm5d5m](U)[Rsp],[fl2r](C)[Ssp],m(U)[n071S].[fl2r](A)p.iri(C)p.[fl2r](U)p.m(U)[n071S].[fl2r](G)p.m(U)p,[fl2r](G) UCUACUUGUGUU 0106481 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[nO71S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[ ACAGCACAGUU

[0422]

[0423] Ssp].m(U)[Ssp].m(U)}$$$$V2.0SSR- RNAl{m(U)[Rsp].[fl2r](C)[Ssp].m(U)[n065S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n065S].[fl2r](G)p.m(U)p.[fl2r](G)p.m(U)p.m( UCUACUUGUGUU 0106482 U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[nO65S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].m(U)[S ACAGCACAGUU sp].m(U)}$$$$V2.0

[0424] SSR- RNAl{m(U)[Rsp].[fl2r](C)[Ssp].m(U)[n070S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n070S].[fl2r](G)p.m(U)p.[fl2r](G)p.m(U)p.m( UCUACUUGUGUU 0106483 U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n070S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].m(U)[S ACAGCACAGUU sp].m(U)}$$$$V2.0

[0425] SSR- RN Al{m ( U )[Rsp]. [fl2r] (C) [Ssp].m( U ) [nO71S]. [f!2r] (A)p.m(C)p. [f!2r] ( U )p.m( U ) [n07 IS]. [f!2r] (G)p.m( U )p.[fI2r](G)p.m( U )p.m £ UCUACUUGUGUU 0106484 U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[nO71S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].m(U)[S ACAGCACAGUU sp].m(U)}$$$$V2.0

[0426] SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](U)[n069R].m(G)p.[fl2r](U)p.[f!2r](A)p.[fl2r](A)p.m£C)p.[fl2 CUGUGCUGUAAC 0106487 r](A)p.m(C)p.[fl2r](A)[n069R].m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[fl2r](A)}$$$$\ / 2.0 ACAAGUAGA SSR- RNAl{[vped5m](U)[Ssp].[fl2r](C)[Ssp].m(U3[nOOlS].[fl2r](A)p.m(C3p.[fl2r](U)p.m(U3[nOOlS].[fl2r](G)p.m(U)p.[fl2r](G)p.m UCUACUUGUGUU 0106517 (U)p.m(U)p.m(A)[Ssp].[f!2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(C)[Ssp].[fl2r](A)[Ssp].nn(G)[Ssp] ACAGCACAGUU

[0427]

[0428] .m(U)[Ssp].m(U)}$$$$V2.0

[0429] Table ID. Example Guide Strand Compositions.

[0430] iD HELM BASE SEQUENCE SSR- RNAl{m(U)[sp].[fi2rj(C)[sp].m(U)p.[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(U)p.m(U)p.m(A)p.[fl2r UCUACUUGUGUU 0104100 ](C)p.m(A)p.[f!2r](G)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(G)[sp].m(U)[sp].m(U)}$$$$V2.0 ACAGCACAGUU SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](U)[Ssp].m(A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001Rj.[fl2r] UUAUAGAGCAAG 0104186 (A)p.m(G)p.m(A)p.[fi2r](A)p.m(C)p.[fi2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl[p.[Rm5d5m](U)[Ssp].[fl2r](U)[Rsp].m(Aj[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001R].[fl2r] UUAUAGAGCAAG 0104189 (A)p.m(G)p.m(A)p.[fl2rHA)p.m(C)p.[fl2rHA)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001R].[fl2r](A)p.m(G) UUAUAGAGCAAG 0104473 p.m(A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001S].[fl2r](A)p.m(G) UUAUAGAGCAAG 0104475 p.m(A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(U)[sp].[fi2r](U)[sp].m(A)p.[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)p.[fl2r](A)p.m(G)p.m(A)p.[fl2r](A UUAUAGAGCAAG 0104717 )p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[sp].m(U)[sp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(U)[Ssp].[fl2r](C)[Rsp].m(C)[n001S].[fl2r](U)p.m(U)p.[fl2r](C)p.m(C)p.m(C)p.m(U)p.m(G)[n001R].[fl2r](A)p.m(A)p UCCUUCCCUGAA

[0431]

[0432] 0105217.m(G)p.[fl2ri(G)p.m(U)p.[fl2r](U3p.m(C)p.iri(C)p.m(U)p.m(C)p.m(C)[Ssp].m(U)[Ssp].m(U)}$$$$V2. O GGUUCCUCCUUSSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)p.[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)p.[fl2r](A)p.m(G)p.m(A)p.[fl2r] UUAUAGAGCAAG 0105245 (A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r](G) UCUACUUGUGUU 0106183 p.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m{C)[Ssp].[fl2r](A)[n001S].m(C)[Sspj.[fl2r](A)[Ssp].m(G)[ ACAGCACAGUU Ssp].m(U)[Ssp].m(U)}$$$$V2.0

[0433] SSR- RNAl{p.[Rm5d5m](U)[Ssp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.iTi(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r](G)p UCUACUUGUGUU 0106224.m(U)p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[S ACAGCACAGUU sp].m(U)[Ssp].m(U)}$$$$V2.0

[0434] SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].[thpyr](A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001S].[fl2r](A)p. UUAUAGAGCAAG 0106453 m(G)p.m(A)p.[fl2rj(A)p.m(C)p.[fl2rj(A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.[thpyr](A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001S].[fl2r](A)p. UUAUAGAGCAAG 0106454 m(G)p.m(A)p.[fl2r](A)p.m(C)p,[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(U)[Ssp].[fl2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[fi2rHG)p.[thpyr](A)p.m(G)p.m(C)p.m(A)in001S].[fi2r](A)p. UUAUAGAGCAAG 0106455 m(G)p.m(A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(U)[Ssp].[fl2rHU)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.in(C)p.[thpyr](A)[n001S].[fl2r](A)p. UUAUAGAGCAAG 0106456 m(G)p.m(A)p.[fl2r](A)p.m(Cjp.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{d([3nU])[Ssp].[fl2r](U)[Rsp].m(A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001S].[fl2r](A)p.m( UUAUAGAGCAAG 0106457 G)p.m(A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(Gjp.m(U)p.m(U)[Ssp].m(U3[Ssp].m(U)}$$$$V2.0 AACACUGUUUU SSR- RNAl{m(U)[Rsp].[fl2r](C)[Ssp].m(U)[n029Sj.[fl2r](A)p.nn(C)p.[fl2r](U)p.m(U)[n029S].[fl2r](G)p.m(U)p.[fl2r](G)p.m(U)p.m( UCUACUUGUGUU 0106512 U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[f!2r](A)[n029S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp].m(U)[S ACAGCACAGUU sp].m(U)}$$$$V2.0

[0435] SSR- RNAl{[d5m](U)[Rsp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r](G)p.m(U) UCUACUUGUGUU 0106514 p.m(U)p.m(A)[Sspj.[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(C)[Ssp].[fl2r](A)[Ssp].! Ti(G)[Ssp].m( ACAGCACAGUU U)[Ssp].m(U)}|CHEMl{[ptz]}$CHEMl, RNAl,l: Rl-l: Rl$$$V2.0

[0436] SSR- RNAl{[d5m](U)[Ssp],[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p. FTi(U)[n001S].[n2r](G)p.m(U)p,[fl2r](G)p.m(U) UCUACUUGUGUU 0106515 p.m(U)p.m(A)[Ssp].[fl2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(C)[Ssp].[fl2r](A)[Sspj.m(G)[Sspj.m( ACAGCACAGUU U)[Ssp].m(U)}|CHEMl{[ptz]}$CHEMl, RNAl,l: Rl-l: Rl$$$V2.0

[0437] SSR- RNAl{[vped5m](U)[Rsp].[fl2r] (C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r] (U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r] (G)p.m UCUACUUGUGUU 0106516 (U)p.m(U)p.m(A)[Ssp].[fi2r](C)[Ssp].m(A)[Sspj.[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp] ACAGCACAGUU.m(U)[Ssp].m(U)}$$$$V2.0

[0438] SSR- RNAl{[vped5m](U)[Ssp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.m(U)p.[fl2r](G)p.m UCUACUUGUGUU 0106517 (U)p.m(U)p.m(A)[Ssp].[f!2r](C)[Ssp].m(A)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[fl2r](A)[n001S].m(C)[Ssp].[fl2r](A)[Ssp].m(G)[Ssp] ACAGCACAGUU

[0439]

[0440] .m(U)[Ssp].m(U)}$$$$V2.0{()()()()()()()()()()()[d][][fl][][][fl][fl][][fl][fl]GG2S2S00S2200S2G2G SS TCUACUUUUU RNAl5TCU1ACUU1UURrsprspmnrpmprpmnrpmprpm-..........

[0441] ()()H)()()()()()()()(G[][fl2[][S][fl2]G[S][S][fl2][0][S][fl2][][]068 ACAGCACAUUUASCSACA01SCASGS 0151 pmpmsprspmsprspmsprnmsprspmspm...........

[0442] $$$$)()}|{}[][]S20 UUCHEMlCHEMlRNAllRllRlVtspmpz::-.,,.

[0443] GG SS UCUACUUUUUR- GG 0069 ACACACAUU151

[0444] GGG SS UUAUAACAAR- G 00620 AACACUUUUU15

[0445] aGGG SS TUAUAACAAR- £ s

[0446] d d d Si a. E £ E E 2 2 2

[0447] " E “ < S G 0062 AACACUUUUU151

[0448] r-.| < C ji <

[0449] L JL E E E CL E CM GGG SS UTAUAACAAR- < < CM <s jd ji. jd 2: g. d G 00622 AACACUUUUU15 <_ 2 2

[0450] CN 3

[0451] a < <2 <zT GGG SS UUATAACAAR- u? £M 3 E? E o <7T <zT UT O O

[0452] o o O O ^7 0 r d ji o A G 00623 AACACUUUUU15 -L O £. CM O o uE. CM JZT 2 0

[0453] o 3 2 rT <77 -_i CM <_ o

[0454] c o o GG SS UUAUAAGCAAR- ” 5£ ^- 0 ~ 0 cj O > 0 A O > r%j a tn. - v> c o A A si 0 H 0 > 0 v> O CM O

[0455] Aj. —. < N —. rsj c? G 0062 AACACUUUUU154 c £ v> c C v> C fN r- -t / y

[0456] > —. v> c V> c: >. C V> —. v> E v> La 71 <r > < > 77 > < > d d 12; V)- v> < w d ^ C <_ 2v>GGG SS UUAUAACAAR- < £ < 77 — - C v> v> XX 3 E to- E d Z v> Si 3T E 3 g. a a E E 12. 2G 0062 AACACUUUUU155 tri E 3 < _d CL ■— CL CM o gp E E £ E O H; E 5: E 3 E CL,-L CX I——) UUAUAGAGCAAG SSR- •X Z)?< Z1O r-L E u E a 2 Ji ci CL -B '2 -— ' CL JZ CL £ 7 E S ~E x — 4-; 4 / j o br C tn — CM £ CL ji o E EG 00626 AACACUUUUU15 E 5 ji £ X ji ~ tn E LD cn, E £ Z) O u. £>i — • c J£L CL 77 a “ E — w 'E 2 2 7: S’ O " CL 2 2GGG SS UUAUAACAAR- - — ' '7T' w E ~ (n < Zi E £ a d £ <9 a O 2 <9 a a E LH, 2 — Si 71 E cQ, E jx 2 d vv G 0062 AACACUUUUU157 E E E A" E a E E g s 7 z> Z) Ji S 5S 3 <2 <2, CL CM £ 2 CL ' d CL CL CX CL r-L dGGG SS UUAUAACAAR- jiC2“ 7: E s£. l / J QL < ~E S E zz? < E S' Q. LO < cAi< <£ tn <£ L^ <s ^2. 2 2 CM XX E,5, 7X CL E -a E G 00628 AACACUUUUU15 C tn ■ ex c ex CL Ji E E S E 5 C Uh § s E S E z? a d £ £ 5' g- £■. CO. tn < r-x ex m GGG SS UUAUAACAAR- ex 71 d - _d c d 2 ji c >& 7 —s 2 T 77 CL <5 Ai E O 2 Pt >»1 / 5£ 5 G 00629 AACACUUUUU15 7? 2 2 3 £ d £ d £ d a d 2 *-. a. CL **— J zr,d a £ £ E d 2 S E S E CM GGG C SS UUAUAACAARM- d CM 2- S' E oj 2 CM 2Z 5 a CL -X £ d — E ±L E ±L E a JJ E ‘g Z) Tf* CL d -S < E 3 £ <1 < Z! d G 00630 AACACUUUUU15 Q. j— d T ^d d d a 77 < Z) d 2 < S < a Ji ji E °z 2. w ZZ7' 2 ~ < «c, E GGG SS UUAUAACAAR- d <2 < E < (£.< (jj 2 1 Si 71 -Q. S'E- E 1_ ’-s S £ E- £ E ji E ji CM ci- E E E E d -S E E d -S: 063 AACACUGUUUU1051 a E £ c? P E d < J. d E d n CX 77" d d T? d d U. JX JX “ E P S s s ii cn ” GGG UUAUAACAA{()()()()()()()()()()()()[][fi][][][fl][fl][][fl]S200S22GG00S2G SS RNAlUURA1UAACA1ARmsprspmnrpmprpmpmpmpmnrpm-........... d d 33: D E CM1“7 E a E 2 3 a E 2 2 2 2 a. 2- " S' < T7 d $$$()()()()()()()()()()()}$[fl][h][][]G2GSS0 00632 AACACUUUUUAACACUUUUUV215t pmprpmppyrpmpmpmpmpmspmspm............ TT a c E — Ji £ ji E — E E tn < Z) CM CM 7 CM ♦ As a S’ £ 5 E x: z) — un a a £ E S a s a 2 -H 2 2 2 2: Z) 77 o £7 “S' _d d d V? £ __i u _x u.7 0 05: 'E a E 7 □: E o a (z, _a c 2 (Z) '7 / (Z1 A- P a o d £ a 'il d £ d £ rH t: 2 2 E £ E 2 E 0 0 E 0 £ 0 2 ° CJ ° CJ CL -A0ci < d? 5 o -ici 0 77 o <i 0 Ji 0 g 0 A: §.a § 7: r’l tn Ct ° d cz — q= a.< A w A, £ A £ A <_ A £ A. <_ a < E C?- “ a 7 ■— 77 C / ) — • a < d <' T7 <x < T7 <■ < TT O 77 f g d £ < -H s < P CM 77 2 & 5 E S ~ 5~. < E " E S E ■E s E <5 cc £ E. a T? 3 o I S CM E £ EL d 2 d CL d 2 d CL rsi 7 a ” a < N 2 £ 3 IX g tn: -, t / i 7V tn S’ d tn r“> _A u tn 7*7 rr O d rr Si DC a. & Si Si ££ d CL; _ 1, -t / > a g tn H 77 c t, d — d d d Af £ s T S ZD. 3 >. t- tn V> a CL Si u, Si a a a CL d a 77{-Z1. 77 °- 77 a E c?10. Q. g 2 2: D -- 77 TT < rj E CM CM CM jjj, CL -CL 5 £ “ <. Z) ~ 7; r-j T7 W i — i S " d;*-. d 2 2 a d ±i d d g, CM a <_ E E E" XI CM XX 3 XX < N i — t ef ^ <5 Id CM CL SC CL ZX CL CL 5= Q. 2 <, a PT 73 CL. — ■ ( / ) r— i tn1— -• un —7' tn1— / tn T-1tnrT~ ’ LD “*» ^L < ZJ TT < X 3 ^2, CL ^ 2 V2, jx rxi 2 81 a a 5 77 ex 77 rM u < S < Z) 3 <, z) 3 2 f a ~5.2- p a < 2 ^L r— CL r~ ^L £ £ £ ir -~? d r ^E, g E E sT E 2 E 7: £ d £ < CL ■^7 CL ^7 CL pq" CL S £ 15. XT < XT 2 a < ^7 i- < -S: < 2 < ■£ < o < E < E < E < 0 < c < E < C £ z E a 35» z: Z 7T H 5 z E >s~. tn 772d2d cc At DC E C£ c cc At CC CL DC CL cc d DC CL < Z) DC CM ££ E 0£ Pt

[0457]

[0458] GGG SS UUAUAACAAR- G0633 AACACUUUUU 015

[0459] {()()()()()()()()()()()()[][flj[][][fl][fl][][fl]GGGS200S22GG00S2G SS UUAUAACAA RNAlUURA1UAACA1ARmsprspmnrpmprpmpmpmpmnrpm-,.,........

[0460] $$$()()()()()()()()()()()}$[fl][fl][h][][]G 006322GSS20 AACACTUUUU154AACACTUUUUVt pmprpmprpmppyrpmpmpmspmspm............

[0461] GGG SS UUAUAACAAR- G 0063 AACACUUUUU155

[0462] GGG SS UUAUAACAAR- G 00636 AACACUTUUU15

[0463] £N

[0464] ID < D GG SS UUAUAAGCAAR- d d

[0465] 2 2 £ 15 15

[0466] E E E E G 0063 AACACUUTUU157 E 2 -UT- £ d £ d _CL d. jd. ji jGGG SS UUAUAACAARd- E S' E E

[0467] d 3 d £ <_ < £ o

[0468] G 006 o E E 38 AACACUUUTU15 2 E o c o _ 2 jd 2 rsi Ai c A

[0469] 15 15 {()()()()()()()()()()()()[][fl][][][fl][fl][j[fl]GG SSS200S22GG00S2 UUAUAAGCAAR RNAlUURA1UAACA1AGmsprspmnrpmprpmpmpmpmnrpm-........... E r< (N £ E £ rS '-i < > < > d £ d o o $$$$()()()()()()()()()()()}[f!][fi][l[][h]G 0063922GSS20 AACACUUUUT15AACACUUUUTVt pmprpmprpmpmpmpmpmspmsppyr............ E E 2 el 2 ™ ST A

[0470] rH fN <zT A Z7? A

[0471] rH r-4 2 S C -U7 £ - v w> 2 d 3 jd o > o > O > GGS UCUACUUUUUR- 2 v>

[0472] O V> O Vf o O v > o > S

[0473] > -& 2 15 £ 15 £. C V> c o v> o -tn- u

[0474] C v> c: < / y GG 0068 ACACACAUU154 ■2 TS —. < / >1— • v> “ v> v> S CL E 2.

[0475] < <_ 12 < < v> Q- tdL E • E d 15 d CL d. e CL »

[0476] £ E S 3 E S 3 <□ CL 2 _d 2 £ 2 £ 2 {()()()()()()()()()()[d][][fl][][i[fl][fl][][fl][fl]G200S2200S22G SS UCCUUCCCUAA RNAlR55URCRC1UUCC1CURpmmsprspmnrpmprpmnrpmprp-.,........ ‘Z! 2 S- E,a 1 E ji £ £ E E E 2 s o < >. o d u U r- CAL O _A U) r-2, U,-2, ()()()()()()()()()()()[][fl][][][fl][][][fl][][][fl][][ GGUCCUCCUU 0106587AAGS2GSUS2USCS2C001SUS2CSCSUmpmpmsprspmsprspmsprnmsprspm........... £ S:

[0477] "7^” £X» LZ" Q Ji S £- CM ji ~ £ o

[0478] E C V r- CX. 3z S’

[0479] E E E $$$$()()}][]S20UUV spmspm... 2 vT v> £ fN E iz)

[0480] CL “ C £ jd ■ tn 2 “

[0481] L jd T7 < 2 ^ < E E 3z 5 >

[0482] GGG SS 2 -uy J9 v> UUAUAACAAR- CJ v> d vy r-J Si £ s o 2 o 2 2. ■3 v> £ LO- £ d. S’ 2 CL W 2 G 006620 AACACUUUUU1 £ £ £? E 5 d. v> E E E E £ E E E £. 2 ^ E ‘X d S d. a. ji J9- d CL tD " ZD J-L oZ ’ E •i EGGG SS UUAUAACAAR- -H: CL « O S < D < <% < ii <£ 3 2 E < <3 c ^. ■2^ 2 £ E g E " Q. G 00662 AACACUUUUU11 &• E Zz> vi t~ E " C | — ’ CL E 3 d. O- __L E 5" £ a • LO CL —. CL V)GGG SS UUAUAACAAR- CL £ “ ex f“ Q. 2 “ < Z|, H S Ji 'zz < 3-. £“ < •— <* Sz < 2 o £ JI, G 00662 AACACUUUUU15 £ 2 2 2 | 2 s CL 2 d. E d r-H E £ 2 E ji ZJ ~£ - £ i’**1* CD CM Ai H. 1 d. Sz GGG SS UUATAACAAR- £ 2 d- E S’ 2 o £J E Q.2 E c d s- E S 2 S 2 s °- o SG 006626 AACACUUUUU1 s $ jd d ss xr a. °- “ E jd Z) _9r g < £ CM 1- IZl „ V) CL ji S GGG SS UUAUAACAAR-.< £ <_ CM ■ d rxi 2. tn ’"■J di 2 E,d dq <.E*-. ZD E ZT E d E d E d. E E S E 0662 AACACUGUUUU107 d S c. U >-■ jd ^d d E Ji d. ji E S’ E <. H S.£■ S jj <5 £ GGG UUAUAACAA SSR- <, d. J2z z) r ZD ZD o d. zi 'S’ E S 2 o d 2 & £ CL Q_ 2 g G 006628 AACACUUUUU1? O _d £ E d £ 2? 2 CN DC rxi di S3 S’ s <f g 2 d Q- ji M=, Z> M= ZD < N t. < £ £ 2 E < d — 1- O ±L <£ 2 < D r-A C 2 fN • >. d. E " < Z) “ ST E ST E < S) ST E E < TT <_D CL £X £3 CL rxi E rx -— • V) O. r—. < J3 S S 3 Ji 3 °z O > o Vi — j 2 jd 5= a- o £*- o o o u o o o w 2 £ S 3 c, •£ C 7T c c " c: Dx' £ S d ZZD 2 d & g- J: O E £r 7^ E 2 Si E Ji CL

[0483] CL g <_ CJ <, d. ZD d < d. <, d d < j± 2 ^d < E £N

[0484] 5 d TT (J T? tJ E <, E <_ E <_ E,2 E 2 E 5--, 2 ■7=' CM r-A CD S 2 E S CL s -i S' 3 S’ < N V) c S’ S’ < N S’. S:,-2, d d 2 ce o= C£ CC uC er: C£ az C£ H 6 £ <■ CL CL s- < “ Ey:< 3 3 S S' cc S S E S £ < 2 T? 2 < 2 ji ” d. 2 £ £ “ 22 2 2 TT < 5 “ S — rsi < rsi — 15 E 15 E 15 E 5 E 5 E <5 Ai S ^5 (N v)

[0485] d d •—Z £N 3" d 3* d Ji -H: <r in <r a itz, lx < ’’CL < d d _d a < 2 "d 2 < ■o -zi. “O -Zx Vi v) 77 Vi v) tSs CL ' — > m c < Z) TT <z; T7 LH TT <zi TT tn T7 LH 2 S 2 E u—* 2 d CM r=! < N — o 21 £ ■—■j £ 7 _S ZD CJ ±L 2 S, 2- 2 sz d “ < D T*' “ ID “ E E r> — E d CL - ^E E E J9 E.9: J: E £. _d. V) n < — < 77 < ^7 E 77 < 7^7 < dr < £7 < 77 £ 7^7 d

[0486] < < d. < < cz c < < _ < <<tJi 2 — o 1 E E 1 £ 2 9

[0487] or or d § < CC ±L cc d C£ CL CC CL or £ r-j Zi- CC ±L DC TT az rj

[0488]

[0489] GGGGG SS UUUAAUAR- 02G 01747 AUUAAAAUUU

[0490] GGGGG SS UUUAAUAR- G 0028 AUUAAAAUUU174

[0491] G SS UAACAAUAAAUR- GGG 008082 ACCAAAUU1

[0492] aG SS UAACAAUAAAUR- i CL 2 ° < 0

[0493] E E > C Q. GGG 00808 ACCAAAUU14

[0494] d — VI E §

[0495] — d 12 d v> {()()()(()(())()[d][][fl][][][fl])[fl])[][fl]([fl])G2SG00S2200S22 SS UAACAAUAAAU RNAlR55URA1ACAA1UAAR(pmmsprspmnrpmprpmnrprpmp-..........

[0496] £ £ 12 < < 2 ■$■ $$$$()()()()()()()())()()()()}[fl][fl][][]GGG22GGGSS20 00808 ACCAAAUUAUACCAAAUUV15(mpmpmprpmprpmpmpmpmpmspinspm.............. £

[0497] s S

[0498] s=. s-E

[0499] £ E E. £ s i £ v>. 0

[0500] CL a <§ CX kD d _d < CM < q a a / ! d ^

[0501] z> 2. =) 5 3 '^ £ q Zs r^J 3 < Z - C inL

[0502] E g E a rN

[0503] _£X > -S 0 --7 (Z) a §SSQC NAME BAEEUENE HELM_ a 2 S’ IN S' _< CM A] E C--J ■—• v -S £7 ZD Z) LU d1- ' < Z! E.2:5— • X S E as x & a GG SS WV AACAUUUCUUR-- a. —. < Z) 22;, - ( in 2T O

[0504] £ £ CM — < Z) E g: -tX 2 2 3 a 828 G 0096 41CUCUAUAA115 < < Z! g eg 0 i cj 0 a. d. 0 12 tn § £ E £ 15? E ji o' \ _ a 0 ZD c E£L E? a “ 13 3 — <_l < N XS CL d aGG SS wv AACAUUUCUUR-- 3 2 m O 3 y- £ ^ 2 <77 a 7T J ■g? CL CM CM TT <-> T 0 (7? w T? 2! a CD2080 G 0099 4CUCUAUAA115 E 0 E d 2 <3 EE. E 0 L) <3 E £ 3 i±. < d JE g. ri d ° d a m 5 1 a a jX d 2 zz? < z f-. tn < < or 'as Q if < “ < ID E (D un < E Z) H3 CJ ro?-M E — 0 GGGG SS wvAAACCUUCR-- £ 0 E T? £ 0 J " E 3 5 CM ♦ ™ a £ S 0 Ji < N Q. r—, TO 2! E 2 296GGGGG 0023 415 AAAA154 rq g. < (N 75” ±L CL DC ZZ • m S’ 2! £ d. S SECL LfJ d E d tn \ _ O;x: 0 d CL LU d S Aj 0 ax 0 £2 £ J2, < s 2 a s GG SS CUGUCUUAACR- -F- ^d. a 2 LU £ CM a 5 E 0 E <_ E £ J CL £ E a x GG 006206 ACAAUAA1 S < N (U. S' 2- £ S CL -A CL d 2 a? X5 2D £ H. Cl d < „ a s q E < ig < L in < c GGG SS CUUCUUAACR- d; C; 2 a (Z) < E IN d <_ E £ E ri 20 < q GG 00603 ACAAUAA15 —r;rj S' 35 < N 52 S 22! E Js CL VS- S S ex r E 5 2^ v> VL S 2 a S' ±7 E “ d S £ {()[]()[fl]()()[fl]()()[hd]()()[fl]()[fl]()[fl](GGS2G2G222 SS CUGUCUUAAC RNAlCUUCUGU2AARmspmprpmprpmpexecrpmprprpr-..........2! ” v> ±L £, V> 0 £ u xs E. £ v> y, ~4", v> <zT <77 <_ <77 <?7 a d. < a <3 9: GG 00606 ACAAUAA15$$$ )()()()()()()()()()()}$[fl][fl][fl][fl][][fi]222G2GS220CACAAUAAVpmprpmprpmprpmprpmspr........... r-ir-.; E ±x E " E 0 " X 0 SL 0 E ex d £ d a 2 2 < E z 0 ■ § a a GGG SS CUUCUUAACR- c n. C u (N CZ r— i? E tn c ex rz < izC CL gg CL XS LH a s £ 2 aEz GG 0060 ACAAUAA157 XS < j n4 / 1E a. <'-< ZD a S' TT E.0 a £ rH £ c CL • — ■ CC CM £ 13 Al si d Z) ’I 13 a E ~- E 2! E —

[0505] r-A E 2. S r-L rsl " E rH E d 5 d S E d - CL _L Cl. tp: CL E • — LU S' d inEd 2 a < - a < x SE< A S’ CX LU 2. T? s: “ 3D a £ —. cn a < C 13 a a " E r~< E ™ ID xs 2f 5 E < Si 12 E CL c ex XS ZD d ex xs 7T E jd < LU d E d ™ CL s s CM ZD? s qc Z CM S a < ~E 6 = 2 £ 2 5 JX £ a £.£ £ 1 E ex IN E E d. “ f’M CL dS d c E W CL -A CL ±L CL S' y in £ d CL d. u u a 0 DC ±L CC br < N d a ££ Q. O XS LH > a 'g a s < E Z £ 2 "> XS V> £ 23 Es E < 2 £ a 13 £ E -i Si E S a a => E E" E E 22 s, 2 m r—l LD “• XX LT) 5 m c v> c: CL — > " J vi ■7=' _r a 2 d in rZ. Z..73 CL X5 ■ vy Q- ~D XJ - 12 E < a £ < in in fN <n CM Lf) CL c^ UH CL LH qx < Z) qx CX 2 y y. <■ £ < G" a 1 E £ E S J E E ZD 3- u* a a 2 ££ — a £ a S E s 2 £ £ E gr cs d jj — E “ E n CL <zs d E S ■7Z fx? CL ji d ~ 5 y y a J“2 < Z) ■S d £ d ex < 11 yr 0 a <.;5 a a Z E ‘Z7‘ tn H 5 <£ CL < < >':::> < -S. < < 0 < i < TT < £ 5 d C- z E Z “ V> 2Z JS 2 7^ X 2 CM C c

[0506] DC d oc E zi ££ E er is. 0 S E Kh DC < <z: DC CL E tr E d

[0507] a

[0508]

[0509]

[0510] SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[hexdec2r](U)p.m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A CUGUGCUGUAAC 0106508 )p.m (C)p.[fl2rj (A)p.m (C)p.[fl2r] (A)[n001 R].m(A)p.[f l2r](G )p.m(U)p.[fl2r] (A)p.m (G) [Ssp],[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[hexdec2r](U)[n001R].m(G)p.[fl2r](U)p.[fl2r](A)p.[ CUGUGCUGUAAC 0106509 f!2r](A)p.m(C)p.[f!2r](A)p.m(C)p.[f!2r](A)[n001R].m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[f!2r](A)}$$$$ ACAAGUAGA V2.0

[0511] SSR- RNAl{m(C)[Ssp].m(U)[Ssp].[fi2r](G)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[hexdec2r](U)[n001R].m(G)p.[fl CUGUGCUGUAAC 0106510 2r](U)p.[fl2r](A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)[n001R].m(A)[Ssp].[fi2r](G)[Ssp].m(U)[Ssp].[fl2r]( ACAAGUAGA A)[Ssp].m(G)[Ssp].m(A)}$$$$V2.0

[0512] SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](U)[n029R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r]( CUGUGCUGUAAC 0106511 A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)[n029R].m(A)p.[fl2r](G)p.m(U)p.[fi2r](A)p.m(G)[Ssp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)[Ssp].[f!2r](G)[Ssp].m(U)[Ssp].[fl2r](G)[Ssp].m(C)[Ssp].[f!2r](U)[n029R].nn(G)p.[fl2r](U) CUGUGCUGUAAC 0106513 p. [f!2r]( A)p. [fl2r](A)p,m (C)p. [fl2r](A)p.m (C)p.[fl2r](A)[nO29R].m(A) [Ssp].[fl2r](G)[Ssp].iri(U) [Ssp]. [fl2r] (A) [Ss ACAAGUAGA p].m(G)[Ssp].m(A)}$$$$V2.0

[0513] SSR- RNAl{m(C)[sp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](U)[n001].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A)p CUGUGCUGUAAC 0106563.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)[ri001].m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)]sp].m(A)}$$$$V2.0 ACAAGUAGA SSR- RNAl[m(G)[Ssp].m(G)p.[f!2r](A)p.m(G)p.[fl2r](G)p.m(A)p.[fl2r](A)[n001R].m(C)p.[fi2r](C)p.[fl2r](U)p.[fl2r](U GGAGGAACCUUC 0106589 )p.m(C)p.[fl2r](A)p.m(G)p.[fl2r](G)[n001R].m(G)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(G)[Ssp].m(A)}$$$$V2.0 AGGGAAGGA SSR- RNAl{m(C)[sp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[hexdec2r](U)p.m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r](A) CUGUGCUGUAAC 0106597 p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[sp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[sp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[hexdec2r](U)[n001R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl CUGUGCUGUAAC 0106598 2r](A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[sp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[sp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[hexdec2r](U)[n009R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl CUGUGCUGUAAC 0106599 2r](A)p.m(C)p.[fl2r](Ajp.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[sp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[sp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[hexdec2r](U)[n033R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl CUGUGCUGUAAC 0106600 2r](A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[sp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[hexdec2r](U)[n009R].m(G)p.[fl2r](U)p.[fl2r](A)p.[ CUGUGCUGUAAC 0106601 f I2r] (A)p.m [C)p.[fl2r] (A)p.m [C)p.[fl2r] (A)p.m(A)p.[fl2r] (G)p.m(U)p.[fl2r](A)p.m (G) [Ssp]. [fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[hexdec2r](U)[n033R].m(G)p.[fl2r](U)p.[fl2r](A)p.[ CUGUGCUGUAAC 0106602 fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](U)[n031R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r]( CUGUGCUGUAAC 0106603 A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](U)[n037R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r]( CUGUGCUGUAAC

[0514]

[0515] 0106604 A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[fi2r](A)}$$$$V2.0 ACAAGUAGASSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2r](G)p.m(C)p.[fl2r](U)[n046R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r]( CUGUGCUGUAAC 0106605 A)p.m(C)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fi2r](G)p.m(C)p.[fl2r](U)[n047R].m(G)p.[fl2rHU)p.[fl2r](A)p.[fl2r]( CUGUGCUGUAAC 0106606 A)p.m(C)p.[fi2r](A)p.m(C)p.[fl2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[fl2r](A)}$$$$\ / 2.0 ACAAGUAGA SSR- RNAl{m(C)[Ssp].m(U)p.[fl2r](G)p.m(U)p.[fl2rKG)p.m(C)p.[fl2r](U)[n043R].m(G)p.[fl2r](U)p.[fl2r](A)p.[fl2r]( CUGUGCUGUAAC 0106607 A)p.m(C)p.[fi2r](A)p.m(C)p.[fi2r](A)p.m(A)p.[fl2r](G)p.m(U)p.[fl2r](A)p.m(G)[Ssp].[fl2r](A)}$$$$V2.0 ACAAGUAGA SSR- RNAl{p.m(A)[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fl2r](U)p.[fl2r](C)p.[fl2r](U)p.m(U)p. AACAGUGUUCUU 0106629 m(G)p.m(C)p.m(U)p.m(C)p.m(U)p.m(A)p.m(U)p.m(A)[Ssp].[thpyr](A)}|CHEMl{[nC6o]}|CHEM2{[GalNAc3Cl GCUCUAUAA 2oyl]}$CHEMl, RNAl,l: Rl-l: Rl| CHEMI, CHEM2,1: R2-1: R1$$$V2. O

[0516] SSR- RNAl{p.nn(A)[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fl2r](U)p.[fl2r](C)p.[fl2r](U)p.nn(U)p. AACAGUGUUCUU 0106630 m(G)p.m(C)p.m(U)p.m(C)p.m(U)p.[thpyr](A)p.m(U)p.m(A)[Ssp].m(A)}|CHEMl{[nC6o]}|CHEM2{[GalNAc3Cl GCUCUAUAA 2oyl]}$CHEMl, RNA1,1: R1-1: R11 CHEMI, CHEM2,1: R2-1: R1$$$V2. O

[0517] SSR- RNAl{p.m(A)[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fi2rHU)p.[fl2r](C)p.[fl2r](U)p.m(U)p. AACAGUGUUCUU 0106631 m(G)p.m(C)p.m(U)p.m(C)p.m(U)p.m(A)p.[thpyr](T)p.m(A)[Ssp].m(A)}|CHEMl{[nC6o]}|CHEM2{[GalNAc3Cl GCUCUATAA 2oyl]}$CHEMl, RNAl,l: Rl-l: Rl| CHEMI, CHEM2,1: R2-1: R1$$$V2. O

[0518] SSR- RNAl{p.m(Aj[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fl2rHU)p.[fl2r](C)p.[fl2r](U)p.! Ti(U)p. AACAGUGUUCUU 0106632 m(G)p.m(C)p.m(U)p.m(C)p.m(U)p.m(A)p.m(U)p.[thpyr](A)[Ssp].m(A)}|CHEMl{[nC6oj}|CHEM2{[GalNAc3Cl GCUCUAUAA 2oyl]}$CHEMl, RNA1,1: R1-1: R11 CHEMI, CHEM2,1: R2-1: R1$$$V2. O

[0519] SSR- RNAl{p.m(A)[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fl2rHU)p.[fl2r](C)p4fl2r](U)p.m(U)p. AACAGUGUUCUU 0106633 m(G)p.m(C)p.m(U)p.m(C)p.[thpyr](T)p.m(A)p.m(U)p.m(A)[Ssp].m(A)}|CHEMl{[nC6o]}|CHEM2{[GalNAc3Cl GCUCTAUAA 2oyl]}$CHEMl, RNAl,l: Rl-l: Rl| CHEMI, CHEM2,1: R2-1: R1$$$V2. O

[0520] SSR- RNAl{p.m(A)[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fl2rHU)p.[fl2r](C)p.[fl2r](U)p.! Ti(U)p. AACAGUGUUCUU 0106634 m(G)p.[thpyr](C)p.m(U)p.m(C)p.m(U)p.m(A)p.m(U)p.m(A)[Ssp].m(A)}|CHEMl{[nC6oj}|CHEM2{[GalNAc3Cl GCUCUAUAA 2oyl]}$CHEMl, RNAl,l: Rl-l: Rl| CHEMI, CHEM2,1: R2-1: R1$$$V2. O

[0521] SSR- RNAl{p.!n(Aj[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fl2rHU)p.[fl2r](C)p.[fl2r](U)p.iTi(U)p. AACAGUGUUCUU 0106635 m(G)p.m(C)p.[thpyrHT)p.m(C)p.m(U)p.m(A)p.m(U)p.m(A)[Ssp].m(A)}|CHEMl{[nC6o]}|CHEM2{[GalNAc3Cl GCTCUAUAA 2oyl]}$CHEMl, RNA1,1: R1-1: R11 CHEMI, CHEM2,1: R2-1: R1$$$V2. O

[0522] SSR- RNAl{p.m(A)[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fl2rHU)p.[fl2r](C)p.[fl2r](U)p.m(U)p.[ AACAGUGUUCUU 0106637 thpyr](G)p.m(C)p.m(U)p.m(C)p.m(U)p.m(A)p.m(U)p.m(A)[Ssp].m(A)}| CHEMl{[nC6o]}| CHEM2{[GalNAc3Cl GCUCUAUAA 2oyl]}$CHEMl, RNAl,l: Rl-l: Rli CHEMI, CHEM2,1: R2-1: R1$$$V2. O

[0523] SSR- RNAl{p.m(A5[Ssp].m(A)p.m(C)p.m(A)p.m(G)p.m(U)p.[fl2r](G)p.m(U)p.[fl2rHU)p.[fl2r](C)p.[fl2r](U)p.[thpyr]( AACAGUGUUCUT 0106638 T)p.m(G)p.m(C)p.m(U)p.m(C)p.m(U)p.m(A)p.m(U)p.m(A)[Ssp].m(A)}|CHEMl{[nC6o]}|CHEM2{[GalNAc3C12 GCUCUAUAA

[0524]

[0525] oyl]}$CHEMl, RNAl,l: Rl-l: Rl| CHEMI, CHEM2,1: R2-1: R1$$$V2. O{()()()()()()()()()()()()[][fl][fl][fl][fl]GGSG2G222 SS AACAUUUCUU RNAlAACAUUUCUURpmspmpmpmpmpmprpmprprprpmp-.............

[0526] ()()()()()()()()()}|{}|{ G0660G[h][S][6]2[GlCUCUAUAA 014CUCUAUAACHEMlCCHEMNA3Clt mpmpmppyrpmpmpmpmspmnoac........

[0527] $$$$} |l] 222OCHEMlRNAllRllRl CHEMI CHEM21R1R1Voy::::--,,,,.

[0528] {()()()()()()()()()()()([][fl][][fl][fl][fl]S20022 RNAlCAUUUUA1RAUC2Cmspmpmpmpmpmprnmprprprpm...........

[0529] $$$$)()()()()()()()()()}[][]0S20 UCAC01RUCUAAAVpmpmpmnmpmpmpmpmspm..........

[0530] X X

[0531] f

[0532] i t

[0533] g

[0534] i |

[0535] H X

[0536] X X X

[0537] J| | J|

[0538] ^ J

[0539] X

[0540] 1 1 I I j| 4

[0541] t l

[0542] X

[0543] ^

[0544]

[0545] dl Gi P Dassengerueupex

[0546] SS0096 SS0020S006R115R147 DR1151--- SS0S026 SS099R014474 DR0111R1015--- SS099 SS0S0022R1015R014475 DR154--- SS009 SS0036S002630R1047R014 DR1--- SS0009 SS00066S00263R147R14 DR11--- SS0002 SS0002S002632R141R145 DR1--- SS02 SS0082S002633R01401R104 DR1--- SS099 SS0003S00263R0140R141 DR14--- SS00099 SS0006S00263R14R141 DR15--- SS00026 SS0003S002636R14R147 DR1--- SS0026 SS06S0263R104R01407 DR017--- SS0030 SS003S002638R104R0145 DR1--- SS00030 SS0006S002639R14R145 DR1--- SS00092 SS0003S00260R14R144 DR14--- S092 SS003S0026SR0140R1047 DR141--- SS022 SS00S00266R0140R10454 DR11---

[0547]

[0548]

[0549] DSR-0102662 SSR-0104084 SSR-0104022 DSR-0102663 SSR-0104053 SSR-0104022 DSR-0102664 SSR-0104083 SSR-0104022 DSR-0102665 SSR-0104039 SSR-0104027 DSR-0102666 SSR-0104069 SSR-0104027 DSR-0102667 SSR-0104038 SSR-0104027 DSR-0102668 SSR-0104068 SSR-0104027 DSR-0102669 SSR-0104045 SSR-0104094 DSR-0102670 SSR-0104075 SSR-0104094 DSR-0102671 SSR-0104044 SSR-0104094 DSR-0102672 SSR-0104074 SSR-0104094 DSR-0103133 SSR-0106188 SSR-0104027 DSR-0103134 SSR-0106190 SSR-0104027 DSR-0103135 SSR-0106180 SSR-0104027 DSR-0103136 SSR-0106181 SSR-0104027 DSR-0103137 SSR-0104101 SSR-0104029 DSR-0103138 SSR-0106185 SSR-0104026 DSR-0103139 SSR-0106189 SSR-0104026 DSR-0103140 SSR-0106195 SSR-0104026 DSR-0103141 SSR-0106172 SSR-0104026 DSR-0103142 SSR-0106184 SSR-0104026

[0550]

[0551] DSR-0103143 SSR-0106189 SSR-0104027 DSR-0103144 SSR-0106170 SSR-0104027 DSR-0103145 SSR-0106172 SSR-0104027 DSR-0103146 SSR-0106178 SSR-0104027 DSR-0103147 SSR-0106194 SSR-0104026 DSR-0103148 SSR-0106178 SSR-0104026 DSR-0103149 SSR-0106181 SSR-0104026 DSR-0103150 SSR-0106187 SSR-0104027 DSR-0103151 SSR-0106197 SSR-0104027 DSR-0103152 SSR-0106174 SSR-0104027 DSR-0103153 SSR-0106191 SSR-0104026 DSR-0103154 SSR-0106179 SSR-0104026 DSR-0103155 SSR-0106182 SSR-0104026 DSR-0103156 SSR-0106186 SSR-0104027 DSR-0103157 SSR-0106173 SSR-0104027 DSR-0103158 SSR-0106176 SSR-0104027 DSR-0103159 SSR-0106184 SSR-0104027 DSR-0103160 SSR-0106187 SSR-0104026 DSR-0103161 SSR-0106188 SSR-0104026 DSR-0103162 SSR-0106176 SSR-0104026 DSR-0103163 SSR-0106177 SSR-0104026

[0552]

[0553] DSR-0103164 SSR-0106183 SSR-0104026 DSR-0103165 SSR-0106185 SSR-0104027 DSR-0103166 SSR-0106193 SSR-0104027 DSR-0103167 SSR-0106175 SSR-0104027 DSR-0103168 SSR-0106186 SSR-0104026 DSR-0103169 SSR-0106190 SSR-0104026 DSR-0103170 SSR-0106192 SSR-0104026 DSR-0103171 SSR-0106175 SSR-0104026 DSR-0103172 SSR-0104039 SSR-0104026 DSR-0103173 SSR-0106180 SSR-0104026 DSR-0103174 SSR-0106192 SSR-0104027 DSR-0103175 SSR-0106195 SSR-0104027 DSR-0103176 SSR-0106171 SSR-0104027 DSR-0103177 SSR-0106179 SSR-0104027 DSR-0103178 SSR-0106197 SSR-0104026 DSR-0103179 SSR-0106171 SSR-0104026 DSR-0103180 SSR-0106174 SSR-0104026 DSR-0103181 SSR-0106194 SSR-0104027 DSR-0103182 SSR-0106196 SSR-0104027 DSR-0103183 SSR-0106182 SSR-0104027 DSR-0103184 SSR-0106193 SSR-0104026

[0554]

[0555] DSR-0103185 SSR-0106170 SSR-0104026 DSR-0103186 SSR-0106191 SSR-0104027 DSR-0103187 SSR-0106177 SSR-0104027 DSR-0103188 SSR-0106183 SSR-0104027 DSR-0103189 SSR-0104037 SSR-0104027 DSR-0103190 SSR-0104100 SSR-0104028 DSR-0103191 SSR-0106196 SSR-0104026 DSR-0103192 SSR-0106173 SSR-0104026 DSR-0103199 SSR-0106222 SSR-0104026 DSR-0103200 SSR-0106225 SSR-0104026 DSR-0103201 SSR-0106188 SSR-0106208 DSR-0103202 SSR-0106188 SSR-0106210 DSR-0103203 SSR-0106188 SSR-0106212 DSR-0103204 SSR-0106188 SSR-0106218 DSR-0103205 SSR-0106224 SSR-0104026 DSR-0103206 SSR-0106183 SSR-0106447 DSR-0103207 SSR-0106188 SSR-0106211 DSR-0103208 SSR-0106188 SSR-0106215 DSR-0103209 SSR-0106188 SSR-0106217 DSR-0103210 SSR-0104100 SSR-0106445 DSR-0103211 SSR-0104100 SSR-0106446

[0556]

[0557] DSR-0103212 SSR-0106188 SSR-0106207 DSR-0103213 SSR-0106188 SSR-0106213 DSR-0103214 SSR-0106188 SSR-0106214 DSR-0103215 SSR-0106188 SSR-0106219 DSR-0103216 SSR-0106223 SSR-0104026 DSR-0103217 SSR-0106183 SSR-0106448 DSR-0103218 SSR-0106183 SSR-0106452 DSR-0103219 SSR-0106188 SSR-0106206 DSR-0103220 SSR-0106188 SSR-0106216 DSR-0103221 SSR-0106220 SSR-0104026 DSR-0103222 SSR-0106221 SSR-0104026 DSR-0103223 SSR-0106183 SSR-0106449 DSR-0103224 SSR-0106183 SSR-0106450 DSR-0103225 SSR-0106183 SSR-0106451 DSR-0103226 SSR-0106188 SSR-0106209 DSR-0103227 SSR-0106266 SSR-0101599 DSR-0103228 SSR-0106267 SSR-0101599 DSR-0103229 SSR-0106268 SSR-0101599 DSR-0103230 SSR-0106269 SSR-0101599 DSR-0103231 SSR-0106270 SSR-0101599 DSR-0103232 SSR-0106271 SSR-0101599

[0558]

[0559] DSR-0103233 SSR-0106272 SSR-0101599 DSR-0103234 SSR-0106273 SSR-0101599 DSR-0103235 SSR-0106274 SSR-0101599 DSR-0103236 SSR-0106275 SSR-0101599 DSR-0103237 SSR-0106276 SSR-0101599 DSR-0103238 SSR-0106277 SSR-0101599 DSR-0103239 SSR-0106278 SSR-0101599 DSR-0103240 SSR-0106279 SSR-0101599 DSR-0103241 SSR-0106280 SSR-0101599 DSR-0103242 SSR-0106281 SSR-0101599 DSR-0103243 SSR-0106282 SSR-0101599 DSR-0103244 SSR-0106283 SSR-0101599 DSR-0103245 SSR-0106284 SSR-0101599 DSR-0103246 SSR-0106285 SSR-0101599 DSR-0103247 SSR-0106286 SSR-0101599 DSR-0103248 SSR-0106287 SSR-0101599 DSR-0103249 SSR-0106288 SSR-0101599 DSR-0103250 SSR-0106289 SSR-0101599 DSR-0103251 SSR-0106290 SSR-0101599 DSR-0103252 SSR-0106291 SSR-0101599 DSR-0103253 SSR-0106292 SSR-0101599

[0560]

[0561] DSR-0103254 SSR-0106293 SSR-0101599 DSR-0103255 SSR-0106294 SSR-0101599 DSR-0103256 SSR-0106295 SSR-0101599 DSR-0103257 SSR-0106296 SSR-0101599 DSR-0103258 SSR-0106297 SSR-0101599 DSR-0103259 SSR-0106298 SSR-0101599 DSR-0103260 SSR-0106299 SSR-0101599 DSR-0103261 SSR-0106300 SSR-0101599 DSR-0103262 SSR-0106301 SSR-0101599 DSR-0103263 SSR-0106302 SSR-0101599 DSR-0103264 SSR-0106303 SSR-0101599 DSR-0103265 SSR-0106304 SSR-0101599 DSR-0103266 SSR-0106305 SSR-0101599 DSR-0103267 SSR-0106306 SSR-0101599 DSR-0103268 SSR-0106307 SSR-0101599 DSR-0103275 SSR-0106183 SSR-0106206 DSR-0103276 SSR-0106183 SSR-0106209 DSR-0103299 SSR-0106222 SSR-0106206 DSR-0103300 SSR-0106222 SSR-0106209 DSR-0103301 SSR-0106222 SSR-0106447 DSR-0103319 SSR-0106480 SSR-0106206

[0562]

[0563] DSR-0103320 SSR-0106481 SSR-0106206 DSR-0103321 SSR-0106482 SSR-0106206 DSR-0103322 SSR-0106483 SSR-0106206 DSR-0103323 SSR-0106484 SSR-0106206 DSR-0103324 SSR-0106487 SSR-0106206 DSR-0103325 SSR-0104037 SSR-0106206 DSR-0103326 SSR-0106479 SSR-0106206 DSR-0103337 SSR-0104103 SSR-0104107 DSR-0103338 SSR-0104104 SSR-0104024 DSR-0103339 SSR-0104102 SSR-0104106 DSR-0103340 SSR-0104105 SSR-0104025 DSR-0102626 SSR-0104473 SSR-0101599 DSR-0102657 SSR-0105217 SSR-0105234 DSR-0103028 SSR-0104717 SSR-0101596 DSR-0103099 SSR-0105245 SSR-0101599 DSR-0103327 SSR-0106453 SSR-0101599 DSR-0103328 SSR-0106454 SSR-0101599 DSR-0103329 SSR-0106455 SSR-0101599 DSR-0103330 SSR-0106456 SSR-0101599 DSR-0103331 SSR-0106457 SSR-0101599 DSR-0103342 SSR-0106584 SSR-0106563

[0564]

[0565] DSR-0103343 SSR-0104100 SSR-0106563 DSR-0103359 SSR-0104186 SSR-0101599 DSR-0103360 SSR-0106620 SSR-0101599 DSR-0103361 SSR-0104189 SSR-0101599 DSR-0103362 SSR-0106621 SSR-0101599 DSR-0103363 SSR-0106530 SSR-0101599 DSR-0103364 SSR-0104475 SSR-0106630 DSR-0103365 SSR-0106527 SSR-0101599 DSR-0103366 SSR-0106528 SSR-0101599 DSR-0103367 SSR-0106533 SSR-0101599 DSR-0103368 SSR-0106536 SSR-0101599 DSR-0103369 SSR-0106625 SSR-0101599 DSR-0103370 SSR-0106520 SSR-0101596 DSR-0103371 SSR-0104475 SSR-0106629 DSR-0103372 SSR-0106532 SSR-0101599 DSR-0103373 SSR-0104475 SSR-0106640 DSR-0103374 SSR-0104475 SSR-0106637 DSR-0103375 SSR-0104475 SSR-0106638 DSR-0103376 SSR-0106522 SSR-0101599 DSR-0103377 SSR-0106523 SSR-0101599 DSR-0103378 SSR-0106524 SSR-0101599

[0566]

[0567] DSR-0103379 SSR-0106526 SSR-0101599 DSR-0103380 SSR-0106534 SSR-0101599 DSR-0103381 SSR-0106627 SSR-0101599 DSR-0103382 SSR-0104475 SSR-0106632 DSR-0103383 SSR-0104475 SSR-0106633 DSR-0103384 SSR-0104475 SSR-0106634 DSR-0103385 SSR-0104717 SSR-0106643 DSR-0103386 SSR-0106626 SSR-0101599 DSR-0103387 SSR-0106628 SSR-0101599 DSR-0103388 SSR-0104475 SSR-0106635 DSR-0103389 SSR-0106525 SSR-0101599 DSR-0103390 SSR-0106535 SSR-0101599 DSR-0103391 SSR-0106537 SSR-0101599 DSR-0103392 SSR-0106538 SSR-0101599 DSR-0103393 SSR-0106539 SSR-0101599 DSR-0103394 SSR-0104475 SSR-0106631 DSR-0103395 SSR-0106521 SSR-0101599 DSR-0103396 SSR-0106529 SSR-0101599 DSR-0103397 SSR-0106531 SSR-0101599 DSR-0103404 SSR-0106183 SSR-0106603 DSR-0103408 SSR-0106183 SSR-0106510

[0568]

[0569] DSR-0103409 SSR-0106183 SSR-0106604 DSR-0103411 SSR-0106183 SSR-0106506 DSR-0103412 SSR-0106183 SSR-0106511 DSR-0103414 SSR-0106183 SSR-0106508 DSR-0103415 SSR-0106183 SSR-0106513 DSR-0103416 SSR-0106183 SSR-0106503 DSR-0103418 SSR-0106183 SSR-0106598 DSR-0103419 SSR-0106183 SSR-0106600 DSR-0103420 SSR-0106183 SSR-0106602 DSR-0103424 SSR-0106183 SSR-0106509 DSR-0103425 SSR-0106183 SSR-0106601 DSR-0103426 SSR-0106183 SSR-0106605 DSR-0103427 SSR-0106183 SSR-0106606 DSR-0103428 SSR-0106183 SSR-0106607 DSR-0103429 SSR-0106183 SSR-0106507 DSR-0103430 SSR-0106183 SSR-0106597 DSR-0103431 SSR-0106183 SSR-0106599 DSR-0103432 SSR-0106587 SSR-0106589 DSR-0103712 SSR-0106224 SSR-0106206 DSR-0103713 SSR-0106516 SSR-0106206 DSR-0103714 SSR-0106517 SSR-0106206

[0570]

[0571] DSR-0103715 SSR-0106514 SSR-0106206 DSR-0103716 SSR-0106515 SSR-0106206 DSR-0103717 SSR-0106518 SSR-0106206 DSR-0103718 SSR-0106512 SSR-0106206 DSR-0103719 SSR-0106519 SSR-0106206 DSR-0103966 SSR-0107427 SSR-0107400 DSR-0103967 SSR-0107427 SSR-0107419 DSR-0103968 SSR-0107427 SSR-0107420 DSR-0103969 SSR-0107427 SSR-0107412 DSR-0103970 SSR-0107427 SSR-0107413 DSR-0103971 SSR-0107427 SSR-0107414 DSR-0103972 SSR-0107427 SSR-0107421 DSR-0103973 SSR-0107427 SSR-0107415 DSR-0104104 SSR-0107428 SSR-0107412 DSR-0104105 SSR-0108082 SSR-0108086 DSR-0104106 SSR-0108084 SSR-0108086 DSR-0104107 SSR-0108085 SSR-0108086

[0572]

[0573] bS SQ SS BAEE HELMR numer

[0574] GGG UUAUAACAAAACA G SS00 CUUUUUR14475- []3GGGUUAUAACAAAn

[0575] G SS0 ACACUUUUUR106457- GGG UUAUAACAAAACA G SS003 CUUUUUR1447- rH W 0 <n- CN <_ 0 v> r 0~3<VO>‘ O v>

[0576] g= o O V> O Vr

[0577] 0 vGGG UUAUAACAAAACA> 0 v> 0 v>

[0578] O V O> ee v> C ii C V> C V> uE. ^_L Aj G SS0086 CUUUUUR141- q- O GO > <■ a a a < cd fc. q a a a,-A O V> E E E E O V> E E E E d -5> G W a CL CL ex ex < / y d d-GGG UUAUAACAAAACA O. —. VI. in.a >x a a 0 o V> O O vn U m a a a a Fx c £2. G SS06620 CUUUUUR01- a <3 — • < Z) E — E S • £ s ’e S E E — - E E <" 73 ex PL- jd £ Ji d a d a E P "a s q.d Or-:. O _x O E X c £GGG UUAUAACAAAACA ex E 'd C Ji. in C m £ m E « E a E Pi aG SS089 CUUUUUR0141- S’ • m • in d a Si d a d P JX <7^ Q. tn,

[0579] c Px: E 5* <_ 73 p < 2. § E < a < s a a d B CL E c E E E £ E -g E a £ £ GGG UUAUAACAAAACA. in p b E d d P d r> _d _d _d ex a d Ji d C CL G SS00662 CUUUUUR11- E a O =5 a 2. a a a a S S a s a s d El £X GO " E ” E 5 £ £ £ E ~E Aj E Aj E < => a S?*-. d qc g d £. S Ji d GGG UUAUAACAAAACA —.a — a d E £ E £ < J S' d a d a d a n ” a a. d G SS00890 CUUUUUR11- — Q, ’< E < E < E < £ < c rj E a S 5 " d E ji{()()()()()()()()()()[][j[fl][][j[fl][fl][][fS200S22GG00 RNAl5UURA1UAACA1Rpcmsprspmnrpmprpmpmpmpmn........... E a e " «-. d E d a jd 73 Ai £ d a d a d a d d (J $$$()()()()()()()()()()()()()}$ l][fl][fl][][] UUAUAGAGCAAGAACA2AGA2AC2ACUGUSUSUV2Urpmpmprpmprpmpmpmpmpmspmspm............. a a 5: £2. Sr.. d ^3 E a s E a < E a “E a ^7 d G 0 SS0892 CUUUUUR011- >- d 7— d d Ji 19 S a s- d E _d r-3 CJ £N CJ 0^ g o (N < a E d g a a a p E E d a: £ a: co “ E a GGG UUAUAACAAAACA n 73 03 tn '• / " J ex cn d GT).

[0580] r-d »-i CL ” d.0rf" 0 < 0 < 0 < O CUGUUUU SSR0104205- O S. a00 xc; 0 xc; 0 g a O 0 a T” " d c >- C - — ■ a T7 E c*'1“ <5 < s s s a < ±L < ±L < 2 d < g a S 5: -x E E d E a GO. E E d § < jq r-L O CL _L O c £ a a O 0 < ’aa’ V> (- CX '7”" "d Si 9- a O < in t cz in v" fX LZ0P ec 9 a a Gn ‘ c octoc E Eu-—’ ex CL a d 3^' fN E jd <.< M=. a.a 3 < S J: a <, § S a a a Si E d J CM Aj £N rxj (N Aj < N TT fN cc E qc £N ^ a ex o 5C CS tZ) E eg E "d d "dr'd ex CL d S’ *“*“ m <r GH < I t i ct -Cx fXJ s~ GH <£ < T a < £ ™ £* — E a s £ a £ a d ex ex ’ —; d a s S' ~~ ’ ex d c,9: E Si LO id Si £ a E S' < A <f LD LH id d E £ E " C3 r- T3 £ 2 E S’ d ~O c c in in c un < d d d z> E £ a “ <. G CL E d a S E 5 s a < a a <£ ■*£T £X S a

[0581] E d ^7 d ^7.d S E s rH 5= 0 dr rH gg. O a ° < ^-. 0 <.£ < _ L rsj < a q < r-L r-j < -7 rsj < E < P f< £Z 2 < Z P. CN Z ff: > 2: ££ > z: oc >,-X rsi

[0582] X! Z > tX r-R v> ct: d ne cc > cc " §.i X H v> C£ t-1 v> □c >-< v>

[0583]

[0584] GGG UUAUAACAAAACA

[0585] G SS0082 CUUUUUR117- GGG UUAUAACAAAACA G SS0893 CUUUUUR101- GGG UUAUAACAAAACA G SS008299 CUUUUUR1- CM CM CJ CM Q • (N

[0586] CC > o <+- GGG L UUU M- LU rf r-L CM £M O tn- AUAACAAAACA er >

[0587] O tn- 7q ° Jfl; ££ O

[0588] c tn- d 2 o1

[0589] Xx1[]G3 SS089 CUUUUUR0144n- s’ O 0 tn- * 2* < 3 o O O C 0 tn- T— i Lid o CJ O Z) 0 2 c O X 2 5 E E? ZD £Z -— ' r~ 1 O O de, c: < 3

[0590] P- — E E C ~~ E £ 2

[0591] <■ s a < g < rGGG UUAUAACAAAACA 2

[0592] -L < -A < T 2 -H “EL £ £ Q. £X d '"c? E E

[0593] U < / > E E E E £- U7 []G3 SS0080 CUUUUUR145n- H uo

[0594] E £ LT). in 2 ”d E E E - cS •i (J dZ jqrd’. Cl. CL id <n,

[0595] — LJ - m V)

[0596] Ji 'd: C (Z) J IDi ~E E" cd- <n ii ii a. 5 • — E ~ 0 « ■ C C GGG UUAUAACAAAACA d 3 £ m £ cn E S E S C CL o 'E

[0597] CL SS008R1451-[][]G33 CUUUUUnn d 3 i s S: £ C Q» d XL E lx • <z; t / i <3 E C £ £ E ErE Q. tn <_ Z) £ d dL £ CL £ Q. £ 'd Q. CD E p-. un U7 E ~E < s < s GGG UUAUAACAAAACA _d.9; 3 d izi £ 3 ■— “ — “,d d E " E ZD 3 zj £ z> 3 z) G SS08 CUUUUUR01454- n £ 5 £ 2. ~ CL J3: i E g" E g tg ~ £ E £ ~E CL 77 g!= E £ 3 CM H jj d d. Ji d Ji d £ 5 d 3.2: CM E £ 5 TUAUAGAGCAAGAACA (NES 5 £ S £ S £ S CM E d £ ±L d g. a.

[0598] M-, d G CUUUUU SS008R1455- CM E S’ X S’ s’ O < E d £ S’ x “: £ *-. d M-. a. u-. d M-. CL ^d (£ Sc. d E d < E {)()()(())))()(()[dl]([][fl][][fl])[fl]((()[][fl]S200S22GG002 RNAl5UUA1UAACARA1rsprpmnrpmprpmpmpmpnnnr.......... £ E d £ d 0 ji < D CL CD d 0 £ E d 3 E d E. Sr < c ()()()()()()()()()()()()}|{}[fl][fl][][][]GGGG22GSS UUAUAACAAAACAAACACUUUUUCHEMlt pmpmprpmprpmpmpmpmpmspmspmpz............ E d £ E < E < ~E d 23 £ " E jd 33 E E ji E £ E, H E 1 CUGUUUU SSR0108724- $$$$CHEM1RNA11R11R1V2O::-,,. E d " d Ji 32 £ a.: D d a.: D d 3> 3 E 3 E ZT d. H T? d 5 £ CM 3 E 3 E a. E 3 E V d qr (J E 3 E g £ 77 d T? d TT d d GGG UUAUAACAAAACA 2 d -i E S: S £ CM g g < Z), g O g O 2,uO < G SS00823 CUUUUUR17- r-4 CL 2 -d o <=- °z £ g 3 E = E nA E CD ZZ?" r-i CL c Z7 0 <r — E 0 zq in • rq CM 0 -- - 'D d q Z _d c: O < L, £ T-i CL c: ZZ < g=. 0 <r <-M AA CM § dL 0 0 3 0 O £ a S. > O < < g E d q TT CM < 3 £Z < —.kE! CM 2 £= n-. CM vy c? CJ > -q £! E CL ^ 3 E ji < v> < q= V> < M- V> £ o <■ s E d >—. tn* " EL LJ 'q’ tn- 6 E ’CL £ T A E _d a: E d £M c _CL v> c CL tn- < Z! zz a. m c d 2 C- CL fxj ac E ry £ "d £ v> "a £ oc "d £ T 'CL H- CC ct E 3 < d unctn- W C- rH s CT C V> d E C£ sc CC £ tn- £ET — <£ 2- < Z) cd E tn- ■ — ■ CL r-l Q- — - d. —; < S CM ZT ZJ ct 3. < ” 3 < 3 < £ £ < * qr £N S 5 TZ id < ji CM Z? X CM 5 < Z) <_ CL CL B, g | g <5 < g s <' (Z! -H Ji ±L ~ OC ii z “ tL Z 9* d VT, < un <rS E a d. lx d CL d q, d d LQ, < a W n-». »S2 CL d,1? in -it 5 E ZD in < UJ in, < in, < q IO <r < E t. E q" d in ■—'■ q £ cr ") E Li-J S' E tjj F? ji 5 E u ■q _d Z3 E ar E M £ u7 £ id d in - d sj dd dxqz (j ZT 0 7 Ji u x> c: CZ CL zz1d < H 0 E E Tn S E 3 77 0 ” 0) ~ c: (H v> £ O v> iZj V LU cc < in 0 5 in id -*-• Ln id LTi -„ 0 E g- d -a Md LU d c x ■a £ q LU cc; p £ T7 p £ 77 £ d E o “ < 5: E I £ ^7. 0 T-i CL t / > 77 d < N qr d q T-J d q < JJ v> < 77 < >-. q < < q; < < s i < 9 21 ci- LU z z ■-■ 7: 2 a — 1, < N Z < ZT rv' q CC ZT 2 Ct cc > er T7 > er Z? Z ar 0 ar “ 2

[0599]

[0600]

[0601] []3GGUUAUAGACAAAn

[0602] G SS0088 ACACUUUUUR117- []3GGGUUAUAACAAAn

[0603] G SS08 ACACUUUUUR10477- GGG UUAUAACAAAACA G SS0869 CUUUUUR104- fN, _, Ct CM GGG UUAUAACAAAACA

[0604] LU qc O rsi AJ 3

[0605] Di >

[0606] o ’ csj G SS00868 CUUUUUR14- o

[0607] 3" o o c o v> AJ r-f LU c — c > o v>- O ZE G or > 3

[0608] O 3 C V~v GG UUAUAACAAAACA S o u O ■L o v> r-\ c — < S '* Ct c 3 o G SS0088 CUUUUUR147- > E 3 3 J2 V> rd V> E o v> < T 3 < 3 E E OGGG UUAUAACAAAACA LH- 3 3 < 2 c v> E E 3 Co E E 3 3 CUGUUUU SSR0108479- LJ

[0609] 3 C un E E

[0610] 3 3 L S 3 u 1 1 1 j < Z) 3 Q.

[0611] • LD id on E E E ~ i l id tn

[0612] E 3 3 d 2 E z)

[0613] J2L — - 3 I S id tn, <g E E S*

[0614] fS tn E c 3 H E, E UHL? E O Lf s s d 3 « 2 tn C CL l

[0615] 2 S. en. C tnL

[0616] CL un E sc CLt-r><. 5 S JI 2 D i S E c — c 5 H 3 Z S S. < * tn i d

[0617] E • 2 E 2 o E S d CL dl E E ”£

[0618] Sb si d 2- z:i 3 3 (D ZJ A; E ~ d

[0619] !*-. d o. S Aj E < D S £ E 3 Al £ d o Aj E!*-. _d d. ±L d S 2

[0620] E gz. Jl d < J ' X S < P=. a.

[0621] d < D E d d (j <_ g Ai E d ID w £ d -, d < £ d — ' < E

[0622] E d < E d ip 2 E E 5:

[0623] d z>

[0624] " S d 2 E E 2: <. £ i T? d. d 2 £ _d 2- E

[0625] i £ id 2 £

[0626] - d 2

[0627] E TT d £ 5 E _d 2.

[0628] T? d

[0629] £ u y d S S -t E 2 E -A £ o d £ 2 TT d o S. Xt E

[0630] J d 3 d S E

[0631] H § < £ d S 2 ° < c

[0632] o < <■ ~ a

[0633] c o ~ O <f Lz E: '7^ —:■ rxi C T? <tj O CL d s

[0634] 5~ £ CL > y: <-N c ■z?

[0635] r s < S £ r-L ry v> < M=. %i O Ci- q CL 3 V> Jij 3 3 C ■72’ E ci IN £ E •£ JS: <-J. —. rN E a. 2 2 d u 2 E -2- U 5T- ~ d a= d. u. £ d ZD c v> 'i- d 3 _< 1H 2 £ v; rsi ~ 2 E.d tj 2 E 5Z S- Q, 2J T? d T7 d CM — ~ < N 2 E s < E i 72 Ai S < ” d tn r~ LT) ctC. “ET Aj CL Ai 5 s < S' 2 < < / ) " J: S’ ■. / < 2. < Z)!_ ’ 1 / 5 S3 2. dz Z-c! " cc ji Ai d. 3 i=S CL y ic y M= ’c’ -9: => < < 2 <£ E E c d 2> 5 < d1 / 1rl m £ ct T3 1^* TS <- 1 / 1 <= S £ I ‘g ID £ < d. Zl £ -9: 7 P u £ d s "7^ E m S H £ ct h £ d 2 LT) 3 LUct:. 13

[0636] " O c X > ji d 3: 2 < £ — E H u 2 £ y (D ■y o. rH CL LH- d. <

[0637] < si <t Ai y < < < E < T7 < < 7? z: CM 2 <

[0638] ct qc o a: - cc T7 cc TT 3 a: 2 S § 5:

[0639]

[0640]

[0641] RNAl{p.[Rm5d5m](U)[Rsp3.[fl2r](Cj[Ssp].m(U)[n001S].[fl2r](A)p.m(Cjp4fl2r](U)p.m(Uj[n001S].[fl2r](G)p.[fi2r](U) p.m(G)p.m(U)p.m(U)p.m(A)p.[fl2r](C)p.m(A)p.[fl2r](G)p.m(C)p.m(A)[n001S].m(C)p.m(A)p.m(G)[Ssp].m(U)[Ssp]. UCUACUUGUGUUACA SSR-0108208 m(U)}$$$$V2.0 GCACAGUU RNAl{p.[Rm5d5m](U)[Ssp].[fl2r](C)[Rsp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.[fl2r](U) p.m(G)p.m(U)p.m(U)p.m(A)p.[fl2r](C)p.m(A)p.[fl2r](G)p.m(C)p.m(A)[n001S].m(C)p.m(A)p.m(G)[Sspj.m(U)[Ssp]. UCUACUUGUGUUACA SSR-0108209 m(U)}$$$$V2.0 GCACAGUU RNAl{p.[Rm5d5m]( U)[Rsp].[fl2r](C)[Rsp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.[fl2r](U )p.m(G)p.m(U)p.m(U)p.m(A)p.[fl2r](C)p.m(A)p.[fl2rHG)p.m(C)p.m(A)[n001S].m(C)p.m(A)p.m(G)[Ssp].m(U)[Ssp]. UCUACUUGUGUUACA SSR-0108210 m(U)}$$$$V2.0 GCACAGUU RNAl{p.[Rm5d5m](U)[Ssp].[fl2r](C)[Ssp].m(U)[n001S].[fl2r](A)p.m(C)p.[fl2r](U)p.m(U)[n001S].[fl2r](G)p.[fl2r](U) p.m(G)p.m(U)p.m(U)p.m(A)p.[fl2r](C)p.m(A)p.[fl2r](G)p.m(C)p.m(A)[n001S].m(C)p.m(A)p.m(G)[Ssp].m(U)[Ssp]. UCUACUUGUGUUACA SSR-0108211 m(U)}$$$$V2.0 GCACAGUU RNAl{p.[Rm5d5m](U)[Rsp].[fl2r](U)[Ssp].m(U)[n001S].[fl2r](A)p.m(G)p.[fi2r](A)p.m(G)[n001S].[fl2r](U)p.[fl2r](G )p.m(A)p.m(G)p.m(G)p.m(A)p.[fl2rHU)p.m(U)p.[fl2rHA)p.m(A)p.m(A)p.m(A)p.m(U)p.m(G)[Ssp].m(U)[Ssp].m(U)} UUUAGAGUGAGGAUU SSR-0108216 $$$$V2.0 AAAAUGUU RNAl{p.[Rm5d5m](U)[Rsp].[fl2i'](U)[Ssp].m(U)[n001S].[fl2rHA)p.m(G)p.[fl2r](A)p.m(G)[n001S].[fl2rnu)p.[fl2r](G )p.m(A)p.m(G)p.m(G)p.m(A)p.[fl2r](U)p.m(U)p.[fl2r](A)p.m(A)p.m(A)[n001S].m(A)[Ssp].m(U)[Ssp].m(G)[Ssp].m! UUUAGAGUGAGGAUU SSR-0108298 U)[Ssp].m(U)}$$$$V2.0 AAAAUGUU RNAl{[d5m](U)[Ssp].[fl2r](U)p.m(A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001R].[fl2r](A )p.m(G)p.m(A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[n001R].m(U)[n001R].m(U)}| CHEMI UUAUAGAGCAAGAACA SSR-0111125 {[tz]}|CHEM2{[cp]}$CHEMl, RNAl,l: Rl-l: Rl|CHEMl, CHEM2,l: R2-l: Rl$$$V2.0 CUGUUUU RNAl{[d5m](U)[Ssp].[fl2r](U)p.m(A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.m(A)p.m(G)p.m(C)p.m(A)[n001R].[fl2r](A )p.m(G)p.m(A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.nn(U)[Ssp].m(U)[n001R].m(U)}|CHEMl{[tz UUAUAGAGCAAGAACA SSR-0111123 ]}| CHEM2{[cp]}$CHEMl, RNA1,1: R1-1: R11 CHEMI, CHEM2,1: R2-1: R1$$$V2. O CUGUUUU RNAl{[d5m](U)[Ssp].[fl2r](U)p.m(A)[n001S].m(U)p.m(A)p.[f!2r](G)p.nn(A)p.m(G)p.m(C)p.m(A)p.m(A)p.m(G)p.m( A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p.m(U)[Ssp].m(U)[Ssp].m(U)}|CHEMl{[tz]}|CHEM2{[cp] UUAUAGAGCAAGAACA SSR-0111034 }$CHEM1, RNA1,1: R1-1: R1| CHEMI, CHEM2,1: R2-1: R1$$$V2. O CUGUUUU RNAl{[d5m](U)[Ssp].[fl2r](U)p.[fl2r](A)[n001S].[fl2r](U)p.m(A)p.[fl2r](G)p.fTi(Ajp.m(G)p.m(C)p.m(A)p.m(A)p.m(G )p.m(A)p.[fl2r](A)p.m(C)p.[fl2r](A)p.m(C)p.m(U)p.m(G)p.m(U)p,m(U)[Ssp].iTi(U)[Ssp].m(U)}|CHEMl{[tz]}|CHEM UUAUAGAGCAAGAACA

[0642]

[0643] SSR-0111098 2{[cp]}$CHEMl, RNAl,l: Rl-l: Rlj CHEMI, CHEM2,1: R2-1: R1$$$V2. O CUGUUUU

[0644]

[0645] GGG UCCUUCCCUAAUU

[0646] SS00898 CCUCCUUR17- GGG UUAUAACAAAACA G SS088 CUUUUUR1070- {()()()()()()()()()()[dhd][][fl][][][fl][fl][2S200S22GG00 RNAl5UURA1UAACAexecrsprspmnrpmprpmpmpmpmn.........

[0647] ()()()()()()()()()()()()()} |][fl][fl][fl][][]GGGG22GSS UUAUAACAAAACA lR2AAACACUUUUU Crpmpmprpmprpmpmpmpmpmspmspm.............

[0648] $$$${}|}|[]{[]G2222O SS022 CUUUUU HEMlCHEMCHEMlRNAllRllRl CHEMI CHEM1R1R1VR1114tzcp::::---,,,,.

[0649] CM CM, _ > ‘Z? f'J CM, _, CM _ _,

[0650] GGG UUAUAACAAAACA CM +±

[0651] 2 5 ry1r7

[0652] d1x1d LU G SS333 CUUUUUR0111- o SF LU cc LLJ o o o 3Z O 3Z 0 o u o CJ o U O o u O LJ c c; —~ x

[0653] ™ O O c ~~ C “ GGG U

[0654] S' 2- <■ s <. =? AUAACAAAACAC < s < s < s

[0655] T J| E E E E G SS009 UUUUUR1114- E J[ E E d _S lx 2 a E {()())()()()()()()[][d][][fl][][][fl][fl][]00SS200S2(2GG00 RNAl1URA1UAACA1Rnpsprspmnrpmprpmpmpmpmn........... 2 a 2 a rzr t < / ni i LdJ tn i cjd in 2: a 2 CA

[0656] $$$$()()()()()()()()()()()()()} [fl][fi][fl][][]GGG2G22GSS2 UAUAACAAAACACAAACACUUUUUVrpmpmprpmprpmpmpmpmpmspmspm............ E S id tn,

[0657] E S E ■ —

[0658] i s _Q. E ej' Z)

[0659] G0 SS000 UUUUUR1115-. a a. i s 2 E

[0660] o • o E < D E o d ID E

[0661] c to ( / ) E a g a nj £ S E a E e lx

[0662] jd id 2 81 •er v> “ Ul a, GGG< UUAUAACAAAACAA d tn 0 2 ZD < -tn y>,

[0663] U O < ~ q

[0664] 'X - C G SS0886 CUUUUUR011- < s gN

[0665] E E ■ T = ° CM- S-. S' r he: —

[0666] E g er c > c <” tn* E T S r. w {()()())()()()()()()[dd][][fl][][][fl]([fl][][flS200S22GG00 RNAl5UURA1UAACA1Rmsprspmnrpmprpmpmpmpmn........... c w CX d ji a. >.2 d S 5 E CM d x v> ex d 2 a > 0 5- 2 kJ 2 ()()()()()()()()()()()()()}|{][fi][fi][][] UUAUAGAGCAAGAACA 2AGA2AC2ACUGUUSUSUCHEMlrpmpmprpmprpmpmpmpmpmspmspm............ S 5$ in er v>

[0667] id 4- C E $$$$}|}| []{[]G2222O CUUUUUl41 CHEMCHEMl RNA11R11R1 CHEMI CHEM1R1R1Vtmzcp::::--,,,,. SS00886R15- £ E CM t o-l • 3 qz a CL ' M-, a rv~ i= a „ g E 2 ‘-T CM {()()())(()(()()[d])[][fl]([][][fl]([fl]))[][flS200S22GG002 RNAl5UURA1UAACA1Rmsprspmnrpmprpmpmpmpmn.......... a < D 7-i ci ID r d. 3 S t! id CM

[0668] < c ce GGG UUAUAACAAAACA()()()()()()()()()()()()()}|{][fl][fl][][][G22GSSAAACACUUUUUCHEMl rpmpmprpmprpmpmpmpmpmspmspm............ E 2 S CM < E S < E d < E? E Q. Hl E" E E 2 E 2 s CUGUUUU SSR0108863-$$$$}|{}jl][]CHEM2CHEMlRNAllRllRlCHEMlCHEM2lR2lRlV20 pyrcp::::--,,,,. H * CM • cr • e? Q. Z> <c^ CL ”j CX ZJ 11 i E 2 r d. 3 2 S S T:rX ’g’’ LU S E 2 3 s 3 E < M zd £ O a. x H a 'U a. u z d " a 5GGG UUAUAACAAAACA '— • - T S 2 u CM ID g U g o 77 g o S 2 2 o S S XG SS00886 CUUUUUR17- —. " LU S S H 2 2 e s E 3 = s' * > —. E x tn in 2 7 E § a. a u in U LU

[0669] o Z> g < er 2 I 2 < j-j 2 <f id 0 -1. ~i o rH c er d c: XT1o S. — £ <5 — CM CC C X? 5 n < g q. —. fs,| CZT < 5= — 1 '< S < kJ ±L

[0670] " ‘-z' tr d c d A E a E i g E 2;; “ 2. er 77 U •; 2 2 a ii H 2 y a Z A I / ) c— t / 5 C V> a 3 U £ C £ £ E 2 ry C v> E g d E 2 E 2 JX g. < — d r-? ■"• ~T~ s 2 5 a H ad- < cc a < 3 or

[0671] uj 2- S § CM >- x g CM £ g S 2 g CM > g CM Tx g 0 Z ~ i~. g “ ±L Lu O s SS S' u "--e £X CL LO a a. X d fM " S d 'd < Z! m -er tn, < v> tn < rfE in < H un. < VT, O < 2 w ^=1 LU in < v> S E £ "n ± X S E i Z) £ re S £ g re d E a re d re d — d id d gj m “J o c; — E (t-j CM _a tjs E O E < s LO DO tn id ’s un id LO S 5 g ID g -O C X -0 £ id 2 E di 2. E £ re r 0 S £ £ 77 E 77 a O dr d -£• -H CL — df d E? rH CX CM

[0672] < >. < <r < < 2 < a =5 < < ~ < < E Z Q. tn

[0673] § £ er Z er Z nj CT Z LT] cc TT

[0674]

[0675]

[0676] s 3 Q

[0677]

[0678]

[0679] DSR-0102524 SSR-0104475 SSR-0101599 7822.32 7822.5 8686.45 8685.6 DSR-0103331 SSR-0106457 SSR-0101599 7792.30 7790.2 8686.45 8685.6 DSR-0102626 SSR-0104473 SSR-0101599 7822.32 7823.9 8686.45 8685.6 DSR-0103359 SSR-0104186 SSR-0101599 7914.36 7912.4 8686.45 8685.6 DSR-0103360 SSR-0106620 SSR-0101599 7914.36 7912.9 8686.45 8685.6 DSR-0103361 SSR-0104189 SSR-0101599 7914.36 7914.3 8686.45 8685.6 DSR-0103362 SSR-0106621 SSR-0101599 7914.36 7913.0 8686.45 8685.6 DSR-0104162 SSR-0108190 SSR-0101599 7914.36 7914.7 8686.45 8685.6 DSR-0104164 SSR-0108192 SSR-0101599 7900.33 7900.3 8686.45 8685,6 DSR-0104165 SSR-0104205 SSR-0101599 7912.34 7912.5 8636.45 8685.6 DSR-0104166 SSR-0108217 SSR-0101599 7898.31 7900.1 8685,45 8685.6 DSR-0104167 SSR-0108193 SSR-0101599 7953.35 7951.3 8686.45 8685.6 DSR-0104168 SSR-0108299 SSR-0101599 7970.46 7971.2 8636.45 8685.6 DSR-0104471 SSR-0108449 SSR-0101599 7953.35 7953.2 8686.45 8685.6 DSR-0104472 SSR-0108450 SSR-0101599 7953.35 7953.0 8686.45 8685,6 DSR-0104473 SSR-0108451 SSR-0101599 7953.35 7953.0 8636.45 8685.6 DSR-0104492 SSR-0108454 SSR-0101599 7997.45 7998.2 8685,45 8685.6 DSR-0104493 SSR-0108455 SSR-0101599 7981.46 7981.9 8686.45 8685.6 DSR-0104499 SSR-0108724 SSR-0101599 7935.27 7934.2 8636.45 8685.6 DSR-0104498 SSR-0108723 SSR-0101599 7951.34 7949.5 8686.45 8685.6 DSR-0104497 SSR-0108722 SSR-0101599 7981.34 7980.5 8686.45 8685.6 DSR-0104496 SSR-0108721 SSR-0101599 7997.41 7996.3 8636.45 8685.6 DSR-0104495 SSR-0108720 SSR-0101599 7925.25 7924.8 3686.45 8685.6 DSR-0104494 SSR-0108719 SSR-0101599 7941.32 7940.2 8686.45 8685.6 DSR-0104508 SSR-0108470 SSR-0101599 7937.28 7936.5 8636.45 8685.6

[0680] 7951.34 7951.0 8686.45 8685.6

[0681]

[0682] DSR-0104502 SSR-0108733 SSR-0101599DSR-0104501 SSR-0108732 SSR-0101599 7997.41 7996.8 8686.45 8685.6 DSR-0104500 SSR-0108731 SSR-0101599 7941.32 7941.7 8686.45 8685.6 DSR-0104506 SSR-0108836 SSR-0101599 7953.35 7952.8 8686.45 8685.6 DSR-0104505 SSR-0108816 SSR-0101599 7953.35 7937.2 8686.45 8685.6 DSR-0104504 SSR-0108817 SSR-0101599 7937.28 7953.0 8686.45 8685.6 DSR-0104503 SSR-0108477 SSR-0101599 7953.35 7953.0 8686.45 8685.6 DSR-0104509 SSR-0108469 SSR-0101599 7882.24 7880.3 8686.45 8685.6 DSR-0104507 SSR-0108468 SSR-0101599 7898.29 7896.7 8686.45 8685.6 DSR-0104524 SSR-0108478 SSR-0101599 7882.22 7881.3 8686.45 8685,6 DSR-0104525 SSR-0108479 SSR-0101599 7866.17 7864.6 8636.45 8685.6 DSR-0104526 SSR-0108480 SSR-0101599 7921.21 7920.3 8685,45 8685.6 DSR-0104147 SSR-0107428 SSR-0107400 8078.53 8080.3 6991.69 6991.1 DSR-0104311 SSR-0104067 SSR-0106206 7922.77 7922.5 7116.69 7115.6 DSR-0104314 SSR-0106220 SSR-0106206 8014.81 8013.2 7116.69 7115.6 DSR-0104315 SSR-0108200 SSR-0106213 8038.87 8036.1 7188.87 7189.4 DSR-0104316 SSR-0108201 SSR-0106213 8038.87 8037.3 7138.87 7189.4 DSR-0104317 SSR-0108202 SSR-0106213 8038.87 8036.7 7188.87 7189.4 DSR-0104318 SSR-0108203 SSR-0106213 8038.87 8037.3 7188.87 7189.4 DSR-0104319 SSR-0108208 SSR-0106213 7926.38 7924.8 7133.87 7189.4 DSR-0104320 SSR-0108209 SSR-0106213 7926.38 7924.8 71.88.87 7189.4 DSR-0104321 SSR-0108210 SSR-0106213 7926.38 7924.4 7188.87 7189.4 DSR-0104322 SSR-0108211 SSR-0106213 7926.38 7924.9 7138.87 7189.4 DSR-0104465 SSR-0108298 SSR-0107412 8110.67 8110.2 7300.29 7299.9 DSR-0104466 SSR-0108216 SSR-0107412 7983.38 7981.3 7300.29 7299.9 DSR-0104467 SSR-0108298 SSR-0107400 8110.67 8110.2 6991.69 6991.1

[0683] 7983.38 7981.3 6991.69 6991.1

[0684]

[0685] DSR-0104468 SSR-0108216 SSR-0107400DSR-0104469 SSR-0108298 SSR-0107413 8110.67 8110.2 6896.54 6895.7 DSR-0104470 SSR-0108298 SSR-0108195 8110.67 8110.2 6801.39 6800.4 DSR-0105723 SSR-0111125 SSR-0101599 8095.44 8093.2 8686.45 8685.6 DSR-0105722 SSR-0111124 SSR-0101599 8016.36 8015.1 8686.45 8685.6 DSR-0105721 SSR-0111123 SSR-0101599 8016.36 8014.9 8686.45 8685.6 DSR-0105720 SSR-0111034 SSR-0101599 7866.19 7866.9 8686.45 8685.6 DSR-0105719 SSR-0111098 SSR-0101599 7842.13 7842.1 8686.45 8685.6 DSR-0105718 SSR-0111033 SSR-0101599 7854.16 7854.4 8686.45 8685.6 DSR-0105717 SSR-0111032 SSR-0101599 7854.16 7854.4 8686.45 8685,6 DSR-0105716 SSR-0111064 SSR-0101599 7842.13 7841.9 8636.45 8685.6 DSR-0105715 SSR-0111031 SSR-0101599 7842.13 7841.9 8685,45 8685.6 DSR-0105714 SSR-0111030 SSR-0101599 7961.34 7961.6 8686.45 8685.6 DSR-0105713 SSR-0111092 SSR-0101599 7913.22 7913.0 8636.45 8685.6 DSR-0105712 SSR-0111063 SSR-0101599 7937.28 7937.4 8686.45 8685.6 DSR-0105683 SSR-0111126 SSR-0101599 7925.25 7924.6 8686.45 8685,6 DSR-0105711 SSR-0111029 SSR-0101599 7937.28 7937.2 8636.45 8685.6 DSR-0105710 SSR-0111028 SSR-0101599 7937.28 7937.3 8685,45 8685.6 DSR-0104672 SSR-0108987 SSR-0105234 7770.13 7770.2 8944.72 8947.3 DSR-0105761 SSR-0111126 SSR-0111096 7925.25 7924.6 8844.61 8843.7 DSR-0105728 SSR-0111028 SSR-0111096 7937.28 7937.3 8844.61 8843.7 DSR-0105682 SSR-0108470 SSR-0111096 7937.28 7936.5 8844.61 8843.7 DSR-0105692 SSR-0108193 SSR-0111096 7953.35 7951.3 8844.61 8843.7 DSR-0105727 SSR-0111028 SSR-0111095 7937.28 7937.3 8765.53 8765.2 DSR-0105681 SSR-0108470 SSR-0111095 7937.28 7936.5 8765.53 8765.2 DSR-0105691 SSR-0108193 SSR-0111095 7953.35 7951.3 8765.53 8765.2

[0686] SSR-0111126 SSR-0111094 7925.25 7924.6 8766.3

[0687]

[0688] DSR-0105726 8765.53DSR-0105725 SSR-0111028 SSR-0111094 7937.28 7937.3 8765.53 8766.3 DSR-0105680 SSR-0108470 SSR-0111094 7937.28 7936.5 8765.53 8766.3 DSR-0105690 SSR-0108193 SSR-0111094 7953.35 7951.3 8765.53 8766.3 DSR-0106699 SSR-0108870 SSR-0101599 7953.43 7952.6 8686.45 8685.6 DSR-0106695 SSR-0111224 SSR-0101599 8163.76 8162.3 8686.45 8685.6 DSR-0106694 SSR-0111333 SSR-0101599 7969.43 7968.1 8686.45 8685.6 DSR-0106697 SSR-0111049 SSR-0101599 7813.26 7812.9 8686.45 8685.6 DSR-0106696 SSR-0111050 SSR-0101599 7857.36 7856.4 8686.45 8685.6 DSR-0104656 SSR-0108861 SSR-0101599 7953.35 7952.8 8686.45 8685,6 DSR-0104657 SSR-0108865 SSR-0101599 7953.36 7951.2 8686.45 8685.6 DSR-0104659 SSR-0108863 SSR-0101599 7955.41 7956.4 8586.45 8685.6 DSR-0104661 SSR-0108867 SSR-0101599 7955.41 7956.6 8686.45 8685.6 DSR-0104662 SSR-0108869 SSR-0101599 7927.35 7927.3 8686.45 8685.6 DSR-0105324 SSR-0108871 SSR-0101599 7966.47 7968.0 8686.45 8685.6 DSR-0104660 SSR-0108864 SSR-0101599 7898.31 7898.9 8686.45 8685,6 DSR-0107025 SSR-0111958 SSR-0101599 7952.36 7951.9 8686.45 8685.6 DSR-0107026 SSR-0108866 SSR-0101599 7967.38 7968.2 8586.45 8685.6 DSR-0107027 SSR-0112941 SSR-0101599 7862.15 7863.5 8686.45 8685.6 DSR-0107023 SSR-0111959 SSR-0101599 7966.39 7967.1 8686.45 8685.6 DSR-0107024 SSR-0113547 SSR-0101599 7966.39 7967.6 8686.45 8685.6 DSR-0107397 SSR-0114847 SSR-0101599 7682.23 7685.4 8686.45 8685.6

[0689] SSR-0114864 SSR-0101599 7955.37 8686.45 8685.6

[0690]

[0691] DSR-0107398 7957.0Notes:

[0692] HELM notations, due to their length, may be divided into multiple lines in the above tables. As appreciated by those skilled in the art, nucleoside units are unmodified unless otherwise indicated (e.g., with m, [fl2r], etc.); linkages, unless otherwise indicated, are natural phosphate linkages; and acidic / basic groups may independently exist in their salts. If an internucleotidic linkage is not specified, the internucleotidic linkage is a natural phosphate linkage. A natural DNA sugar may also be indicated with “d" as in d(G), d(A), d(C), d(T). etc., and a natural phosphate linkage may be indicated with “p” in Table 1. Oligonucleotides in Table 1 are described using various features of Hierarchical Editing Language for Macromolecules (HELM), which is described in, e.g., Zhang, T. et al. J Cheni Inf Model.

[0693] 2012 Oct 22;52(10):2796-806 and Milton, J. et al. J Cheni Inf Model. 2017 Jun 26;57(6): 1233-1239, which are incorporated herein by reference. As described in Zhang et al., 2012 and Milton et al., 2017, connections between oligonucleotides, linker moieties. GalNAc moieties, etc. may be indicated in HELM, for example, as the following: CHEMI. RNA1,1: R1-1: R1, wherein CHEMI is. e.g., a first linker or moiety, RN Al is an oligonucleotide, and 1: R1-1: R1 indicates that CHEMI is linked via a first attachment point to a first attachment point on a first monomer of RNA 1, as described herein (see, e.g., below ) A double-stranded oligonucleotide may be indicated in HELM, for example, as the following: RNA1, wherein RNA1 is, e g., a first oligonucleotide strand of a double -stranded oligonucleotide, and RNA2, wherein RNA2 is, e.g., a second oligonucleotide strand of the double-stranded oligonucleotide.

[0694] Various moieties and modifications (e.g., internucleotidic linkages, sugars, nucleobases. etc ) are described in the present disclosure including the below':

[0695] m: 2’-OMe (sugar is 2'-O-methylribose);

[0696] [112r]: 2’-F (sugar is 2’-fluoro-2’-deoxyribose);

[0697] PO or p: phosphodiester or phosphate;

[0698] PS or [sp]: phosphorothioate;

[0699] Rp or [Rsp]: phosphorothioate in the Rp configuration;

[0700] Sp or [Ssp]: phosphorothioate in the < Sp configuration;

[0701] [sa]: sulfonamide

[0702] [su]: sulfonic acid

[0703] [cp]: capping phosphate

[0704] [tz]: 1H-1,2,3-triazole (1,4 linkage)

[0705] O HO — P-r^N-^

[0706] I \ _ i

[0707] [ptzj: O N N, (lH-l,2,3-triazol-4-yl)phosphonate or 4-phosphono-l,2,3-triazol-l-yl, bonded to 5 ’-carbon of the 5 '-end nucleoside;

[0708] [4mtz]: 4-methyl-1H-1,2,3-triazole (1,4 linkage)

[0709] [5tz]: 1H-1,2,3-triazole (1,5 linkage)

[0710] [lin41tz]: 1-methyl-1H-1,2,3-triazole (4-1 linkage)[d5hexdec2r]: 2-(?-hexadecyl-5-deoxyribose

[0711] [2Stn2pyrl]: (S)-2 -methyl pyrrolidine (1-2 linkage)

[0712] [dSp]: 1,2-dideoxyribose (5-3 connectivity)

[0713] [d5m]: 5-deox -2-O-metliylribose

[0714] [dd5m]: 5-desmelhyl-5-deoxy-2-O-metliy Iribose

[0715] [Pyra]: 1 / f-pyrazole (1-4 linkage)

[0716] [pyrl]: pyrrolidine (1-3 linkage)

[0717] [2pyrl]: pyrrolidine (1-2 linkage)

[0718] [azir]: aziridine (1-2 linkage)

[0719] [vped5mj: 5-(fi)-vinylpbosphonate-5-deoxy-2-O-methylribose;

[0720] [Zvped5m |: 5 -(Z)-viny lphosphonate-5 -deoxy -2-methy Iribose

[0721] [m41nii]: 4-methyl-lH-imidazol (1-4 linkage)

[0722] [m5Imi]: 5 -methyl- 127-imidazol (1-5 linkage)

[0723] [d5unam]: 5-deoxy-2,3-unlocked-2 -methoxyribose

[0724] n

[0725]

[0726] OOl: (A-( 1,3 -dime thylimidazolidin-2-y lidene)phosphoramidate);

[0727] [nOOIR]: nOOl in Rp configuration;

[0728] [nOO IS]: nOOl in Sp configuration;

[0729] x.o

[0730] N p)

[0731] n

[0732]

[0733] 003: p' or N-( 1.3 -dimethylimidazoiidin-2-v lidene)phosphoramidate;

[0734] [n003R]: n003 in Rp configuration;

[0735] [n003S]: n003 in Sp configuration;

[0736] N

[0737] n

[0738]

[0739] 004:

[0740] [n()04R]: n004 in ^p configuration;

[0741] [nOO4S]: n004 in Sp configuration;

[0742] / >— N.^ PCXO

[0743] N i

[0744] J

[0745] 1

[0746]

[0747] 1OO8:0or AL(di(morpholin-l-yl)melhylene)phosphorainidale;[11OO8R]: n008 in Rp configuration;

[0748] [11008S]: 11OO8 in Sp configuration;

[0749] 4-0 / 9 T_z

[0750] p-o

[0751] i

[0752] N

[0753] n

[0754]

[0755] 009: or Af-(l-docecyl-3-methylimidazolidin-2- y lidene)phosphoram idate;

[0756] [nOO9R]: n009 in Rp configuration;

[0757] [n009S]: n009 in Sp configuration;

[0758] /

[0759] / hi -~A,

[0760] \ 'O

[0761] - N 7^

[0762] '' o.y

[0763] n

[0764]

[0765] 025: or A'-(l,3-diniethyltetrahydro yriinidin-2(lH)-ylidene)pliosphoramidate;

[0766] [n025R]: n025 in 7?p configuration;

[0767] [11025S]: n025 in Sp configuration;

[0768] [11026R]: 11026 in A’p configuration;

[0769] [nO26Sj: 11026 in 5p configuration;

[0770] „-N

[0771] > / O

[0772] " N

[0773] A O' jo

[0774] N

[0775]

[0776] 11029: or A'-(l -(4-dimcthylaniino-butyl)-3-nietliy1imidazolidin-2- y 1 idene )phosphoram idate;

[0777] [nO29R]: n029 in Rp configuration;

[0778] [n029S]: n029 in 5'p configuration;

[0779] P-0 ’’

[0780] /

[0781] N

[0782] " N N'

[0783]

[0784] n031:v--yor; V-( I -hexyl-3-methyliniidazolidin-2-ylidene)phosphoramidate;

[0785] [nO31R]: n031 in Jtp configuration;

[0786] [n03 IS]: 11031 in Sp configuration;P-O

[0787] N N N

[0788]

[0789] or A-(l-hexadecyl-3-metliylimidazolidin-2- y lidene)pho sphoram idate;

[0790] [nO33R]: n033in p configuration;

[0791] [nO33Sj: n033 in. S‘p configuration;

[0792] n

[0793]

[0794] 037: —1or A. A-(i.3-dihexyIimidazolidin-2-y lideneiphosphoramidate;

[0795] [nO37R]: n037in p configuration;

[0796] [nO37S]: n037 in Sp configuration;

[0797] p-o

[0798] N

[0799] / N A N " ^ n

[0800]

[0801] 039: \ orAJV-(l,3-didodecylimidazolidin-2-ylidene)phosphoramidate;

[0802] [nO39RJ: n039 in Rp configuration;

[0803] [11039S]: n039 in 5p configuration;

[0804] +°.? X

[0805] P-o

[0806] N

[0807] n

[0808]

[0809] 043: '' — / orjV-(1.3-di(2-inethoxyethyl)imidazolidin-2- y tide ne) pho sphoram idate;

[0810] [nO43R]: n043 in Rp configuration;

[0811] [11043S]: n043 in Sp configuration;

[0812] p-o

[0813] N

[0814] n

[0815]

[0816] 046: —!or Ar, Ar-(l,3-di[(2£)-pent-2-en-l-yl]imidazolidin-2- y liden e)pho spho ram id ate;

[0817] [11046R]: n046in Rp configuration;

[0818] [nO46S]: 11046 in, Sp configuration;

[0819]

[0820] or N, N-(\ _3-di[(2Z)-pent-2-en-l-yl]imidazolidin-2- y lidene)pho sphoram idate;

[0821] [nO47R]: n047in Rp configuration;

[0822] |nO47S]: n047 in Sp configuration;

[0823]

[0824] nX: stereorandoin n058;

[0825] [nO58R]: n058 in p configuration;

[0826] [nO58S]: n058 in 5'p configuration;

[0827] 4°s f \

[0828] / p-oz

[0829] n

[0830]

[0831] 065: f ' I or A'^r-(l,3-dimethyl-l,3-dihydro-2H-benzo[d]imidazol-2- ylidene)pliosphoramidate;

[0832] [n065R]: n065 in Rp configuration;

[0833] [nO65SJ: n065 tn Sp configuration;

[0834] ' — \,N

[0835] < N-—. n

[0836]

[0837] 069: or Ar, A-(l-(3-aminopropyl)-3-inethylinndazolidm-2- ylidene)phosphoramidate;

[0838] [nO69R]: n069 in Rp configuration;

[0839] [nO69Sl: n069 in 5p configuration;4-Q P / -0X

[0840] \ N

[0841] N-4

[0842] n

[0843]

[0844] 070: or ArjV-(3aR,7aS'-l,3,diniethyloctahydro-2Jf-benzo[d]iniidazol- 2y iidene phosphorami date;

[0845] [11070R]: n070 in 7?p configuration;

[0846] [n070S]: n070 in 5 configuration;

[0847] p-0

[0848] \, N

[0849] N-^(

[0850] "N""

[0851] n

[0852]

[0853] 071: or / V,. V-(3a7?t7a7?-l,34in’ethyloctahydro-27f-benzo[d]imidazol- 2y 1 id e n e)ph osph oram i date;

[0854] [nO71R]: n071 in Rp configuration;

[0855] [n07 IS]: n071 in Sp configuration;

[0856]

[0857]

[0858] 0 N=N

[0859] OH

[0860] pzt-d5moe-U or 5pzt-d5moe-U

[0861]

[0862] wherein ' p / l" or “5pzt” corresponds to the triazolepbosphonate moiety, and ‘ d5moe” corresponds to the 5 ’-deoxy -2 -0- methoxy ethyl moiety’;

[0863] O F

[0864] pzt-d5

[0865]

[0866] fl2r-U or 5pzt-d5fl2r-U, wherein “pzt” or “5pzt” corresponds to the triazolepbosphonate moiety, and “d5f!2r” corresponds to the 5’-deoxy-2’-fluoro moiety.pzt-d

[0867]

[0868] 51r-U or 5pzt-d51r-U the triazolephosphonate moiety, and “dolr” corresponds to the 2’-O,4’C-methylene-bridged or locked nucleic acid (2’.4’-BNA or LNA) moiety

[0869] O

[0870] O OMe

[0871] pzt-d

[0872]

[0873] 5m-U or 5pzt-d5m-U5, wherein “pzt” or '5pzt” corresponds to the triazolephosphonate moiety, and "d5m” corresponds to the 5’-deoxy-2’-0-methyl moiety;

[0874] O OMe

[0875] pzt-d

[0876]

[0877] 5m-3nU or 5pzt-d5m-3nU, wherein “pzt” or “5pzG corresponds to the triazolephosphonate moiety, “d5m” corresponds to the 5’-deoxy-2’-( -niethyl moiety, andl’3nU”

[0878] G ' M • ' ( corresponds to the N-3-uridine (N3U) base modification moiety having the structure

[0879]

[0880] [GalNAc3C12oyI]: triantennary GalNAc with C12 linker or

[0881]

[0882] [GalNAc3C 12oy 1] [nC6o]:

[0883]

[0884] NHAc

[0885] [GalNAc3C12oylj[nC6o] conjugated to the 5‘-end of an oligonucleotide (using phosphate):

[0886]

[0887] wherein

[0888]

[0889] is or represents an oligonucleotide chain.

[0890] [nC6o]: -NH-(CH2)6- linker (C6 linker. C6 amine linker or C6 amino linker), connected to one moicly. e.g.. CHEM2 (e g. [GalNAc3C12oyl]). through --NH- (e.g.. forming an amide group -C(0)---NH--), and. in various cases, the 5’-end of the oligonucleotide chain through a linkage (e.g., if the 5’-end of an oligonucleotide contains p. through a phosphate linkage (the end -CH2- bonded to an oxygen atom which is bonded to linkage phosphorus): if the 5 ’-end of an oligonucleotide contains [sp], through a phosphorothioate linkage (the end -CH2- bonded to an oxygen atom which is bonded to linkage phosphorus). [11C60] may also be properly considered as 6 -aminohexanol wherein, as indicated, its amino group forms an amide and its hydroxy forms a linkage such as a phosphate linkage.

[0891] Double Stranded Oligonucleotide Lengths

[0892] As appreciated by those skilled in the art, ds oligonucleotides can be of various lengths to provide desired properties and / or activities for various uses. Many technologies for assessing, selecting and / or optimizing ds oligonucleotide length are available in the art and can be utilized in accordance with the present disclosure. As demonstrated herein, in certain embodiments, dsRNAi oligonucleotides are of suitable lengths to hybridize with their targets and reduce levels of their targets and / or an encoded product thereof. In certain embodiments, a ds oligonucleotide is long enough to recognize a target nucleic acid (e.g.. a target mRNA). In certain embodiments, a ds oligonucleotide is sufficiently long to distinguish between a target nucleic acid and other nucleic acids (e.g., a nucleic acid having a base sequence which is not a target sequence) to reduce off-target effects. In certain embodiments, a dsRNAioligonucleotide is sufficiently short to reduce complexity of manufacture or production and to reduce cost of products.

[0893] In some embodiments, the present disclosure provides an oligonucleotide, wherein the oligonucleotide is a sense strand as described herein. In some embodiments, the present disclosure provides an oligonucleotide, wherein the oligonucleotide is an antisense strand as described herein. In some embodiments, the present disclosure provides oligonucleotides useful as sense strands. In some embodiments, the present disclosure provides oligonucleotides useful as antisense strands. In some embodiments, the present disclosure provides oligonucleotides useful as dsRNAi agents.

[0894] Intemucleotidic Linkages

[0895] Among other things, the present disclosure provides various internucleotidic linkages, including various modified internucleotidic linkages, that may be utilized together with other structural elements, e.g.. various sugars as described herein, to provide oligonucleotides and compositions thereof.

[0896] Various internucleotidic linkages may be utilized in oligonucleotides (e.g.. ss oligonucleotides, ds oligonucleotides, etc.) in accordance with the present disclosure. In some embodiments, an oligonucleotide comprises one or more types of internucleotidic linkages. In some embodiments, an oligonucleotide comprises two or more types of internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least three ty pes of internucleotidic linkages. In some embodiments, a linkage contains a linkage phosphorus atom bonded to an oxygen atom which oxygen atom is not bonded to or is not part of a backbone sugar (“a PO linkage”, e.g., a natural phosphate linkage). In some embodiments, a linkage contains a linkage phosphorus atom bonded to a sulfur atom which sulfur atom is not bonded to or is not part of a backbone sugar (“a PS linkage”, e.g., a phosphorothioate internucleotidic linkage). In some embodiments, a linkage contains a linkage phosphorus atom bonded to a nitrogen atom which nitrogen atom is not bonded to or is not part of a backbone sugar (“a PN linkage”, e.g., nOO 1, MsPA, etc.). In some embodiments, an oligonucleotide comprises one or more PS linkages. In some embodiments, an oligonucleotide comprises one or more PO linkages. In some embodiments, an oligonucleotide comprises one or more PN linkages. In some embodiments, an oligonucleotide comprises one or more PS and one or more PO linkages. In some embodiments, an oligonucleotide comprises one or more PS and one or more PN linkages. In some embodiments, an oligonucleotide comprises one or more PS. one or more PN and one or more PO linkages. In some embodiments, a PS linkage is a phosphorothioate linkage. In some embodiments, each PS linkage is independently a phosphorothioate linkage. In some embodiments, a PO linkage is a natural phosphate linkage. In some embodiments, each PO linkage is independently a natural phosphate linkage. In some embodiments, a PN linkage is a phosphoramidate linkage. In some embodiments, each PN linkage is independently' a phosphoramidate linkage In some embodiments, a PN linkage is a phosphoryl guanidine linkage. In some embodiments, each PN linkage is independently a phosphoryl guanidine linkage. In some embodiments, a PN linkage is a non-negatively charged internucleotidic linkage. Insome embodiments, each PN linkage is independently a non-negatively charged internucleotidic linkage. In some embodiments, a PN linkage is a neutral internucleotidic linkage. In some embodiments, each PN linkage is independently a neutral internucleotidic linkage.

[0897] In certain embodiments, ds oligonucleotides comprise base modifications, sugar modifications, and / or internucleotidic linkage modifications. Various internucleotidic linkages can be utilized in accordance with the present disclosure to link units comprising nucleobases. e.g., nucleosides. In certain embodiments, provided ds oligonucleotides comprise both one or more modified internucleotidic linkages and one or more natural phosphate linkages.

[0898] As widely known by those skilled in the art, natural phosphate linkages are widely found in natural DNA and RNA molecules; they have the structure of -OP(O)(OH)O-, connect sugars in the nucleosides in DNA and RNA, and may be in various salt forms, for example, al physiological pH (about 7.4). natural phosphate linkages are predominantly exist in salt forms with the anion being -OP(O)(O )O~. A modified internucleotidic linkage, or a non-natural phosphate linkage, is an internucleotidic linkage that is not natural phosphate linkage or a salt form thereof. Modified internucleotidic linkages, depending on their structures, may also be in their salt forms. For example, as appreciated by those skilled in the art. phosphorothioate internucleotidic linkages which have the structure of ~OP(O)(SH)O~ may be in various salt forms, e g, at physiological pH (about 7.4) with the anion being -OP(O)(S )O-.

[0899] In some embodiments, an oligonucleotide comprises an internucleotidic linkage which is a modified internucleotidic linkage, e.g., phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, thiophosphate, 3 ’-thiophosphate, 5 ’-thiophosphate, etc. In some embodiments, a modified internucleotidic linkage is a PN linkage. In some embodiments, a modified internucleotidic linkage is a PS linkage. In some embodiments, a modified internucleotidic linkage is a PO linkage (e.g., other than a natural phosphate linkage). In some embodiments, each modified internucleotidic linkage is independently a PN internucleotidic linkage or a PS internucleotidic linkage. In some embodiments, an oligonucleotide comprises one or more PN internucleotidic linkages, one or more PS internucleotidic linkages, and one or more PO internucleotidic linkages. In some embodiments, one or more PN internucleotidic linkages are independently phosphoryl guanidine internucleotidic linkages. In some embodiments, one or more PN internucleotidic linkages are independently nOOl. In some embodiments, one or more PS internucleotidic linkages are independently phosphorothioate internucleotidic linkages. In some embodiments, each PS internucleotidic linkage is independently a phosphorothioate internucleotidic linkage. In some embodiments, one or more PO internucleotidic linkages are independently natural phosphate linkages. In some embodiments, each PO internucleotidic linkage is independently a natural phosphate linkage.

[0900] In some embodiments, a modified internucleotidic linkage is a chiral internucleotidic linkage which comprises a chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is a phosphorothioate linkage. In some embodiments, a chiral internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, a chiral internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a chiral internucleotidic linkage is chirally controlled with respect to its chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is stereochemically pure with respect to its chiral linkage phosphorus. In some embodiments, a chiral internucleotidic linkage is not chirally controlled. In some embodiments, a pattern of backbone chiral centers comprises or consists of positions and linkage phosphorus configurations of chirally controlled internucleotidic linkages (Rp or Sp) and positions of achiral internucleotidic linkages (e.g,, natural phosphate linkages).

[0901] Without wishing to be bound by any particular theory, the present disclosure notes that a neutral internucleotidic linkage can be more hydrophobic than a pho sphoroth ioate internucleotidic linkage, which can be more hydrophobic than a natural phosphate linkage. Typically, unlike a phosphorothioate internucleotidic linkage or natural phosphate linkage, a neutral internucleotidic linkage bears less charge. Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more neutral internucleotidic linkages into a ds oligonucleotide may increase the ds oligonucleotides’ ability to be taken up by a cell and / or to escape from endosomes. Without wishing to be bound by any particular theory', the present disclosure notes that incorporation of one or more neutral internucleotidic linkages can be utilized to modulate melting temperature of duplexes formed between a ds oligonucleotide and its target nucleic acid.

[0902] Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more non-negatively charged internucleotidic linkages, e.g., neutral internucleotidic linkages, into a ds oligonucleotide may be able to increase the ds oligonucleotide’s ability' to mediate a function such as direction of a decrease in expression, activity or level of a gene or a gene product thereof, mediated, for example, by RNAi. In some embodiments, a non-negatively charged internucleotidic linkage can improve the delivery and / or activities (e.g.. RNAi). In some embodiments, a non-negatively' charged internucleotidic linkage is a PN internucleotidic linkage. In some embodiments, it is a methylphosphonate internucleotidic linkage.

[0903] In some embodiments, an oligonucleotide comprises a modified internucleotidic linkage (e.g,, a modified internucleotidic linkage having the structure of Formula I, I-a. I-b, or 1-c, 1-n-l, I-n-2, 1-n-3. 1-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2. II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof) as described in US 9394333, US 9744183, US 9605019. US 9598458, US 9982257, US 10160969, US 10479995, US 2020 / 0056173, US 2018 / 0216107, US 2019 / 0127733, US 10450568, US 2019 / 0077817, US 2019 / 0249173, US 2019 / 0375774, WO 2018 / 223056. WO 2018 / 223073, WO 2018 / 223081. WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357. WO 2019 / 200185. WO 2019 / 217784, and / or WO 2019 / 032612 the internucleotidic linkages (e.g., those of Formula I, I-a, I-b, or I-c, I-n-1, I-n-2, 1-n-3, 1-n-4, II. Il-a-1, Il-a-2. II-b-1, II-b-2. Il-c-1. II-c-2. Il-d-1, II-d-2. etc,.) of each of which are independently incorporated herein by reference. In some embodiments, a modified internucleotidic linkage is a non-negatively charged internucleotidic linkage In some embodiments.provided oligonucleotides comprise one or more non-negatively charged intemucieotidic linkages. In some embodiments, a non-negatively charged intemucieotidic linkage is a positively charged intemucieotidic linkage. In some embodiments, a non-negatively charged intemucieotidic linkage is a neutral intemucieotidic linkage. In some embodiments, the present disclosure provides oligonucleotides comprising one or more neutral intemucieotidic linkages. In some embodiments, a non-negatively charged intemucieotidic linkage or a neutral intemucieotidic linkage (e.g., one of Formula I-n-1, l-n-2, l-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc.) is as described in US 9394333, US 9744183, US 9605019, US 9598458. US 9982257, US 10160969, US 10479995, US 2020 / 0056173, US 2018 / 0216107. US 2019 / 0127733. US 10450568. US 2019 / 0077817, US 2019 / 0249173. US 2019 / 0375774, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357. WO 2019 / 200185. WO 2019 / 217784. and / or WO 2019 / 032612. In some embodiments, a non-negatively charged intemucieotidic linkage or neutral intemucieotidic linkage is one of Formula I-n-1. 1-n-2, 1-n-3. I-n-4. II. II-a-1, II-a-2. II-b-1, II-b-2. II-c-1, II-c-2, II-d-1. II-d-2, etc. as described in WO 2018 / 223056, WO 2019 / 032607. WO 2019 / 075357. WO 2019 / 032607. WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784, and / or WO 2019 / 032612, such intemucieotidic linkages of each of which are independently incorporated herein by reference

[0904] As appreciated by those skilled in the art, many other types of intemucieotidic linkages may be utilized in accordance with the present disclosure, for example, those described in U. S. Pat. Nos.

[0905] 3,687.808; 4,469,863; 4,476,301; 5,177,195; 5,023,243; 5.034,506; 5,166,315; 5.185.444; 5,188,897; 5,214.134; 5,216,141; 5,235.033; 5,264,423; 5,264,564; 5,276,019; 5,278,302; 5.286.717; 5,321,131; 5,399.676; 5,405,938; 5,405,939; 5,434,257; 5,453,496; 5,455,233; 5.466,677; 5.466.677; 5,470.967; 5,476.925; 5,489,677; 5,519,126; 5,536,821; 5,541,307; 5,541,316; 5,550,111; 5.561,225; 5,563,253; 5.571,799; 5,587,361; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618.704; 5.623,070; 5,625,050; 5,633,360; 5,64,562; 5,663,312; 5.677,437; 5,677,439; 6,160,109; 6,239,265; 6,028.188; 6,124,445; 6,169,170; 6,172,209; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6.683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321.029; orRE39464. In certain embodiments, a modified intemucieotidic linkage is one described in US 9982257, US 20170037399. US 20180216108. WO 2017192664, W’O 2017015575, WO2017062862, WO 2018067973, WO 2017160741. WO 2017192679, WO 2017210647, WO 2018098264, PCT7US18 / 35687. PCT7US18 / 38835. or PCT / US18 / 51398, the nucleobases, sugars, intemucieotidic linkages, chiral auxiliaries / reagents, and technologies for oligonucleotide synthesis (reagents, conditions, cycles, etc.) of each of which is independently incorporated herein by reference.

[0906] In certain embodiments, a ds oligonucleotide comprises one or more intemucieotidic linkages that improve one or more pharmaceutical properties and / or activities of the oligonucleotide. It is well documented in the art that certain oligonucleotides are rapidly degraded by nucleases and exhibit poor cellular uptake through the cytoplasmic cell membrane (Poijarvi-Virta et al., Curr. Med, Chem. (2006),13(28);3441-65; Wagner et al., Med. Res. Rev. (2000), 20(6):417-51; Peyrottes et al., Mini Rev. Med. Chem. (2004), 4(4):395-408; Gosselin et al., (1996), 43(1): 196-208; Bologna et al., (2002), Antisense & Nucleic Acid Drug Development 12:33-41). Vives et al. (Nucleic Acids Research (1999), 27(20):4071-76) reported that tert-butyl SATE pro-oligonucleotides displayed markedly increased cellular penetration compared to the parent oligonucleotide under certain conditions.

[0907] Various types of internucleotidic linkages may be utilized in combination of other structural elements, e.g,. sugars, to achieve desired ds oligonucleotide properties and / or activities. For example, the present disclosure routinely utilizes modified internucleotidic linkages and modified sugars, optionally with natural phosphate linkages and natural sugars, in designing ds oligonucleotides. In certain embodiments, the present disclosure provides a ds oligonucleotide comprising one or more modified sugars. In certain embodiments, the present disclosure provides a ds oligonucleotide comprising one or more modified sugars and one or more modified internucleotidic linkages, one or more of which are natural phosphate linkages.

[0908] Among other things, the present disclosure demonstrates that incorporating certain modified internucleotidic linkages, e.g.. PN linkages such as phosphorami date linkages (e.g., phosphoryl guanidine linkages), at certain positions, e g, between the first and second nucleosides from the 5 ’-end of a sense strand, can provide various benefits and advantages, e.g., for reducing target RN levels, for maintaining target mRNA reduction for a longer period of time, etc. In some embodiments, an oligonucleotide, e.g., a passenger strand, comprises a modified, e.g., PN, internucleotidic linkage connecting the 5’ terminal (+1) nucleoside and the immediately downstream (+2) nucleoside. In some embodiments, a modified internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a neutral charged internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a charged internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a PN linkage. In some embodiments, a PN linkage is a phosphoramidate internucleotidic linkage. In some embodiments, a PN linkage is a phosphoryl guanidine internucleotidic linkage. In some embodiments, a PN linkage is MsPA. In some embodiments, a PN linkage is nOOl. In some embodiments, a PN linkage is a non-negatively charged internucleotidic linkage. In some embodiments, a PN linkage is a neutral internucleotidic linkage. Certain data demonstrating various benefits and advantages of modified internucleotidic linkages, e.g., PN linkages, between the first and second nucleosides from the 5'-end of an oligonucleotide are presented in the Examples (e.g., when utilized as a sense strand in a double stranded RNAi oligonucleotide). In some embodiments, the internucleotidic linkage between the last two nucleosides (from the 5’-end) of an oligonucleotide, e g., a sense strand, is a modified internucleotidic linkage. In some embodiments, it is a PN linkage as described herein. In some embodiments, it is a PS linkage, e.g., a phosphorothioate internucleotidic linkage. In some embodiments, the present disclosure provides a dsRNAi agent comprising such an oligonucleotide, e.g., a sense strand. In some embodiments, tire present disclosure provides a method for improving an oligonucleotide, e.g., a sense strand, a dsRNAi agent, etc..comprising incorporating a modified internucleotidic linkage as described herein, e.g., a PN linkage such as a phosphorami date linkage (e.g., a phosphoryl guanidine linkage) between the first and second nucleosides from the 5’-end of the oligonucleotide (e.g., a sense strand). In some embodiments, the present disclosure provides a method for improving a dsRNAi agent, comprising incorporating a modified internucleotidic linkage as described herein, e.g., a PN linkage such as a phosphoramidate linkage (e.g., a phosphoryl guanidine linkage) between the firstand second nucleosides from the 5’ -end of the sense strand. In some embodiments, the present disclosure provides an improvement in an oligonucleotide or a method thereof (e.g., a method comprises administering or delivering an oligonucleotide for RNAi. for preventing or treating a condition, disease or disorder, etc.), the improvement comprises that the oligonucleotide comprises a modified internucleotidic linkage as described herein, e.g., a PN linkage such as a phosphoramidate linkage (e.g., a phosphoryl guanidine linkage) between the first and second nucleosides from the 5’-end of the oligonucleotide (e.g.. a sense strand). In some embodiments, the present disclosure provides an improvement in a dsRNAi agent or a method thereof (e.g., a method comprises administering or delivering a dsRNAi agent for RNAi, for preventing or treating a condition, disease or disorder, etc.), the improvement comprises that the dsRNAi agent comprises a modified internucleotidic linkage as described herein, e.g., a PN linkage such as a phosphoramidate linkage (e g. a phosphoryl guanidine linkage) between the first and second nucleosides from the 5’-end of the sense strand. In some embodiments, benefits, advantages, improvements, etc. of provided technologies comprise improved durability, e.g.. when administered or delivered into a subject. See, e.g., the Examples. In some embodiments, the present disclosure provides a method, comprising assessing activity and / or durability of an oligonucleotide comprising a modified iiiternucleotidic linkage as described herein, e g.. a PN linkage such as a phosphoramidate linkage (e.g., a phosphoryl guanidine linkage) between the first and second nucleosides from the 5 -end of the oligonucleotide. In some embodiments, the present disclosure provides a method, comprising assessing ac tivity and / or durability' of a dsRNAi agent comprising a modified internucleotidic linkage as described herein, e.g., a PN linkage such as a phosphoramidate linkage (e.g., a phosphoryl guanidine linkage) between the first and second nucleosides from the 5’-end of the sense strand. In some embodiments, a method comprises assessing activity and / or durability of a reference oligonucleotide. In some embodiments, in a reference oligonucleotide the internucleotidic linkage between the first and second nucleosides from the 5 ‘-end of the oligonucleotide is not a PN linkage (e.g., a phosphorothioate internucleotidic linkage (e.g., a Sp phosphorothioate internucleotidic linkage)). In some embodiments, in a reference dsRNAi agent, the internucleotidic linkage between the first and second nucleosides from the 5’-end of the senses strand is not a PN linkage (e.g., a phosphorothioate internucleotidic linkage (e.g., a S’p phosphorothioate internucleotidic linkage)). In some embodiments, a reference oligonucleotide or dsRNAi agent is otherwise identical. In some embodiments, improved activity' is observed, e.g., relative to a reference oligonucleotide. In some embodiments, improved durability is observed, e.g., relative to a reference oligonucleotide. In some embodiments, improved durability isobserved, e.g., relative to a reference oligonucleotide, and comparable or improved durability is observed, e.g., relative to a reference oligonucleotide. In some embodiments, improved durability in a subject is observed, e.g., relative to a reference oligonucleotide. In some embodiments, improved durability in a subject is observed, e.g., relative to a reference oligonucleotide, and comparable or improved durability in a subject is observed, e.g., relative to a reference oligonucleotide. In some embodiments, when assessed in animals (e.g., mice), improved durability is observed, e.g., relative to a reference oligonucleotide. In some embodiments, when assessed in animals (e.g, mice), improved durability is observed, e.g., relative to a reference oligonucleotide, and comparable or improved durability is observed, e.g.. relative to a reference oligonucleotide. In some embodiments, benefits, advantages, improvements, etc. is relative to a reference oligonucleotide, e.g., which does not have the modified internucleotidic linkage between the first and second nucleosides from the 5 ’-end as described herein (e.g., instead having a natural phosphate linkage, a phosphorothioate internucleotidic linkage, etc.) but is otherwise identical. In some embodiments, a reference oligonucleotide has a phosphorothioate internucleotidic linkage instead of a PN internucleotidic linkage between the first and second nucleosides from the 5 ’-end of the sense strand. As described herein, configuration of linkage phosphorus can be controlled For example, in some embodiments, a PN linkage such as a phosphoramidate linkage (e g, a phosphoryl guanidine linkage) between the first and second nucleosides from the 5’-end of an oligonucleotide (e.g., a sense strand) is Rp. In some embodiments, a PN linkage such as a phosphoramidate linkage (e.g.. a phosphoryl guanidine linkage) between the last two nucleosides (from the 5’-end) of an oligonucleotide (e.g., a sense strand) is Rp. In some embodiments, a phosphorothioate linkage between the last two nucleosides (from the 5 ’-end) of an oligonucleotide (e.g., a sense strand) is Sp.

[0909] As described herein, internucleotidic linkages and patters thereof, e.g., modified internucleotidic linkages such as N linkages such as phosphoramidate linkages (e.g., phosphoryl guanidine linkages) bcm. ecu the first and second nucleosides from the 5‘-end of sense strands, can be and are routinely utilized in combination with other structural features such as sugars and patterns thereof, many of which have been repotted for RNAi. For example, in some embodiments, most sugars, e.g., about 80%-I00% (e.g., about or at least about 80%, 85%, 90%, or 95%) sugars in a sense strand is independently a modified sugar. In some embodiments, each sugar in an oligonucleotide, e.g., a sense strand, is independently a modified sugar. In some embodiments, each modified sugar is independently a 2’-F modified sugar or 2’-OR2samodified sugar, wherein R2sais optionally substituted Ci-e aliphatic. In some embodiments. each 2'-OR ’ ’ modified sugar is independently a 2’-MOE or 2’-OMe modified sugar. In some embodiments, each 2’-OR2samodified sugar is independently a 2’-OMe modified sugar. In some embodiments, each sugar in a sense strand is independently a 2’-F modified sugar, a 2’-OMe modified sugar or a 2’-MOE modified sugar. In some embodiments, each sugar in an oligonucleotide, e.g., a sense strand, is independently a 2’-F modified sugar or a 2’-OMe modified sugar. In some embodiments, an oligonucleotide, e.g.. a sense strand, comprises one or more 2’-F modified sugar. Insome embodiments, the number of 2’-F modified sugar in an oligonucleotide, e.g., a sense strand, is about or no more than about 6. In some embodiments, the number is about or no more than about 5, In some embodiments, the number is about or no more than about 4. In some embodiments, a region in an oligonucleotide, e.g., in a sense strand, comprises one or more 2'-F modified sugars. In some embodiments, a region in an oligonucleotide, e.g., in a sense strand, comprises one or more 2’-F modified sugars, and the rest of the oligonucleotide does not contain a 2’-F modified sugar. In some embodiments, a region is nucleosides 6-12 from the 5’-end of an oligonucleotide, e.g.. a sense strand. In some embodiments, a region is nucleosides 6-11 from the 5’-end of an oligonucleotide, e.g., a sense strand. In some embodiments, a region is nucleosides 7-11 from the 5’-end of an oligonucleotide, e.g., a sense strand. In some embodiments, length of a region is about 5, 6, 7. 8, or 9 nucleosides. In some embodiments, it is about 5 nucleosides. In some embodiments, it is about 6 nucleosides. In some embodiments, it is about 7 nucleosides. In some embodiments, a region comprises a nucleoside that is complementary to the 11* nucleoside from the 5 ’-end of an antisense strand.

[0910] As demonstrated herein, many modified internucleotidic linkages comprises chiral linkage phosphorus. In some embodiments, a composition is stereorandom with respect to a chiral linkage phosphorus. In some embodiments, an oligonucleotide composition is stereorandom. In some embodiments, chiral linkage phosphorus in an internucleoti ic linkage is chirally controlled, e.g., to be enriched for one configuration over the other, to provide various benefits and advantages, e.g., potency, durability, manufacturing control, etc. For example, in some embodiments, as demonstrated herein, an oligonucleotide, e.g.. a sense strand, comprises a Rp PN linkage such as a phosphorami date linkage (e.g., a phosphoryl guanidine linkage such as nOOl) between the first and the second nucleosides from the 5 -end; in some embodiments, as demonstrated herein, an oligonucleotide, e.g., a sense strand, comprises a Rp PN linkage such as a phosphoramidate linkage (e.g., a phosphoryl guanidine linkage such as nOOl) between the first and the second nucleosides from the 5 ’-end, and a Tip PN linkage such as a phosphora idate linkage (e.g., a phosphor l guanidine linkage such as nOOl) between the last and the second last nucleosides from the 5 ’-end; in some embodiments, as demonstrated herein, an oligonucleotide, e.g., a sense strand, comprises a p PN linkage such as a phosphoramidate linkage (e.g., a phosphoryl guanidine linkage such as nOOl) between the first and the second nucleosides from the 5’-end, and a Sp PS linkage such as a phosphorothioate linkage between the last and the second last nucleosides from the 5’-end. In some embodiments, an improvement comprises chirality of an internucleotidic linkage (e.g., a A’p PN linkage) as described herein. In some embodiments, most or all (e.g,, 80%-100% (e.g., about or at least about 80%, 85%. 90%, or 95%)) other internucleotidic linkages are independently PO linkages such as natural phosphate linkages. In some embodiments, a configuration of linkage phosphorus is enriched. In some embodiments, level of a configuration of chiral linkage phosphorus in an internucleotidic linkage is within a certain range, e.g, about 40%-60%.

[0911] 45-55%, etc. In some embodiments, it is about 50%. In some embodiments, level of a configmation of chiral linkage phosphorus in an internucleotidic linkage is about 90%-100%. In some embodiments,diastereopurity of a linkage phosphorus is about 90%-I00%. In some embodiments, diastereopurity of a linkage phosphorus is about 95%-100%, In some embodiments, it is about 90% or more. In some embodiments, it is about 91% or more. In some embodiments, it is about 92% or more. In some embodiments, it is about 93% or more. In some embodiments, it is about 94% or more. In some embodiments, it is about 95% or more. In some embodiments, it is about 96% or more. In some embodiments, it is about 97% or more. In some embodiments, it is about 98% or more. In some embodiments, it is about 99% or more In some embodiments, diastereopurity of each chiral linkage phosphorus is independently about 90%-100% as described herein (e.g., about 95%-100%, about or at least about 90%. 91%, 92%, 93%. 94%. 95%, 96%, 97%, 98%, 99%. etc.).

[0912] In some embodiments, diastereopurity of an oligonucleotide, e g. a sense strand, an antisense strand, a dsRNAi agent, etc., is about or at least about (DS)nc, wherein DS is about 90%-100%, and nc is the number of chiral linkage phosphorus in the oligonucleotide. In some embodiments. DS is about 95%-100%. In some embodiments, it is about 90% or more. In some embodiments, it is about 91% or more. In some embodiments, it is about 92% or more. In some embodiments, it is about 93% or more. In some embodiments, it is about 94% or more. In some embodiments, it is about 95% or more. In some embodiments, it is about 96% or more. In some embodiments, it is about 97% or more. In some embodiments, it is about 98% or more. In some embodiments, it is about 99% or more. In some embodiments, nc is about 1-30 (e.g., about 2-30. 1-25, 2-20, 1, 2. 3, 4, 5, 6. 7, 8, 9, 10, 11, 12. 13, 14, 15, 16, 17. 18, 19, 20, 21, 22. 23. 24, 25, 26. 27, 28, 29, 30, etc.) In some embodiments, nc is about 1-20. In some embodiments, nc is about 1-10. In some embodiments, nc is about 1-5.

[0913] Double Stranded Oligonucleotide Compositions

[0914] Among other things, the present disclosure provides various ds oligonucleotide compositions. In certain embodiments, the present disclosure provides ds oligonucleotide compositions of ds oligonucleotides described herein. In certain embodiments, a ds oligonucleotide composition, e.g., a dsRNAi oligonucleotide composition, comprises a plurality of a ds oligonucleotide described in the present disclosure. In certain embodiments, a ds oligonucleotide composition, e.g,, a dsRNAi oligonucleotide composition, is chirally controlled. In certain embodiments, a ds oligonucleotide composition, e.g., a dsRNAi oligonucleotide composition, is not chirally controlled (stereorandom).

[0915] In certain embodiments, the present disclosure encompasses technologies for designing and preparing chirally controlled ds oligonucleotide compositions. In certain embodiments, a chirally controlled ds oligonucleotide composition comprises a controlled / pre-determined (not random as in stereorandom compositions) level of a plurality of ds oligonucleotides, wherein the ds oligonucleotides share the same linkage phosphorus stereochemistry at one or more chiral internucleotidic linkages (chirally controlled internucleotidic linkages). In certain embodiments, ds oligonucleotides of a plurality share the same pattern of backbone chiral centers (stereochemistry of linkage phosphorus). In certain embodiments, a pattern of backbone chiral centers is as described in the present disclosure. Incertain embodiments, ds oligonucleotides of a plurality share a common constitution. In certain embodiments, they are structurally identical.

[0916] For example, in certain embodiments, the present disclosure provides a ds oligonucleotide composition comprising a plurality of ds oligonucleotides, wherein ds oligonucleotides of the plurality share:

[0917] 1) a common base sequence, and

[0918] 2) the same linkage phosphorus stereochemistry independently at one or more (e.g., about 1-50, 1-40, 1-30, 1-25. 1-20, 1-15, 1-10, 5-50, 5-40. 5-30, 5-25, 5-20, 5-15. 5-10. 1. 2. 3, 4, 5. 6. 7, 8, 9, 10, 11, 12, 13. 14, 15, 16, 17. 18. 19, 20, 21, 22. 23, 24, or 25 or more) chiral internucleotidic linkages ("chirally controlled internucleotidic linkages”): wherein level of ds oligonucleotides of the plurality in the composition is non-random (e.g., controlled / pre- determined as described herein).

[0919] Common patterns of backbone chiral centers, as appreciated by those skilled in the art, comprise at least one p or at least one. Sp. Certain patterns of backbone chiral centers are illustrated in, e.g.. Table 1.

[0920] In certain embodiments, a chirally controlled ds oligonucleotide composition is enriched, relative to a substantially racemic preparation of ds oligonucleotides share the same common base sequence and a common pattern of backbone linkages, for ds oligonucleotides of the particular ds oligonucleotide ty pe.

[0921] In certain embodiments, ds oligonucleotides of a plurality, e.g., a particular ds oligonucleotide type, have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In certain embodiments, ds oligonucleotides of a plurality have a common pattern of sugar modifications. In certain embodiments, ds oligonucleotides of a plurality have a common pattern of base modifications. In certain embodiments, ds oligonucleotides of a plurality have a common pattern of nucleoside modifications. In certain embodiments, ds oligonucleotides of a plurality' have the same constitution. In certain embodiments, ds oligonucleotides of a plurality' are identical. In certain embodiments, ds oligonucleotides of a plurality are of the same ds oligonucleotide (as those skilled in the art will appreciate, such ds oligonucleotides may each independently exist in one of the various forms of the ds oli onucleotide, and may be the same, or different forms of the ds oligonucleotide). In certain embodiments, ds oligonucleotides of a plurality' are each independently of the same ds oligonucleotide or a pharmaceutically acceptable salt thereof.

[0922] In certain embodiments, a chirally controlled ds oligonucleotide composition is chirally pure (or stereopure. stereochemically pure) ds oligonucleotide composition, wherein the ds oligonucleotide composition comprises a plurality’ of ds oligonucleotides, wherein the ds oligonucleotides are independently of the same stereoisomer (including that each chiral element of the ds oligonucleotides, including each chiral linkage phosphorus, is independently defined (stereodefined)), A chirally pure (or stereopure, stereochemically pure) ds oligonucleotide composition of a ds oligonucleotidestereoisomer does not contain other stereoisomers (as appreciated by those skilled in the art, one or more unintended stereoisomers may exist as impurities from, e.g., preparation, storage, etc,).

[0923] Sugars

[0924] Various sugars, including modified sugars, can be utilized in accordance with the present disclosure. In certain embodiments, the present disclosure provides sugar modifications and patterns thereof optionally in combination with other structural elements (e.g, internucleotidic linkage modifications and patterns thereof, pattern of backbone chiral centers thereof, etc.) that when incorporated into oligonucleotides can provide improved properties and / or activities.

[0925] The most common naturally occurring nucleosides comprise ribose sugars (e.g., in RNA) or deoxyribose sugars (e.g., in DNA) linked to the nucleobases adenosine (A), cytosine (C), guanine (G), thymine (T) or uracil (U). In certain embodiments, a sugar, e.g.. various sugars in many oligonucleotides in Table 1 (unless otherwise notes), is a natural DNA sugar (in DNA nucleic acids or

[0926] oligonucleotides, har ing the structure of

[0927]

[0928] , wherein a nucleobase is attached to the 1’ position, and the 3’ and 5’ positions are connected to internucleotidic linkages (as appreciated by those skilled in the art, if at the 5’-end of a ds oligonucleotide, the 5’ position may be connected to a 5’-end group (e.g., -OH), and if at the 3’-end of a ds oligonucleotide, the 3’ position may be connected to a 3 '-end group (e.g.. -OH). In certain embodiments, a sugar is a natural RNA sugar (in RNA nucleic

[0929] acids or oligonucleotides, having the structure of

[0930]

[0931] , wherein a nucleobase is atached to the 1 ’ position, and the 3’ and 5’ positions are connected to internucleotidic linkages (as appreciated by those skilled in the art, if at the 5 ’-end of a ds oligonucleotide, the 5’ position may be connected to a 5’-end group (e.g., -OH), and if at the 3 -end of a ds oligonucleotide, the 3’ position may be connected to a 3 ’-end group (e.g., -OH). In certain embodiments, a sugar is a modified sugar in that it is not a natural DNA sugar or a natural RNA sugar. Among other things, modified sugars may provide improved stability'. In certain embodiments, modified sugars can be utilized to alter and / or optimize one or more hybridization characteristics. In certain embodiments, modified sugars can be utilized to alter and / or optimize target recognition. In certain embodiments, modified sugars can be utilized to optimize Tm. In certain embodiments, modified sugars can be utilized to improve oligonucleotide activities.

[0932] Sugars can be bonded to internucleotidic linkages at various positions. As non-limiting examples, internucleotidic linkages can be bonded to the 2’, 3‘, 4’ or 5‘ positions of sugars. In certain embodiments, as most commonly in natural nucleic acids, an internucleotidic linkage connects with one sugar at the 5’ position and another sugar at the 3" position unless otherwise indicated.In certain embodiments, a sugar is an optionally substituted natural DNA or RNA sugar. In

[0933] certain embodiments, a sugar is optionally substituted

[0934]

[0935] In certain embodiments, the 2’

[0936] position is optionally substituted. In certain embodiments, a sugar is

[0937]

[0938] In certain

[0939] embodiments, a sugar has the structure of

[0940]

[0941] wherein each of 8. R's. R3s. R4s, and Rv is independently -H, a suitable substituent or suitable sugar modification (e.g.. those described in US 9394333, US 9744183, US 9605019, US 9982257, US 20170037399. US 20180216108. US 20180216107, US 9598458. WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647. WO 2018 / 098264. WO 2018 / 022473, WO 2018 / 223056, WO 2018 / 223073. WO 2018 / 223081. WO 2018 / 237194. WO 2019 / 032607, WO2019 / 032612, WO 2019 / 055951, and / or WO 2019 / 075357, the substituents, sugar modifications, descriptions of Rls, R2s, R3s, R4s. and R35, and modified sugars of each of which are independently

[0942] incorporated herein by reference). In certain embodiments, a sugar has the structure of

[0943]

[0944] . In certain embodiments, R4sis -H. In certain embodiments, a sugar has the structure of

[0945]

[0946] , wherein R2sis ~H. halogen, or -OR, wherein R is optionally substituted Ci-s aliphatic. In certain embodiments, R2sis -H. In certain embodiments, R2'3is -F. In certain embodiments, R2sis -OMe. hi certain embodiments, R2sis -OCIUCIUOMe.

[0947] Use of modified sugars in oligonucleotides for various applications has been wide reported in the art. For example, many modified sugars and patterns thereof have been reported and utilized in RNAi. Certain modified sugars and patterns of sugars are reported in US11993774, US12442002, US20250170161, W02013075035, WO2016028649, WO2021026150, W02023003922. WO2023104677, WO2023138689, WO2023183876, W02023245060, WO2024044542. WO2024059165, WO2024120412, WO2024138105, WO2024138111, WO2024148329, W02024168010. WO2024173520. WO2024179573. WO2024182580, WO2024186171. WO2024187190. WO2024188164. WO2024192379, WO2024196753, WO2024197017, WO2024226789. WO2024249240, W02025021034, WO2025034422, W02025034560,WO2025040002, WO2025049773, WO2025059466, WO2025064821, WO2025074157, WO2025082057, WO2025128589, W02025131019, WO2025137384, WO2025137390, WO2025166455, WO2025184301, WO2025185730, WO2025199356, WO2025201277, WO2025212467, WO2025228441. and W02025260042. the sugars and patterns thereof in RNAi agents are incorporated herein by reference.

[0948] Various additional sugars useful for preparing oligonucleotides or analogs thereof are known in the art and may be utilized in accordance with the present disclosure.

[0949] Various patterns of sugars and modifications thereof may be utilized in provided oligonucleotides (e.g.. ds oligonucleotides) in accordance with the present disclosure. In some embodiments, one or more of tire sugars of an oligonucleotide or portion thereof (e.g., a guide strand, a passenger strand) are modified. In some embodiments, a modified sugar comprises a 2 '-modification. In some embodiments, each modified sugar independently comprises a 2’-modification. In some embodiments, a 2 -modification is 2’-OR. In some embodiments, a -modification is a 2 -OMe. In some embodiments, a 2’ -modification is a 2’-MOE. In some embodiments, a 2 ’-modification is an LNA sugar modification. In some embodiments, a 2 ’-modification is 2’-F In some embodiments, each sugar modification is independently a 2’ -modification. In some embodiments, each sugar modification is independently 2’-OR or 2’-F. wherein R is optionally substituted Ci-6 alkyd. In some embodiments, each sugar modification is independently 2’-OR or 2’-F, wherein R is optionally substituted Ci e alkyl, and wherein at least one is 2’-F. In some embodiments, each sugar modification is independently 2’- OR or 2’-F, wherein R is optionally substituted C\-& alky 1, and wherein at least one is 2’-OR. In some embodiments, each sugar modification is independently 2 ’-OR or 2’-F, wherein R is optionally substituted Cj.f1alkyl, and wherein at least one is 2’-F and at least one is 2 -OR. In some embodiments, each sugar modification is independently 2’-OMe or 2’-F. In some embodiments, each sugar modification is independently 2’-OMc or 2’-F, wherein at least one is 2’-F. In some embodiments, each sugar modification is independently 2’-OMe or 2’-F, wherein at least one is 2’-C)Me. In some embodiments, each sugar modification is independently 2'-OMe or 2’-F, wherein at least one is 2’-F and at least one is 2’-OMe.

[0950] In some embodiments, an oligonucleotide or a portion thereof (e.g., a guide strand, a passenger strand) comprises a high level of 2’-OMe modified sugars, e.g., about 10%-100% (e.g., about 20%, 25%, 30%, 35%, 40%, 45%. 50%. 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, or more, or about 100%) of sugars in an oligonucleotide or a portion thereof comprises 2’-OMe. In some embodiments, about 50% or more of sugars in an oligonucleotide or a portion thereof comprises 2’-OMe. hi some embodiments, about 60% or more of sugars in an oligonucleotide or a portion thereof comprises 2’-OMe. In some embodiments, about 70% or more of sugars in an oligonucleotide or a portion thereof comprises 2’-OMe. In some embodiments, about 80% or more of sugars in an oligonucleotide or a portion thereof comprises 2’-OMe. In some embodiments, an oligonucleotide or aportion thereof (e.g., a guide strand, a passenger strand) also comprises one or more sugars comprising no 2’-OMe (e.g., sugars comprising no modifications and / or sugars comprising other modifications).

[0951] In some embodiments, a guide strand comprises a pattern of sugar modifications comprising one or more 2'-OR modified sugars, wherein R is optionally substituted Ci s alkyl. In some embodiments, a guide strand comprises a pattern of sugar modifications comprising one or more 2’-OMe modified sugars. In some embodiments, a guide strand comprises a pattern of sugar modifications comprising one or more 2’-F modified sugars. In some embodiments, a guide strand comprises a pattern of sugar modifications comprising one or more 2’ -OR modified sugars and one or more 2’-F modified sugars, wherein R is optionally substituted Ci 6 alkyl. In some embodiments, a guide strand comprises a pattern of sugar modifications comprising one or more 2 -OMe modified sugars and one or more 2 -F modified sugars.

[0952] In some embodiments, a third nucleoside, counting from 5’ to 3’. in a guide strand comprises a 2’-F modified sugar. In some embodiments, a third nucleoside, counting from 5’ to 3’, in a guide strand comprises a 2’-OR modified sugar, wherein R is optionally substituted Ci 6 alkyl. In some embodiments, a third nucleoside, counting from 5’ to 3 in a guide strand comprises a 2’ -OMe modified sugar. In some embodiments, a fourth nucleoside, counting from 5’ to 3’. in a guide strand comprises a 2’-F modified sugar. In some embodiments, a fourth nucleoside, counting from 5’ to 3’, in a guide strand comprises a 2’-OR modified sugar, wherein R is optionally substituted Ci e alkyl. In some embodiments, a fourth nucleoside, counting from 5’ to 3’. in a guide strand comprises a 2’-OMe modified sugar.

[0953] In some embodiments, a third nucleoside, counting from 5’ to 3’, in a guide strand comprises a 2’-F modified sugar and a fourth nucleoside in the guide strand comprises a 2’-OR modified sugar, wherein R is optionally substituted Ci-6 alkyl. In some embodiments, a third nucleoside, counting from 5’ to 3’, in a guide strand comprises a 2’-F modified sugar and a fourth nucleoside in the guide strand comprises a 2’-OMe modified sugar. In some embodiments, a third nucleoside, counting from 5‘ to 3’, in a guide strand comprises a 2 -OR modified sugar, wherein R is optionally substituted Ci-e alkyl, and a fourth nucleoside in the guide strand comprises a 2’-F modified sugar In some embodiments, a third nucleoside, counting from 5‘ to 3’, in a guide strand comprises a 2'-OMe modified sugar and a fourth nucleoside in the guide strand comprises a 2’-F modified sugar.

[0954] In some embodiments, a guide strand comprises a 5‘ end region that comprises a pattern of sugar modifications comprising, from 5’ to 3’. a 2’-OR modified sugar, a 2’-F modified sugar, a 2’-OR modified sugar, a 2’-F modified sugar, a 2’-OR modified sugar, and a 2‘-F modified sugar, wherein each R is optionally substituted Ci-e alkyl. In some embodiments, a guide strand comprises a 5’ end region that comprises a pattern of sugar modifications comprising, from 5’ to 3’, a 2’-OMe modified sugar, a 2’-F modified sugar, a 2’-OMe modified sugar, a 2’-F modified sugar, a 2 -OMe modified sugar, and a 2’-F modified sugar.In some embodiments, a guide strand comprises a 5’ end region that comprises a pattern of sugar modifications comprising, from 5’ to 3’, a 2’-OR modified sugar, two 2’-F modified sugars, two 2’-OR modified sugars, and a 2’-F modified sugar, wherein each R is optionally substituted Ci-6 alkyl. In some embodiments, a guide strand comprises a 5’ end region that comprises a pattern of sugar modifications comprising, from 5’ to 3', a 2’-OMe modified sugar, two 2’-F modified sugars, two T-OMe modified sugars, and a 2‘-F modified sugar.

[0955] In some embodiments, a guide strand comprises a pattern of sugar modifications comprising, from 5’ to 3’, a 2’-OR modified sugar, a 2 -F modified sugar, a 2’-OR modified sugar, a 2’-F modified sugar, a 2’-OR modified sugar, a 2’-F modified sugar, four 2 -OR modified sugars, a 2’-F modified sugar, two 2’-OR modified sugars, a 2 -F modified sugar, a 2’-OR modified sugar, a 2’-F modified sugar, and seven 2 -OR modified sugars, wherein each R is optionally substituted Ci-g alkyl, in some embodiments, a guide strand comprises a pattern of sugar modifications comprising, from 5’ to 3’, a 2‘-OMe modified sugar, a 2’-F modified sugar, a 2’-OMe modified sugar, a 2’-F modified sugar, a 2’-OMe modified sugar, a 2’-F modified sugar, four 2’-OMe modified sugars, a 2’-F modified sugar, two 2’-OMe modified sugars, a 2’-F modified sugar, a 2 -OMe modified sugar, a 2’-F modified sugar, and seven 2’-OMe modified sugars.

[0956] In some embodiments, a guide strand comprises a pattern of sugar modifications comprising, from 5’ to 3’, a 2’-OR modified sugar, two 2’-F modified sugars, two 2'-OR modified sugars, a 2’-F modified sugar, four 2’-OR modified sugars, a 2’-F modified sugar, two 2’-OR modified sugars, a 2’-F modified sugar, a 2 ’-OR modified sugar, a 2’-F modified sugar, and seven 2 ’-OR modified sugars, wherein each R is optionally substituted Ci e alkyl. In some embodiments, a guide strand comprises a pattern of sugar modifications comprising, from 5’ to 3’, a 2’-OMe modified sugar, two 2’-F modified sugars, two 2’-OMe modified sugars, a 2’-F modified sugar, four 2’-OMe modified sugars, a 2'4 modified sugar, two 2’-OMc modified sugars, a 2’-F modified sugar, a 2'-OMc modified sugar, a 2’-F modified sugar, and seven 2’-OMc modified sugars.

[0957] In some embodiments, a passenger strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) 2’-OR modified sugars, wherein R is optionally substituted Ci-s alkyl. In some embodiments, a passenger strand comprises one 2’-OR modified sugars, wherein R is optionally substituted Ci 6 alkyl. In some embodiments, a passenger strand comprises two 2’-OR modified sugars, wherein R is optionally substituted Ci-s alkyl. In some embodiments, a passenger strand comprises three 2’-OR modified sugars, wherein R is optionally substituted Cue alkyl. In some embodiments, a passenger strand comprises four 2’ -OR modified sugars, wherein R is optionally substituted Ci-e alkyd. In some embodiments, a passenger strand comprises five 2 ’-OR modified sugars, wherein R is optionally substituted Ci-e alkyl. In some embodiments, a passenger strand comprises six 2’-OR modified sugars, wherein R is optionally' substituted Cue alkyl In some embodiments, a passenger strand comprises seven 2’ -OR modified sugars, wherein R is optionally substituted Ci-6 alkyl. In some embodiments, a passenger strand comprises eight 2’-OR modifiedsugars, wherein R is optionally substituted Ci-6 alkyl. In some embodiments, a passenger strand comprises nine 2'-OR modified sugars, wherein R is optionally substituted Ci-g alkyl, hi some embodiments, a passenger stand comprises ten 2’-OR modified sugars, wherein R is optionally substituted Ci-s alkyl. In some embodiments, a passenger strand comprises 11 2’ -OR modified sugars, wherein R is optionally substituted Cj.g alkyl. In some embodiments, a passenger strand comprises 12 2’-OR modified sugars, wherein R is optionally substituted Ci-6 alkyl. In some embodiments, a passenger strand comprises 13 2’-OR modified sugars, wherein R is optionally substituted Ci-e alkyl. In some embodiments, a passenger strand comprises 142’-OR modified sugars, wherein R is optionally substituted Ci s alkyl. In some embodiments, a passenger strand comprises 15 2’-OR modified sugars, wherein R is optionally substituted Ch-e alkyl. In some embodiments, a passenger strand comprises 16 2 -OR modified sugars, wherein R is optionally substituted Ci-e alkyl. In some embodiments, a passenger strand comprises 172 -OR modified sugars, wherein R is optionally substituted Ci-6 alkyl. In some embodiments, a passenger strand comprises 182’ -OR modified sugars, wherein R is optionally substituted Cur, alkyl. In some embodiments, a passenger strand comprises 192’ -OR modified sugars, wherein R is optionally substituted Cus alkyl. In some embodiments, a passenger strand comprises 20 or more 2 -OR modified sugars, wherein R is optionally substituted Cue alkyl In some embodiments, a 2’-OR modified sugar is a 2’-OMe modified sugar. In some embodiments, each 2 / -OR modified sugar is a 2’-OMe modified sugar.

[0958] In some embodiments, a passenger strand comprises a pattern of sugar modifications comprising one or more 2’-OR modified sugars, wherein R is optionally substituted Cur alkyl. In some embodiments, a passenger strand comprises a pattern of sugar modifications comprising one or more 2’-OMe modified sugars. In some embodiments, a passenger strand comprises a pattern of sugar modifications comprising one or more 2’-F modified sugars. In some embodiments, a passenger strand comprises a pattern of sugar modifications comprising one or more 2’-OR modified sugars and one or more 2'-F modified sugars, wherein R is optionally substituted Cue alkyl. In some embodiments, a passenger stand comprises a pattern of sugar modifications comprising one or more 2’-OMe modified sugars and one or more 2’-F modified sugars.

[0959] In some embodiments, a passenger strand comprises one or more (e.g., 1, 2, 3, 4, 5, or 6 or more) 2’-OR modified sugars at the 5‘ end, wherein R is optionally substituted

[0960]

[0961] alkyl. In some embodiments, a passenger strand comprises one or more (e.g., 1, 2, 3. 4, 5, 6, 7, 8, or 9 or more) 2‘-OR modified sugars at the 3’ end, wherein R is optionally substituted Cue alkyl. In some embodiments, a passenger strand comprises one or more (e.g., 1, 2. 3, 4, 5, or 6 or more) 2’-OMe modified sugars at the 5’ end. In some embodiments, a passenger strand comprises one or more (e.g., I, 2. 3, 4, 5, 6. 7, 8, or 9 or more) 2’-OMe modified sugars at the 3' end. In some embodiments, a passenger strand comprises one or more (e.g., 1. 2, 3, 4, 5. or 6 or more) 2’-OR modified sugars in a 5’ end region, wherein R is optionally substituted Ci-s alkyl. In some embodiments, a passenger strand comprises one or more (e.g..

[0962] 1. 2, 3. 4, 5, 6. 7, 8, or 9 or more) 2’-OR modified sugars in a 3’ end region, wherein R is optionallysubstituted C;-.s alkyl. In some embodiments, a passenger strand comprises one or more (e.g., 1, 2, 3, 4, 5, or 6 or more) 2’-OMe modified sugars in a 5’ end region. In some embodiments, a passenger strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more) 2’-OMe modified sugars in a 3’ end region.

[0963] In some embodiments, a passenger strand comprises one or more (e.g., I, 2, 3, 4, 5, or 6 or more) 2‘-F modified sugars. In some embodiments, a passenger strand comprises one 2‘-F modified sugar. In some embodiments, a passenger strand comprises two 2’-F modified sugars. In some embodiments, a passenger strand comprises three 2’-F modified sugars. In some embodiments, a passenger strand comprises four 2’-F modified sugars. In some embodiments, a passenger strand comprises five 2’-F modified sugars. In some embodiments, a passenger strand comprises six or more 2’-F modified sugars.

[0964] In some embodiments, a passenger strand comprises one or more (e.g, 1. 2. 3, 4. 5. or 6 or more) 2'-F modified sugars in a middle region between a 5’ end region and a 3’ end region. In some embodiments, a passenger strand comprises one 2’-F modified sugar in a middle region between a 5’ end region and a 3‘ end region. In some embodiments, a passenger strand comprises two 2’-F modified sugars in a middle region between a 5’ end region and a 3’ end region. In some embodiments, a passenger strand comprises three 2’-F modified sugars in a middle region between a 5’ end region and a 3’ end region. In some embodiments, a passenger strand comprises four 2’-F modified sugars in a middle region between a 5’ end region and a 3’ end region. In some embodiments, a passenger strand comprises five 2’-F modified sugars in a middle region between a 5’ end region and a 3’ end region. In some embodiments, a passenger strand comprises six or more 2’-F modified sugars in a middle region between a 5’ end region and a 3’ end region.

[0965] In some embodiments, a passenger strand comprises a pattern of sugar modifications comprising, from 5’ to 3’, a 2’-F modified sugar, a 2' -OR modified sugar, and three 2'-F modified sugars, wherein R is optionally substituted Ci-& alkyd. In some embodiments, a passenger strand comprises a pattern of sugar modifications comprising, from 5' to 3’, a 2'-F modified sugar, a 2'-OMe modified sugar, and three 2’-F modified sugars. In some embodiments, a passenger strand comprises a middle region between a 5’ end region and a 3’ end region, wherein the middle region comprises a pattern of sugar modifications comprising, from 5’ to 3’, a 2’ -F modified sugar, a 2’-OR modified sugar, and three 2‘-F modified sugars, wherein R is optionally substituted CJ -A alkyl. In some embodiments, a passenger strand comprises a middle region between a 5’ end region and a 3’ end region, wherein the middle region comprises a pattern of sugar modifications comprising, from 5’ to 3’, a 2"-F modified sugar, a 2'-OMe modified sugar, and three 2’-F modified sugars. In some embodiments, a passenger strand comprises a middle region between a 5’ end region and a 3" end region, wherein the middle region comprises a pattern of sugar modifications comprising, from 5’ to 3\ a 2’-F modified sugar, a 2’-OR modified sugar, and three 2’-F modified sugars and wherein the 5' end region and 3’ end region only comprise 2’-OR modified sugars, wherein each R is optionally substituted Cj-e alkyl. In someembodiments, a passenger strand comprises a middle region between a 5’ end region and a 3' end region, wherein the middle region comprises a pattern of sugar modifications comprising, from 5‘ to 3’, a 2‘-F modified sugar, 2’-OMe modified sugar, and three 2"-F modified sugars and wherein the 5’ end region and 3’ end region only comprise 2’-OMe modified sugars.

[0966] In some embodiments, a passenger strand comprises a pattern of sugar modifications comprising, from 5" to 3", six 2 "-OR modified sugars, a 2"-F modified sugar, a 2 "-OR modified sugar, three 2"-F modified sugars, and nine 2’-OR modified sugars, wherein each R is optionally substituted Cue alkyl. In some embodiments, a passenger strand comprises a pattern of sugar modifications comprising, from 5’ to 3’, six 2’-OMe modified sugars, a 2’-F modified sugar, a 2"-OMe modified sugar, three 2’-F modified sugars, and nine 2’-OMe modified sugars.

[0967] Nucleobases

[0968] Various nucleobases may be utilized in provided ds oligonucleotides in accordance with the present disclosure. In certain embodiments, a nucleobase is a natural nucleobase, the most commonly occurring ones being A. T, C, G and U. In certain embodiments, a nucleobase is a modified nucleobase in that it is not A. T, C, G or U. In certain embodiments, a nucleobase is optionally substituted A, T. C, G or U, or a substituted tautomer of A T, C, G or U. In certain embodiments, a nucleobase is optionally substituted A, T, C, G or U. e.g., 5mC, 5- hydroxymethyl C, etc. In certain embodiments, a nucleobase is alkyl-substituted A. T, C, G or U. In certain embodiments, a nucleobase is A. In certain embodiments, a nucleobase is T. In certain embodiments, a nucleobase is C. In certain embodiments, a nucleobase is G. In certain embodiments, a nucleobase is U. In certain embodiments, a nucleobase is 5mC. In certain embodiments, a nucleobase is substituted A. T, C, G or U, In certain embodiments, a nucleobase is a substituted tautomer of A, T, C, G or U. In certain embodiments, substitution protects certain functional groups in nucleobases to minimize undcsircd reactions during oligonucleotide synthesis. Suitable technologies for nucleobase protection in oligonucleotide synthesis are widely- known in the art and may be utilized in accordance with the present disclosure. In certain embodiments, modified nucleobases improves properties and / or activities of ds oligonucleotides. For example, in many cases, 5mC may be utilized in place of C to modulate certain undesired biological effects, e.g., immune responses. In certain embodiments, when determining sequence identity, a substituted nucleobase having the same hydrogen- bonding pattern is treated as the same as the unsubstituted nucleobase, e.g., 5mC may be treated the same as C [e.g., a ds oligonucleotide having 5mC in place of C (e.g., AT5mCG) is considered to have the same base sequence as a ds oligonucleotide having C at the corresponding location(s) (e.g., ATCG)].

[0969] In certain embodiments, a modified nucleobase is a modified nucleobase known in the art, e.g., WO2017 / 210647. In certain embodiments, modified nucleobases are expanded-size nucleobases in which one or more aryl and / or heteroaryl rings, such as phenyl rings, have been added.In certain embodiments, a nucleobase is one described in US 9394333, US 9744183, US 9605019, US 9598458, US 9982257, US 10160969, US 10479995, US 2020 / 0056173. US 2018 / 0216107, US 2019 / 0127733, US 10450568, US 2019 / 0077817, US 2019 / 0249173, US 2019 / 0375774, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357, WO 2019 / 200185. WO 2019 / 217784, and / or WO 2019 / 032612, the nucleobases of each of which is incorporated herein by reference.

[0970] Additional Chemical Moieties

[0971] In certain embodiments, a ds oligonucleotide comprises one or more additional chemical moieties. Various additional chemical moieties, e.g., targeting moieties. carbohydrate moieties, lipid moieties, etc. are known in the art and can be utilized in accordance with the present disclosure to modulate properties and / or activities of provided oligonucleotides, e.g., stability7, half-life, activities, delivery, pharmacodynamics properties, pharmacokinetic properties, etc. In certain embodiments, certain additional chemical moieties facilitate delivery of oligonucleotides to desired cells, tissues and / or organs, including but not limited the cells of the central nervous system. In certain embodiments, certain additional chemical moieties facilitate internalization of oligonucleotides In certain embodiments, certain additional chemical moieties increase oligonucleotide stability. In certain embodiments, the present disclosure provides technologies for incorporating various additional chemical moieties into oligonucleotides.

[0972] In certain embodiments, a ds oligonucleotide comprises an additional chemical moiety demonstrates increased delivery to and / or activity in a tissue compared to a reference oligonucleotide, e.g.. a reference oligonucleotide which does not have the additional chemical moiety but is otherwise identical.

[0973] In certain embodiments, non-limiting examples of additional chemical moieties include carbohydrate moieties, targeting moieties, etc., which, when incoiporated into oligonucleotides, can improve one or more properties. In certain embodiments, an additional chemical moiety is selected from: glucose, GluNAc (N-acetyl amine glucosamine) and anisamide moieties. In certain embodiments, a provided ds oligonucleotide can comprise two or more additional chemical moieties, wherein the additional chemical moieties are identical or non-identical, or are of the same category (e.g., carbohydrate moiety, sugar moiety, targeting moiety, etc.) or not of the same category.

[0974] In certain embodiments, an additional chemical moiety is a targeting moiety. In certain embodiments, an additional chemical moiety is or comprises a carbohydrate moiety. In certain embodiments, an additional chemical moiety7is or comprises a lipid moiety7. In certain embodiments, an additional chemical moiety is or comprises a ligand moiety for. e.g.. cell receptors such as a sigma receptor, an asialoglycoprotein receptor, etc. In certain embodiments, a ligand moiety is or comprises an anisamide moiety, which may be a ligand moiety for a sigma receptor. In certain embodiments, a ligand moiety is or comprises a GalN Ac moiety, which may be a ligand moiety for an asialoglycoproteinreceptor. In certain embodiments, an additional chemical moiety facilitates delivery to liver.

[0975] In certain embodiments, a provided ds oligonucleotide can comprise one or more linkers and additional chemical moieties (e.g., targeting moieties), and / or can be chirally controlled or not chirally controlled, and / or have a bases sequence and / or one or more modifications and / or formats as described herein.

[0976] Various linkers, carbohydrate moieties and targeting moieties, including many known in the art, can be utilized in accordance with the present disclosure. In certain embodiments, a carbohydrate moiety is a targeting moiety. In certain e bodiments, a targeting moiety is a carbohydrate moiety.

[0977] Certain additional chemical moieties (e.g., lipid moieties, targeting moieties. carbohydrate moieties), and various linkers for connecting additional chemical moieties to ds oligonucleotide chains, are described in WO 2017 / 062862, WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647. WO 2018 / 098264. WO 2018 / 022473, WO 2018 / 223056, WO 2018 / 223073, WO 2018 / 223081. WO 2018 / 237194. WO 2019 / 032607. WO2019032612. WO 2019 / 055951. WO 2019 / 075357, WO 2019 / 200185, WO 2019 / 217784. and / or WO 2019 / 032612, the additional chemical moieties and linkers of each of which are independently incorporated herein by reference, and can be utilized in accordance with the present disclosure. In certain embodiments, an additional chemical moiety is digoxigenin or biotin or a derivative thereof.

[0978] In certain embodiments, an additional chemical moiety is one described in WO 2012 / 030683. In certain embodiments, a provided ds oligonucleotide comprise a chemical structure (e.g., a linker, lipid, solubilizing group, and / or targeting ligand) described in W'O 2012 / 030683.

[0979] In certain embodiments, a provided ds oligonucleotide comprises an additional chemical moiety and / or a modification (e.g.. of nucleobase, sugar, internucleotidic linkage, etc.) described in: U. S. Pat. Nos. 5,688,941; 6,294,664; 6,320,017; 6,576,752; 5,258,506; 5,591,584; 4,958,013; 5,082,830; 5.118.802; 5,138,045; 6,783,931; 5,254,469; 5,414,077; 5,486,603; 5,112.963; 5.599,928; 6,900,297; 5,214,136; 5,109,124; 5,512,439; 4,667,025; 5,525,465; 5,514,785; 5,565,552; 5,541,313; 5,545.730; 4,835,263; 4,876,335; 5,578,717; 5,580,731; 5,451,463; 5,510,475; 4,904,582; 5,082,830; 4,762,779; 4.789,737; 4,824.941; 4,828,979; 5,595,726; 5,214,136; 5,245,022; 5,317,098; 5,371,241; 5,391,723; 4.948,882; 5,218,105; 5,112,963; 5,567,810; 5,574,142; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 5.585,481; 5,292,873; 5.552,538; 5,512,667; 5,597.696; 5,599,923; 7,037,646; 5,587,371; 5,416,203; 5.262.536; 5,272,250; or 8.106.022.

[0980] In certain embodiments, an additional chemical moiety, e.g., a Mod, is connected via a linker. Various linkers are available in the art and tnay be utilized in accordance with the present disclosure, for example, those utilized for conjugation of various moieties with proteins (e.g., with antibodies to form antibody -drug conjugates), nucleic acids, etc. Certain useful linkers are described in US 9982257, US 20170037399, US 20180216108. US 20180216107. US 9598458. WO 2017 / 062862. WO 2018 / 067973. WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2018 / 223056, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 055951, WO 2019 / 075357. WO2019 / 200185, WO 2019 / 217784, and / or WO 2019 / 032612, the linker moieties of each which are independently incorporated herein by reference.

[0981]

[0982] When used as therapeutics, a provided ds oligonucleotide, e.g., a dsRNAi oligonucleotide, or ds oligonucleotide composition thereof is typically administered as a pharmaceutical composition. In certain embodiments, the present disclosure provides pharmaceutical compositions comprising a provided compound, e.g., a ds oligonucleotide, or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier. In certain embodiments, for therapeutic and clinical purposes, ds oligonucleotides of the present disclosure are provided as pharmaceutical compositions. As appreciated by those skilled in the art, ds oligonucleotides of the present disclosure can be provided in their acid, base or salt forms. In certain embodiments, ds oligonucleotides can be in acid forms, e g, for natural phosphate linkages, in the form of “0P(0)(0H)O-; for phosphorothioate internucleotidic linkages, in the form of -OP(O)(SH)O- etc. In certain embodiments, dsRNAi oligonucleotides can be in salt forms, e.g., for natural phosphate linkages, in the form of ~OP(O)(ONa)O~ in sodium salts; for phosphorothioate internucleotidic linkages, in the form of -OP(O)(SNa)O~ in sodium salts: etc Unless otherwise noted, ds oligonucleotides of the present disclosure can exist in acid, base and / or salt forms.

[0983] In certain embodiments, the present disclosure provides salts of ds oligonucleotides and pharmaceutical compositions thereof. In certain embodiments, a salt is a pharmaceutically acceptable salt. In certain embodiments, a pharmaceutical composition comprises a ds oligonucleotide, optionally in its salt form, and a sodium salt. In certain embodiments, a pharmaceutical composition comprises a ds oligonucleotide, optionally in its salt form, and sodium chloride. In certain embodiments, each hydrogen ion of a ds oligonucleotide that may be donated to a base (e.g., under conditions of an aqueous solution, a pharmaceutical composition, etc.) is replaced by a non-H’ cation. For example, in certain embodiments, a pharmaceutically acceptable salt of a ds oligonucleotide is an all-metal ion salt, wherein each hy drogen ion (for example, of -OH, ~SH, etc.) of each internucleotidic linkage (e.g., a natural phosphate linkage, a phosphory l guanidine internucleotidic linkage, a phosphorothioate internucleotidic linkage, etc.) is replaced by a metal ion. Various suitable metal salts for pharmaceutical compositions are widely known in the art and can be utilized in accordance with the present disclosure. In certain embodiments, a pharmaceutically acceptable salt is a sodium salt. In certain embodiments, a pharmaceutically acceptable salt is magnesium salt. In certain embodiments, a pharmaceutically acceptable salt is a calcium salt. In certain embodiments, a pharmaceutically acceptable salt is a potassium salt. In certain embodiments, a pharmaceutically acceptable salt is an ammonium salt (cation N(R)y ). In certain embodiments, a pharmaceutically acceptable salt comprises one and no more than one types of cation. In certain embodiments, a pharmaceutically acceptable salt comprises two or more types of cation. In certain embodiments, a cation is i.i. Na’, K+, Mg2’ or Ca '. In certain embodiments, a pharmaceutically acceptable salt is an all-sodium salt. In certain embodiments, a pharmaceuticallyacceptable salt is an all-sodium salt, wherein each internucleotidic linkage which is a natural phosphate linkage (acid form ~O~P(O)(OH)~O“), if any, exists as its sodiutn salt form (“O?(O)(ONa)~O~), and each internucleotidic linkage which is a phosphorothioate or a phosphoryl guanidine internucleotidic linkage (acid form -O-P(O)(SH) -O-), if any, exists as its sodium salt form (-O-P(OXSNa)-O-).

[0984] In certain embodiments, dsRNAi oligonucleotides are formulated in pharmaceutical compositions described in WO 2005 / 060697, WO 2011 / 076807 or WO 2014 / 136086.

[0985] In certain embodiments, a composition comprising a ds oligonucleotide is lyophilized. In certain embodiments, a composition comprising a ds oligonucleotide is lyophilized, and the l ophilized ds oligonucleotide is in a vial. In certain embodiments, the vial is back filled with nitrogen. In certain embodiments, the lyophilized ds oligonucleotide composition is reconstituted prior to administration. In certain embodiments, the lyophilized ds oligonucleotide composition is reconstituted with a sodium chloride solution prior to administration. In certain embodiments, the lyophilized ds oligonucleotide composition is reconstituted with a 0.9% sodium chloride solution prior to administration. In certain embodiments, reconstitution occurs at the clinical site for administration. In certain embodiments, in a lyophilized composition, a ds oligonucleotide composition is chirally controlled or comprises at least one chirally controlled internucleotidic linkage and / or the ds oligonucleotide targets.

[0986] II. EXEMPLIFICATION

[0987] Various technologies can be utilized to assess properties and / or activities of provided oligonucleotides and compositions thereof. Some such technologies are described in this Example. Those skilled in the art appreciate that many' other technologies can be readily utilized. As demonstrated herein, provided oligonucleotides and compositions, among other things, can be highly active, e.g., in reducing levels of their target nucleic acids.

[0988] Certain examples of provided technologies (compounds (oligonucleotides, reagents, etc.), compositions, methods (methods of preparation, use, assessment, etc.), etc.) were presented herein. EXAMPLE 1. Oligonucleotide Synthesis

[0989] Various technologies for preparing oligonucleotides and oligonucleotide compositions (both stereorandom and chirally controlled) can be utilized in accordance with the present disclosure, including, for example, methods and reagents described in U. S. 9,394,333, U. S. 9.744.183. U. S.

[0990] 9.605,019, U. S. 9,598,458, U. S. 9,982,257, U. S. 10,160,969, U. S. 10,479,995, U. S. 2020 / 0056173, U. S.

[0991] 2018 / 0216107, U. S. 2019 / 0127733, U. S. 10,450.568. U. S. 2019 / 0077817. U. S. 2019 / 0249173. U. S.

[0992] 2019 / 0375774, U. S. 2017 / 0037399, U S. 2018 / 0216108, WO 2018 / 223056. WO 2018 / 223073. WO 2018 / 223081, WO 2018 / 237194, WO 2019 / 032607, WO 2019 / 0.55951, WO 2019 / 075357. WO 2019 / 200185. WO 2019 / 217784. WO 2019 / 032612, WO 2020 / 191252, WO 2021 / 071858. WO 2017 / 062862. WO 2018 / 067973, WO 2017 / 160741, WO 2017 / 192679, WO 2017 / 210647, WO 2018 / 098264, WO 2022 / 099159. and / or W02023 / 201095. The methods and reagents of each of wdiichare incorporated herein by reference. Stereorandom and chirally controlled guide strand sequences were prepared utilizing the synthetic procedures as exemplified in above mentioned disclosures. Respective passenger strands were designed to have covalently linked GalNAc moiety’ as delivery vehicle at either end of sequences. Oligonucleotides with 5 '-GalNAc modifications were synthesized by coupling C6- amino modifier linker at the 5'-end of sequence. Oligonucleotides with 3’-GaINAc moiety as delivery vehicle were synthesized by utilizing 3 ’-C6 amino modified support. The single strand was cleaved from CPG by using deprotection condition as exemplified in earlier disclosures. Tire resulting amino group containing crude oligonucleotide was purified by ion exchange chromatography on AK. TA pure system using a sodium chloride gradient. Desired product was desalted and further used for conjugation with GalNAc acid. After conjugation reaction was found to be complete the material was further purified by ion exchange chromatography and desalted to achieve desired material. For introduction of PN linkages in guide and passenger strands, specific PN coupling cycles were introduced at desired positions in oligonucleotide sequence utilizing the conditions as exemplified in WO2019 / 200185.

[0993] In certain embodiments, oligonucleotides were prepared using suitable chiral auxiliaries, e.g.. DPSE and PSM chiral auxiliaries Various oligonucleotides, e.g, those in Table li, and compositions thereof were prepared in accordance w ith the present disclosure.

[0994] Various technologies can be utilized to assess properties and / or activities of provided oligonucleotides and compositions thereof. Some such technologies are described in this Example. Those skilled in the art appreciate that many other technologies can be readily utilized. As demonstrated herein, provided oligonucleotides and compositions, among other things, can be highly active, e.g., in reducing levels of their target nucleic acids.

[0995] Abbreviation

[0996] IX reagent: TEA-3HF: TEA: H2O: DMSO = 5.0: 1.8: 15.5: 77.7 (v / v / v / v)

[0997] AC2O: acetic anhydride

[0998] ACN: acetonitrile

[0999] ADIII: 2-azido-l,3-dimethyhmidazohum hexafluorophosphate

[1000] CM1MT: A'-cyanomethylimidazoliura triflate

[1001] CPG: controlled pore glass

[1002] DCM: dichloromethane, CH2Q2

[1003] DIPEA: diisopropvlethylamine

[1004] DMF: dimethylfonnamide

[1005] DMSO: dimethylsulfoxide

[1006] DMTr: 4,4'-dimethoxytrityl

[1007] DS1 reagent: TEA-3HF: TEA: H2O: DMSO - 5.0: 7.0: 14.7: 73.3 (v / v / v / v)

[1008] ETT: 5 -(ethylthio)- 1 H-tetrazoIe

[1009] GalNAc: / -acetylgalactosamine

[1010] HF: hydrogen fluorideHATU: l-[bis(dimethylammo)methylene]-lJ / -l,2,3-triazolo[4,5-b]pyridinium 3 -oxide hexafluorophosphate

[1011] IBN: isobutyronitrile

[1012] MeCN: acetonitrile

[1013] Melm: A-melhyliniidazole

[1014] PC: propylene carbonate

[1015] TCA: trichloroacetic acid

[1016] TEA: triethylamine

[1017] TEA-3HF: triethylamine trihydrofluoride

[1018] THF: tetrahydrofuran

[1019] TMS1: Trimethyisilyl iodide

[1020] XH: xanthane hydride

[1021] General procedure for the synthesis of chiral-oligos (25 fjmol scale):

[1022] The automated solid-phase synthesis of chiral-oligos was performed according to the cycles shown in Table 2 (regular amidite cycle, for PO linkages), 'Table 3 (regular amidite cycle, for stereorandom PS linkages), Table 4 (DPSE amidite cycle, for chiral PS linkages), and Table 5 (PSM amidite cycle, for chiral PN linkages).

[1023] Table 2. Regular Amidite Synthetic Cycle for PO linkages

[1024] Waiting Step Operation Reagents and Solvent Volume

[1025] Time 1 Detritylation 3% TCA / DCM 10111L 65 s 0.2M monomer / 20% IBN-MeCN 0.5 mL

[1026] 2 Coupling 8 min 0.5M CMIMT / MeCN l. OmL

[1027] 3 Oxidation 50mM h / pyridine-HcO (9: 1, v / v) 2.0 mL 1 min 20% AC2O, 30% 2,6-lutidine / MeCN 1.0 mL

[1028] 4 Cap-2 45 s

[1029]

[1030] 20% Melm / MeCN 1.0 mL

[1031] Table 3. Regular Amidite Synthetic Cycle for stereo-random PS linkages

[1032] Waiting Step Operation Reagents and Solvent Volume

[1033] Time 1 Detritylation 3% TCA / DCM 10 mL 65 s 2 Coupling 0.2M monomer / 20% IBN-MeCN 0.5 mL 8 min 0.5M CMIMT / MeCN 1.0 mL

[1034]

[1035] 3 Sulfurization 0.2M XH / pyridine 2.0 mL 6 min 20% Ac? O, 30% 2,6-lutidine / MeCN 1.0 mL

[1036] 4 Cap-2 45 s 20% Melm / MeCN 1.0 mL

[1037]

[1038] Table 4. DPSE Aniidite Synthetic Cycle for chiral PS linkages

[1039] Waiting Step Operation Reagents and Solvent Volume

[1040] Time 1 Detritylation 3% TCA / DCM 10 mL 65 s 0.2M monomer / 20% IBN-MeCN 0.5 mL

[1041] 2 Coupling 8 min 0.5M CMIMT / MeCN 1.0 mL

[1042] 3 Cap-1 20% Ac2O, 30% 2,6-lutidine / MeCN 2.0 mL 2 min 4 sulfurization 0.2M XH / pyridine 2.0 mL 6 min 20% AC2O, 30% 2,6-lutidine / MeCN 1.0 mL

[1043] 5 Cap-2 45 s 20% Melm / MeCN 1.0 mL

[1044]

[1045] Table 5. PSM Aniidite Synthetic Cycle for chiral PN linkages

[1046] Waiting Step Operation Reagents and Solvent Volume

[1047] Time 1 Detritylation 3% TCA / DCM 10 mL 65 s 0.2M monomer / 20% IBN-MeCN 0.5 mL

[1048] 2 Coupling 8 min 0.5M CMIMT / MeCN 1.0 mL

[1049] 3 Cap-1 20% Ac? O, 30% 2,6-lutidine / MeCN 2.0 mL 2 min 4 Imidation 0.5M ADIH reagent / MeCN 2.0 mL 6 min 5 20% Ac O. 30% 2,6-lutidine / MeCN 1.0 mL

[1050] Cap-2 45 s 20% Melm / MeCN 1 0 mL

[1051]

[1052] In some embodiments, preparations include one or more DPSE and / or PSM cycles

[1053] General procedure for the C& D conditions (25 / amol scale):

[1054] After completion of the synthesis, the CPG solid support was dried and transferred into 50 mL plastic tube. The CPG was treated with IX reagent (2.5 mL; 100 pL / umol) for 3 h at 28°C, then added cone. NH3 (5.0 mL; 200 L / umol) for 24 h at 37°C. The reaction mixture was cooled to room temperature and the CPG was separated by membrane filtration, washed w ith 15 mL of H2O. The crude material (filtrate) was analyzed by LTQ and RP-UPLC.General procedure for the purification conditions:

[1055] The crude sense and antisense strands were purified separately by AEX chromatography. The purification run was performed using sodium hydroxide eluents. A sodium chloride gradient was used io elute the oligonucleotide from tire column. The elution profile was monitored by UV spectrophotometry, and the selected fraction pool (sense or antisense strand) was then concentrated and diafiltered against purified water to remove the purification buffer by TFF. The UF / DF process proceeded as follows, the selected pool of fractions was neutralized with sodium phosphate monobasic solution and then concentrated to a target concentration. The concentrated oligonucleotide was diafiltered against purified water before final concentration to the target concentration and collected.

[1056] General procedure for the annealing to form duplex:

[1057] The duplex was formed by combining equal molar quantities of the sense and antisense strands with mixing in an appropriately sized vessel. The formation of the duplex w as confirmed by UPLC that there was no excess of single strands. The duplex oligonucleotide solution was filtered through a 0.2-micron filter and then placed in freeze drying tray for lyophilization After lyophilization, the duplex was isolated as a white to off-white solid powder. The resulting duplexes were then analyzed by LTQ / SQ D-MS and reverse phase UPLC. The observed molecular weight of the guide and passenger strands are shown in 'Table 11.

[1058] General procedure for the GalNAc conjugation conditions (1 mol scale):

[1059] Into a plastic tube, tri-GalNAc (2.0 eq.), HATU (1.9 eq.), and DIPEA (10 cq.) were dissolved in anhydrous MeCN (0.5 mL). The mixture was stirred for 10 min at room temperature, then the mixture was added into the amino-oligo (1 umol) in FLO (1 mL) and stirred for 1 h at 37 °C. The reaction was monitored by LC-MS and RP-UPLC. After the reaction was completed, the resultant GalNAc-conjugated oligo was treated with cone. NH3(2 mL) for 1 h at 37 °C. The solution was concentrated under vacuum to remove MeCN and cone. NH3. The residue wras then dissolved in H2O (10 mL) for reversed phase purification.

[1060] While various embodiments have been described and illustrated herein, those of ordinary skill m the art wall readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described in the present disclosure, and each of such variations and / or modifications is deemed to be included. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be example and that the actual parameters, dimensions, materials, and / or configurations may depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art ill recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments of the present disclosure. Itis, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, claimed technologies may be practiced otherwise than as specifically described and claimed. In addition, any combination of two or more features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included w ithin the scope of the present disclosure.

[1061] General procedure for the 5’-Ethyl phosphonate / 5’Methyl Phosphonate- deprotection conditions (25 pinole):

[1062] To prepare TMS1 solution for 5’-ethyl / methyl phosphonate deprotection, pyridine (0.5 tnL) was added to DCM (23.9 mL) and the resulting solution was cooled in ice-bath for 15 minutes. After that TMSI reagent (0.6 mL) was added to the mixture to get a bright yellow solution (total volume 25.0 mL). TMSI quenching solution (50 L) was prepared by adding 2 -Dodecane thiol (12.0 mL) and TEA (18.0 mL) in acetonitrile (18.0 mL).

[1063] After completion of the synthesis, the CPG solid support was dried and transferred into 50 mL plastic tube. Minimum amount of DCM was added to 5’-ethyl / methyl Phosphonate containing oligonucleotide on CPG and the CPG was vortexed to get a homogenous slurry. To this homogenous slurry, the TMSI solution (10.0 mL) was added slowly and mixed well. After the addition of the total TMSI solution the color of the reaction mixture turns yellow indicating excess of TMSI solution. The resulting reaction mixture was stirred for 30 minutes at room temperature. After 30 rain, the support was promptly washed using excess of acetonitrile followed by addition of quenching solution (10.0 mL; 0.4 mL quenching / umole of CPG solid support) and this process was repeated three times (Total quenching volume 30.0 mL), The total exposure time was limited to 20 rain. The CPG was rinsed thoroughly using acetonitrile and drying under vacuum. Afterwards, CPG was subjected to standard cleavage and deprotection (C& D) conditions.

[1064] General procedure for the C& D conditions (25 mol scale):

[1065] After completion of the synthesis and 5 ’-phosphonate deprotection, die CPG solid support was dried and transferred into 50 mL plastic tube. The CPG was treated with DS1 reagent (2.5 mL; 100 uL / umol) for 3 h at 27 °C, then added cone. NEL (5.0 mL; 200 umol / umol) for 24 h at 37 °C. The reaction mixture w as cooled to room temperature and the CPG was separated by membrane filtration, washed with 15 mL of ILO. The crude material (filtrate) was analyzed by LTQ and RP-UPLC.

[1066] General deprotection procedure for the 5’-POM protected Triazole Phosphonate:

[1067] No additional steps required after synthesis. POM group (pivolyloxy methyl) can be easily cleaved with the treatment of Ammonium hydroxide during standard cleavage and base deprotectionstep as mentioned above.

[1068] General deprotection procedure for the S’-Boronic acid protected Triazole Phosphonate:

[1069] Tetramethyl-dioxaborolan protection (i.e. acetal protection) of boronic acid can be cleaved with final detrityiation step (3% DCA in Toluene) of oligo synthesis. After that the sequence undergoes standard cleavage (DS1) and base deprotection step as mentioned above.

[1070] Example 1A. Example alternative procedure for preparation of oligonucleotide compositions (general cycle)

[1071]

[1072]

[1073] Each B is independently a nncleobase such as BA described herein (e.g., A, C, G, T, U. etc.). Each BPR(Jis independently an optionally protected nucleobase such as BA described herein (e.g.. Abz. Cac, Gbu, T. U, etc. suitable for oligonucleotide synthesis). As shown, various linkages can be constructed to connect monomers to nucleosides or oligonucleotides including those on solid support. As appreciate by those skilled in the art these cycles can be utilized to couple monomers to -OH of various other types of sugars.

[1074] In some embodiments, preparations include one or more DPSE and / or PSM cycles.

[1075] A number of oligonucleotide compositions were synthesized and assessed, including, e.g., those in the Figures and 'Tables.

[1076] As described and confirmed herein, technologies of the present disclosure are useful for preparing various compositions of oligonucleotides comprising various structural features at various scales (e.g., 1 umol, 5 umol, or 50 umol). It is understood that certain parameters will be modified based on the scale, e.g., volumes or equivalents. In some embodiments, as confirmed herein, provided technologies, e.g., those utilizing chiral auxiliaries comprising electron-withdrawing groups (e.g., Rc” comprising electron-withdrawing groups (e.g., -SOzR01, C(O)RC1, etc.)) are particularly useful for preparing chirally controlled compositions of oligonucleotides comprising 2‘-OH sugars (e.g., sugars with R2s= OH, such as sugars typically found in natural RNA), particularly when such sugars are bonded to chirally controlled internucleotidic linkages

[1077] The resulting oligonucleotides can undergo an annealing step to form a duplex.

[1078] Example IB. Example procedure for preparation of oligonucleotide compositions (1 pmol scale) Certain stereopure oligonucleotides were synthesized at 1 umol scale using a MerMadel92 synthesizer and universal CPG. In some embodiments, an amidite approach was used to incorporateGalNAc on the 5’ end. Generally, cyanoethyl amidites w;ere used to prepare the PO linkages, DPSE amidites for the PS linkages, and PSM amidites for the PN linkages. A typical MerMadel92, 1 pmol cycle is outlined in the table below:

[1079] Approx.

[1080] Step Operation Reagents and Solvent V olume

[1081] Total Time 1 Detritylalion 3% dichloroacetic acid in toluene 5x225 uL 6.5 min 0.1 M phosphoramidite in combinations of 95 pL / 11 ()

[1082] 2 Double 6 min (per ACN / 1BN / PC / DMF and 0.5M CMIMT in

[1083] Coupling pl, (per

[1084] coupling) ACN coupling) 80% THF / 10% 2,6-lutidine / 10% acetic

[1085] 3 Cap 1 200 jiL 1.5 min anhydride

[1086] Oxidation 0.02M iodine in 70% THF / 20% pyridine / 200 jiL 1,5 min (PO) 10% water

[1087] 4 Sulfurization 0. IM xanthaue hydride in 50% pyridine / 50% 200 uL 6.5 min (PS) ACN

[1088] PN 0.3M ADIH in ACN 200 uL 7 min

[1089] 80% THF / 10% 2,6-lutidine / 10% acetic 100 pL / 100

[1090] 5 Cap 2 1.5 min

[1091]

[1092] anhydride and 16% n-methylimidazole in THF jrL

[1093] The cycles were performed multiple times until the desired length was achieved.

[1094] The first step of deprotection w as performed on the synthesizer. 200 pL of 20% diethylamine in ACN was added to the column for 3 x 6 min followed by washing with ACN and diving, CPG was transferred to a container and 250 pL of fluoride solution was added. The fluoride solution consisted of dimethylformamide, water, triethylamine trihydrofluoride, and triethylamine (15,5 / 3, 1 / 1.0 / 1.8 volume ratio). After about 4 hours at room temperature, approximately 375 pL of 30% ammonium hydroxide was added, and the reaction incubated at 37 °C overnight. The CPG was filtered and washed with water and the filtrate collected.

[1095] The oligonucleotides were purified by anion exchange purification at room temperature. The oligonucleotide was loaded onto a column packed with Source QI 5 resin after equilibration with a 20 mM sodium hydroxide in 20% acetonitrile mobile phase. The purified oligonucleotide was eluted using a mobile phase of 20 mM sodium hydroxide and 2.5 M sodium chloride in 20% acetonitrile. The desired fraction was desalted using a G-25 Sephadex column against water for injection. Desalted samples were dried, reconstituted and analyzed.

[1096] A useful protocol for GalN Ac conjugation is described below as an example.

[1097]

[1098] - oligonucleotide chain

[1099] For example, pre-conjugation oligo sequence can be represented by the following structure.

[1100]

[1101] The tri-antennary GalNAc acid (hydroxyl groups protected as -OAc) can be represented by the following structure

[1102] o pAs

[1103] HHAc & [

[1104] ^9( 0 I

[1105] ■ ’ ’ n - I wfoA -

[1106]

[1107] HHAc g h 'b

[1108] The tri-antennary GalNAc acid (hydroxyl groups protected as -OAc) and HATU were weighed out in a 50 mL plastic tube and dissolved in anhydrous acetonitrile then D1EA was added into the tube. The resulting mixture was stirred for 10 min at 37 °C. Lyophilized pre-conjugation oligo sequence was reconstituted in water in a separate tube and the GalNAc mixture was added to the oligonucleotide solution and stirred for 60 min at 37 °C. The reaction was monitored by RP-UPLC. Reaction was complete in 1 hr. The reaction mixture was concentrated under vacuum to removethe acetonitrile and the resultant GalNAc -conjugated oligonucleotides is treated with cone, ammonia for 2 hi' at 37°C. The formation of final product was confirmed by mass spectrometry and RP-UPLC. 'The conjugated material was purified by anion exchange chromatography and desalted using tangential flow filtration (TFF) to obtain the final product.

[1109] Additional chemical moieties can also be installed by coupling with phosphoramidites comprising such additional chemical moieties (and optional linkers), e.g., phosphoramidites comprising GalNAc such as those described in Example 1C. Additional technologies for preparing oligonucleotides are illustrated below as examples.

[1110] Example 1C. Example procedure for preparation of oligonucleotide compositions (50 jtmol scale) Certain stereopure oligonucleotides were synthesized at 50 pniol scale using a MerMadel2 synthesizer and standard CPG. In some embodiments, an amidite approach was used to incorporate GalNAc on the 5’ end. Generally, cyanoethyl amidites were used to prepare the PO linkages, DPSE amidites for the PS linkages and PSM amidites for the PN linkages. A typical MerMadel2. 50 pmol cycle is outlined in the table below:

[1111] Approx. Step Operation Reagents and Solvent Volume

[1112] Total Time 1 Detritylation 3% dichloroacetic acid in toluene 4x4.5 mL 5 min Single 0.15M phosphoramidite in combinations of 15 mL / 2.25 2 6-12 min Coupling ACN / IBN / PC and 0.5M CMIMT in ACN mL

[1113] 80% THF / 10% 2,6-lutidine / 10% acetic

[1114] 3 Cap 1 5 mL 1 min anhydride

[1115] Oxidation 0.02M iodine in 70% THF / 20% pyridine / 10%

[1116] 6 mL 2-4 min (PO) water

[1117] 4 Sulfurization 0.1M xanthane hydride in 50% pyridine / 50%

[1118] 6 ml.. 6 min (PS) ACN

[1119] PN 0.3M ADIH in ACN 6 mL 10 min 5 80% THF / 10% 2,6-lutidine / 10% acetic anhydride 2.5 mL / 2.5

[1120] Cap 2 Imin

[1121]

[1122] and 16% n-m ethylimidazole in THF mL

[1123] The cycles were performed multiple times until the desired length was achieved.

[1124] In some embodiments, an amidite approach was used to incorporate GalNAc on the 5 ‘ end. Tire GalNAc amidite, or, or tri-anteimary GalNAc -acetyl derivative C6 phosphoramidite, can be represented by the following structure. The synthesis is disclosed in W02023201095 (Paragraph [001468])

[1125]

[1126] The GalNAc amidite was coupled either as a single 10-15 min or a two x 10 min procedure. For each coupling, 1.5 mL of 0.2M GalNAc amidite and 3 mL of CMIMT in ACN were added.

[1127] The first step of deprotection was performed on the synthesizer. 6 mL of 20% diethylamine in ACN was added to the column for 10 min followed by washing with ACN and drying. CPG was transferred to a tube and 5 mL of fluoride solution was added. The fluoride solution consisted of dimethylsulfoxide, water, triethylamine trihydrofluoride, and triethylamine (1.5 / 3.1 / 1.0 / 1 8 volume ratio). After about 1 hour at room temperature, approximately 10 mL of 30% ammonium hydroxide was added, and the reaction incubated at 37°C overnight. The CPG was filtered off and washed with water and the filtrate collected.

[1128] The oligonucleotides were purified by anion exchange purification at room temperature. The oligonucleotide was loaded onto a column packed with Source Q15 resin after equilibration with a 20 mM sodium hydroxide in water or 20 mM sodium hydroxide with 20% acetonitrile in water mobile phase. The purified oligonucleotide was eluted as fractions by gradient elution with a mobile phase of 20 mM sodium hydroxide and 2.5 M sodium chloride in water or 20 mM sodium hydroxide and 2.5 M sodium chloride with 20% acetonitrile in water. Fractions were analyzed, pooled to the desired purity and desalted using a G-25 Sephadex column against water for injection. Desalted samples were dried, reconstituted and sterile filtered prior to final analysis including UPLC, LC-MS and UV-Vis.

[1129] EXAMPLE 2. Synthesis of WV-NU-040

[1130]

[1131] OH

[1132] WV-NU-040

[1133] 5’-PO(OEt)2-triazolyl phosphonate-dT;Diethyl (l-(((2R,3S.5R)-3-hydroxy'-5-(5-mcthyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)- yl)tetrahydrofuran-2-yl)methyl)-lH-l,2,3-triazol-4-yl)phosphonate General Scheme:

[1134] NaN, DMF

[1135]

[1136] WV-NU-040

[1137] Experimental Procedures:

[1138] 1. Preparation of compound 2A

[1139]

[1140] To a solution of compound 1A (10 g, 57.96 mmol) in THF (20 mL) was added to bromo(ethynyl)magnesium (0.5 M, 117.07 mL) at 0°C under N?. The resulting mixture was stirred at 20 °C for 0.5 hr. TLC showed compound 1 A was consumed completely and two new spots formed. The mixture was quenched by addition sat. NH4CI (aq., 50 mL) at 0°C, then diluted with Ethyl acetate (30 mL) and extracted w ith Ethyl acetate (150 mL*3). The combined organic layers were dried over N32SO4, filtered and concentrated under reduced pressure to give crude. The residue was purified by column chromatography (SiCL, petroleum ether / ethyl acetate = 10:1 to 1:1). Compound 2A (5.2 g, 55.34?^ yield) was obtained as a colorless oil.

[1141] LCMS: (M+H+): 163.3TLC (Petroleum ether / Ethyl acetate = 1:1) Rf 0.43

[1142] 2. Preparation of compound 4

[1143]

[1144] 3 4

[1145] To a solution of compound 3 (10 g, 28,05 mmol) in pyridine (200 ml) was added PPh; (13.24 g. 50.49 mmol) and L (10.68 g, 42.08 mmol). The mixture was stirred at 25°C for 12 hr under N2 atmosphere. LCMS showed most of the starting material was disappeared and one main peak with desired mass was detected. The reaction mixture was quenched by sat. aq. Na SO; (200 mL) and extracted with EtOAc (600 mL*3). The combined organic layers were washed with brine (200 mL*2), dried over Na? SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCL, petroleum ether / ethyl acetate 10:1 to 0:1). Compound 4 (4.8 g, 33.40% yield, 91.041% purity) was obtained as a colorless oil.

[1146] LCMS: (M+H'): 467.0

[1147] TLC (Petroleum ether / Ethyl acetate = 1: 3) Rf = 0.75

[1148] 3. Preparation of compound 5

[1149] 0 0

[1150]

[1151] To a solution of compound 4 (4.8 g, 10.29 mmol) in DMF (48 mL) was added aN s (802.89 mg, 12.35 mmol). The mixture was stirred at 50°C for 12 hr. LCMS showed compound 4 was consumed completely and one main peak with desired MS was detected. The reaction was quenched by I-LO (6 mL), and extracted with TBME (6 mL*3). Compound 5 (3.93 g, crude) in a yellow solution of TBME (18 ml.,) was used into the next step without further purification.

[1152] LCMS: (M+H+): 382.3

[1153] 4. Preparation of compound 6THF

[1154] OTBS

[1155]

[1156] 5

[1157] To a solution of compound 5 (3.93 g, 10.30 mmol) in THF (20 mL) was added N, N-diethylethanamine; trihydrofluoride (6.64 g. 41.21 mmol). The mixture was stirred at 20°C for 12 hr. TLC showed a few of compound 5 was remained and new spot was detected. The reaction mixture was concentrated under reduced pressure and the mixture was neutralized with NajCOs (aq.. sat.) until pH = 7. The mixture was concentrated under reduced pressure to removed most of water. The mixture was added DCM (40 mL) and dried over NazSCL, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCL, Petroleum ether: (ethyl acetate: ethyl alcohol = 3:1) = 1:1). Compound 6 (2.7 g, crude) was obtained as a yellow oil.

[1158] TLC (petroleum ether: (ethyl acetate: ethyl alcohol = 3:1) = 1:1) Rf= 0.24

[1159] 5. Preparation of WV-NU-040

[1160] o OEt

[1161] P=O

[1162] OEt

[1163] DMF, Cui, DIEA

[1164] OH

[1165]

[1166] WV-NU-040

[1167] 5’-PO(OEt)2-Triazolyl phosphonate-dT (WV-NU-040).

[1168] To a solution of compound 6 (2 g. 7.48 mmol) and l-[ethoxy(ethynyl)phosphoryl]oxyethane (1.42 g, 876 mmol) in DMF (20 mL) was degassed and purged with N? for 3 times, then DIF. A (1.93 g, 14.97 mmol), Cui (285.06 mg. 1.50 mmol) was added. The mixture was stirred at 20°C for 4 hr under N2atmosphere. LCMS showed most of the starting material was disappeared and the desired substance was found. The reaction mixture was diluted with TMT solution (8 mL). filtered and the filtrate was diluted with ACN (80 mL), and concentrated under reduced pressure to give a residue The residue was washed with EtOAc (100 ml., *3), filtered and concentrated under reduced pressure to give product. WV-NU-040 (1.8 g, 3.92 mmol, 52.38% yield, 93.513% purity) was obtained as a white solid.

[1169] !H NMR (400 MHz, DEUTERIUM OXIDE) 5 ppm 8.39 (s, 1 H). 6.96 (s, 1 Hi. 6.07 (t.7=64 Hz, 1 H), 4.77 (d, 7=44 Hz, 2 H), 4.37 (q, 7=6.2 Hz, 1 H). 4.19 (q. 7=4.9 Hz. 1 H). 4.01 - 4.14 (m, 4 H). 2.20 - 2.37 (m, 2 H), 1.73 (s, 3 H). 1.19 (s. 6 H)

[1170] 31P NMR (1 2 MHz. DEUTERIUM OXIDE) 8 ppm 8.67 (s, 1 P)

[1171] 13C NMR (101MHz. DEUTERIUM OXIDE) 5 = 166.21, 151.53, 137.29, 136.50, 13408,133.47, 133.14, 111.55, 85.38, 82.53, 70.15, 64.72, 64.66, 50.60, 36.90, 15.47, 15.41, 11.49.

[1172] LCMS: (M+H+): 430.1, LCMS purity: 93.513%

[1173] EXAMPLE 3. Synthesis of WV-NU-306

[1174]

[1175] WV-NU-306

[1176] 2'-OMe-5’-PO(OEt)2 triazoiyi phosphonate uridine;

[1177] Dicthyl(l-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3-hydroxy-4- methoxy tetrahy drofuran-2-yl (methyl)- 1 H- 1,2.3-triazol-4-y l)phosphonate General Scheme:

[1178] zOEtOEt °\\ OEt Gl— R - *- OEt ™FOEt 0-25 °C, 2 h 1A 2A 3A

[1179] l2, PPh3, imidazole NaN3THF DMF

[1180] 0-25 °C, 12 h 0-50 °C, 3 h HO OMe

[1181] %-OEt OEt3ADIEA, Cui, THF 25 °C, 4 h

[1182]

[1183] Experimental Procedure:

[1184] 1. Preparation of compound 2A:

[1185] OEt::::: MgBr OEt

[1186] Cl— P

[1187]

[1188] OEt THF, 0-25 °C, 2 h

[1189] 1A 2A

[1190] To a solution compound 1 (10 g, 63.88 mmol) in THF (100 mL) was added bromo (cthynyl) magnesium (0.5 M, 124.75 mL) at 0c,C under Nb. The resulting mixture was stirred at 25 °C for 2 hr.TLC indicated compound 1A w as consumed completely and two new' spots formed. The reaction was clean according to TLC. The reaction mixture was quenched by sat. aq. NH^Cl (100 mL) at 0°C, then extracted with DCM (50 mL*3). The combined organic layers were dried over NaeSCL, filtered to get the crude. Compound 2A (37.34 g, crude, together 'ith four batches) w>as obtained as a brown liquid and used into the next step without further purification.

[1191] TLC: Petroleum ether: Ethyl acetate = 2:1, Rf = 0.25

[1192] 2. Preparation of compound 3 A:

[1193] 9Bm-CPBA O.,OEf

[1194] p - p

[1195]

[1196] OEt DCM, 0-25 °C, 2 h OEt

[1197] 2A 3A

[1198] To a solution of compound 2A (37 g, 253.21 mmol) in DCM (1000 mL) was added m-CPBA (102.82 g. 506.42 mmol, 85% purity) at 0°C. The mixture was stirred at 0-25°C for 2 hr. TLC indicated compound 2A was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction mixture was quenched by sat. aq. Na2SOj (300 mL) and NaHCCL (300mL), then extracted with DCM (200 ml., *3). The combined organic layers were washed with brine (100 ml., *2). dried over NazSCL, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCL. petroleum ether: ethyl acetate = 1:0 to 0:1). Compound 3A (55 g, 44.66% yield, together with three batches) was obtained as a colorless oil

[1199] *H NMR (400 MHz. CHLOROFORM-d) 5 - 4.16 - 4.07 (m. 4H), 297 (d, J - 13.3 Hz. 1H), 1.31 (dt. J = 0.7, 7 1 Hz, 6H)

[1200] 31P NMR (162 MHz, CHLOROFORM-d) 5 - -8.43 (s, IP)

[1201] TLC: Petroleum ether: Ethyl acetate = 1:1. Rf = 0.4

[1202] 5. Preparation of compound 2:

[1203]

[1204] To a solution of compound 1 (50 g, 193.63 mmol) in THF (700 mL) was added imidazole (34.27 g, 503.43 mmol), L (78.63 g, 309.80 mmol) and PPhs (81.26 g, 309.80 mmol) at 0 °C. The mixture was stirred at 25°C for 12 hr. LCMS showed compound 1 was consumed completely and the desired mass was detected. The reaction was quenched by 10% aqueous sodium thiosulfate solution (500 ml). After removing the solvent and volatiles under reduced pressure, the residue was extracted into EtOAc (200 mL*3) and washed with saturated aqueous NaHCCL solution. The organic layer wasseparated, dried over anhydrous NaJSO;. filtered and concentrated. The residue was purified by column chromatography (SiOj, petroleum ether: ethyl acetate = 1:0 to 0: 1). Compound 2 (270 g, 94.73% yield, together with four batches) was obtained as a white solid.

[1205] ’H NMR (400 MHz, DMSO-d6) 6 = 11.42 (s. 1H), 7.68 (d, J = 8.0 Hz. 1H), 5.86 (d, J - 5.4 Hz, 1H), 5.69 (d, J = 8.0 Hz, 1H), 5.45 (d, J = 6.0 Hz, 1H), 4.06 - 4.01 (m, 1H), 4.00 - 3.96 (m, 1H), 3.85 (td, J = 5.0, 6.2 Hz, 1H), 3.55 (dd, J - 5.4. 10.6 Hz, 1H), 3.40 (dd, J - 6.9, 10.6 Hz, 1H), 3.34 (s, 3H) LCMS (M+H+): 368.9

[1206] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf = 0.5

[1207] 4. Preparation of compound 5;

[1208] o 0

[1209] NaN3

[1210] DMF

[1211] 0-50 °C, 3 h

[1212] HO OMe HO OMe

[1213]

[1214] 2 3

[1215] To a solution of compound 2 (10 g. 27.16 mmol) in DMF (100 mL) was added NaNs (1.86 g, 28.66 mmol) at 0°C. The mixture was stirred at 0-50°C for 3 hr. LCMS showed compound 2 was consumed completely and the desired mass was detected. The reaction was quenched by H? O (1500 mL), and extracted with ethyl acetate (500 mL*3). The combined organic layers were washed with saturated aqueous NaCl 100 mL, dried over NaiSCL, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCL. petroleum ether: ethyl acetate = 1:0 to 0:1). The crude product was purified by re -crystallization from DCM (200 mL) at 25 °C. Compound 3 (57 g. 93.44% yield, together with six batches ) tvas obtained as a white solid.

[1216] TI NMR (400 MHz, DMSO-d6) 6 = 11.41 (br s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 5.83 (d, J = 4.9 Hz, 1H). 5.68 (d. J = 8.0 Hz, 1H). 5.35 (br d. J = 6.0 Hz. 1H). 4.07 (q. J = 5.3 Hz. 1H). 3.95 - 3.89 (m.

[1217] 2H), 3.61 (d. J = 4.9 Hz, 2H). 3.36 (s, 3H)

[1218] LCMS: (M+H+): 284.0, LCMS purity: 100%

[1219] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf = 0.45

[1220] 5. Preparation of WV-NU-306:

[1221] ;h'OEt

[1222] ' OEt 3A

[1223] DIEA, Cui, THF

[1224] 25 °C, 4 h

[1225]

[1226] WV-NU-306

[1227] To a solution of compound 3 (9,31 g, 57.42 mmol) and compound 3A (9.31 g. 57.42 mmol) inTHF (140 niL) was degassed and purged with N2for 3 times, then DIEA (12.69 g, 98.15 mmol), Cui (18.69 g, 98,15 mmol) was added. The mixture was stirred at 25°C for 4 hr under N2atmosphere, LCMS showed compound 3 was consumed completely and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to give product. The residue was purified by column chromatography (SiO2, Petroleum ether: Acetonitrile = 1: 0 to 0; 1 to Dicldoromethane: Methanol =1: 0 to 0: 1). Compound WV-NU-306 (56 g, 62.92% yield, together with two batches) was obtained as a yellow solid.

[1228] Batch 2 (46.43 g):

[1229] ’H NMR (400 MHz, CHLOROFORM-d) 5 = 9.79 (br s, 1H). 8.27 (s. IH), 7.03 (d, J - 8.0 Hz, 1H), 5.72 (d, J = 8.0 Hz. 1H), 5.62 (d, J = 2.4 Hz. 1H), 4.96 - 4.70 (m. 2H), 4.30 - 4.12 (m. 6H), 3.97 (dd. J - 2.4. 4.9 Hz, IH). 3.55 (s. 311), 3.48 (s. IH), 1.35 (t, J - 7.0 Hz, 6H)

[1230] 31P NMR ( 162 MHz. CHLOROFORM-d) 8 = 6.69 (s, 1 P)

[1231] LCMS (M+H+):446.1, purity: 97.42%

[1232] TLC: DCM: MeOH =10:1. Rf= 0.65

[1233] Batch 3 (9.22 g):

[1234] *H NMR (400 MHz, CHLOROFORM-d) 5 = 9.54 (s, 1 H). 8.27 (s. IH), 701 (d, J = 8.0 Hz, IH). 5 73 (dd, J = 1.6, 8.0 Hz. IH), 5.62 (d, J = 2.3 Hz, IH), 4.95 - 4.88 (m, IH). 4.75 (dd, J = 5.6, 14.4 Hz, IH), 4.30 - 4.15 (m, 6H). 3.97 (dd, J = 2.2, 4.8 Hz, IH), 3.56 (s, 3H), 3.52 (br d, J = 6.6 Hz. IH), 1.36 (t, J = 7.0 Hz, 6H)

[1235] 3,P NMR (162 MHz, CHLOROFORM-d) 8 = 6.64 (s, IP)

[1236] LCMS (M+H+): 446.1, purity: 93.76%

[1237] TLC: DCM: MeOH =10:1, Rf = 0.65

[1238] EXAMPLE 4. Synthesis of WV-NU-332

[1239]

[1240] 2’-OMe-5‘-bis(pivaloyloxymetliyl)-triazolyl phosphonate uridine;

[1241] [(((l-(((2R,3R.4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3-hydroxy-4- methoxytetrahydrofuran-2-yl)methyl)-lH-l,2,3-triazol-4-yl)phosphoiyl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoate)]

[1242] General Scheme:

[1243]

[1244] Experimental Procedure:

[1245] 1. Preparation of compound 2C:

[1246] / _ /

[1247] On==— MgBr 0

[1248] CK \\ o THF, 0-25 °C, 2h \\ o

[1249]

[1250] To a solution of compound 1C (TO g, 69.21 mmol, 7.46 mL) in THF (100 mL) was added bromo(ethynyl)magnesium (0.5 M, 166.10 mL) under N?, The mixture was stirred at 0-25°C for 2 hr, TLC indicated compound 1C was consumed completely and one new spot formed. The reaction mixture was quenched by addition NH4CI 50 mL at 0cC, and then diluted with waler 50 mL and extracted with EtOAc (100 mL*3). The combined organic layers were dried over NaeSCL, filtered, and concentrated under reduced pressure to give a residue. Without purification. Compound 2C (9 g, crude) was obtained as a yellow oil.

[1251] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf = 0.34

[1252] 2. Preparation of compound 5T:P0M-l(4eq.), 4A MS

[1253] - »- ACN, 85 °C, 10 h

[1254]

[1255] 2C 5A

[1256] To a solution of compound 2C (9 g, 67.13 mmol) in ACN (200 mL) was added 4A MS (2 g, 67.13 mmol), iodomethyl 2,2-dimethylpropanoate (48.75 g, 201.39 mmol). The mixture was stirred at 82°C for 10 hr. TLC indicated compound 2C was consumed completely and two new spots formed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Compound 5 A (5 g, 22.28% yield) was obtained as a colorless oil

[1257] 'H NMR (400 MHz, CHLOROFORM-d) 6 = 5.72 (d, J = 1.6 Hz, 2H), 5.69 (d, J = 0.9 Hz, 2H), 3.03 (d, J - 14.3 Hz, IH), 1.22 (s, 18H)

[1258] 3IP NMR (162 MHz. CHLOROFORM-d) 0 = 10.31 (s. IP)

[1259] TLC: Petroleum ether: Ethyl acetate 3:1, Rf == 0.38

[1260] 3. Preparation of compound 2:

[1261] o

[1262] l2, PPh3, imidazole

[1263] THF

[1264] 0-25 °C, 12 h OH OMe HO OMe

[1265]

[1266] 1

[1267] To a solution of compound 1 (50 g, 193.63 mmol) in THF (700 mL) was added imidazole (34.27 g, 503.43 mmol), L (78.63 g, 309.80 mmol) and PPh, (81.26 g, 309.80 mmol) at 0°C. The mixture was stirred at 25°C for 12 hr. LCMS showed compound 1 was consumed completely and the desired mass was detected. The reaction was quenched by 10% aqueous sodium thiosulfate solution (500 ml). After removing the solvent and volatiles under reduced pressure, the residue was extracted into EtOAc (200 mL*3) and washed with saturated aqueous NallCOj solution. The organic layer was separated, dried over anhydrous NacSCXi, filtered and concentrated. The residue was purified by column chromatography (SiO.:. petroleum ether: ethyl acetate = 1:0 to 0:1). Compound 2 (270 g. 94.73% yield, together with four batches) was obtained as a white solid.

[1268] 'HNMR (400 MHz. DMSO-d6) 6 = 11 42 (s, IH), 768 (d. J = 8.0 Hz, IH). 5.86 (d, J = 5.4 Hz, IH). 5.69 (d. J - 8.0 Hz, IH). 5.45 (d, J - 6.0 Hz, IH). 4.06 - 4.01 (m, IH). 4.00 - 3.96 (m, IH). 3.85 (Id, J - 5.0, 6.2 Hz, 1H), 3.55 (dd. J - 5.4, 10.6 Hz. 1H), 3 40 (dd. J - 6.9. 10.6 Hz, IH), 3.34 (s, 3H) LCMS (M+H+): 368.9

[1269] TLC: Petroleum ether: Ethyl acetate 0:1. Rf = 0.54. Preparation of compound 3:

[1270] o

[1271] NaNs

[1272] DMF

[1273] 0-50 °C, 3 h

[1274] HO OMe

[1275]

[1276] To a solution of compound 2 (10 g, 27.16 mmol) in DMF (100 mL) was added NaN2(1.86 g, 28.66 mmol) at 0°C. The mixture was stirred at 0-50°C for 3 hr. LCMS showed compound 2 was consumed completely and the desired mass was detected. The reaction was quenched by II2O (1500 mL), and extracted with Ethyl acetate (500 mL*3). The combined organic layers were washed with saturated aqueous NaCl 100 L, dried over Na2SO, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: eth l acetate = 1:0 to 0:1). The cinde product was purified by re-crystallization from DCM (200 mL) at 25°C. Compound 3 (57 g, 93.44% yield, together with six batches) was obtained as a white solid.

[1277] ’H NMR (400 MHz, DMSO-d6) 6 = 11.41 (br s. 1H), 7.70 (d. J - 8.0 Hz. 1 H), 5.8.3 (d, J - 4.9 Hz, 1H), 5.68 (d, J = 8.0 Hz, 1H), 5.35 (br d, J = 6.0 Hz, 1H), 4.07 (q, J = 5.3 Hz, 1H), 3.95 - 3.89 (m, 2H). 3.61 (d. J - 4.9 Hz. 2H). 3.36 (s. 3H)

[1278] LCMS: (M+H-t-): 284.0, LCMS purity: 100%

[1279] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf 0.45

[1280] 5. Preparation of compound WV-NIJ-332:

[1281] sodium ascorbate (1.2 eq.) CuSO4.5H2O (1.2 eq.) THF / H2O(1:1), 65 °C, 6 h OH OMe

[1282]

[1283] WV-NU-332

[1284] To a solution of compound 3 (3 g, 10.59 mmol) and compound 5 A (4.25 g, 12.71 mmol, 1 2 eq) in H2O (10 mL) was degassed and purged with nitrogen for 3 times, sodium ascorbate (2.52 g, 12.71 mmol, 1.2 eq), diacetoxycopper (231 g, 12.71 mmol) was added. The mixture was stirred at 65°C for 6 hr under N? atmosphere. TLC indicated compound 5 A was consumed completely and one new spot formed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate = 15:1 to 0:1 to Ethyl acetate: MeCN = 10:1 to 0:1 to Ethyl acetate: Methanol = 8:1 ). Compound WV-NU-332 (2 g, 46.67% yield, 70% purity) was obtained as a white solid.

[1285] 'H NMR (400 MHz, DMSO-d6) 8 = 11.47 - 11.35 (m, 2H), 8.69 (s, 1H), 7.62 (d, J = 8.1 Hz, 1H), 5.80 (d, J - 5.0 Hz, 1H), 5.70 (s, 2H), 5.67 (s, 2H), 5.51 (d, J - 5.8 Hz, HI), 4.80 (d, J - 3.8 Hz,1H), 4.18 - 4.15 (m, 1H), 3.96 - 3.89 (m, 2H), 3.61 (d, J - 4.8 Hz, 1H), 3.36 (s, 3H), 1.06 (s, 18H)31P NMR (162 MHz. DMSO-de) 8 = 7.08 (s, IP)

[1286] LCMS:(M+H+):618.2, purity:70.8%

[1287] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf= 0.06

[1288] EXAMPLE 5. Synthesis of WV-NU-336

[1289]

[1290] OH OMe

[1291] WV-NU-336

[1292] 2’-OMe-5’-bis(2-cyanoethyl)-triazolyl pbosphonate uridine;

[1293] Bis(2-cyanoethyl)(l-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidiii-l(2H)-yl)-3-hydroxy-4- methoxy'tetrahydrofuran-2-yl)methyl)-lH-l,2.3-triazol-4-yl)phosphonate General Scheme:

[1294] PCI3, TEA mCPBA THF, 25 °C, 2 h THF, 0-25 °C, 3 h DCM1B3B 4B

[1295] o l2, PPh3, imidazole THF

[1296] 0-25 °C, 12 h OH OMe 1 2 3

[1297] sodium ascorbate (1.2 eq.) CuSO45H2O (1.2 eq.)

[1298]

[1299] THF / H2O(1:1), 65 °C, 10 h OH OMe WV-NU-336Experimental Procedure:

[1300] 1. Preparation of compound 3B:

[1301] PCI3, TEA

[1302] CI~PS

[1303]

[1304] THF, 25 °C, 2 h o

[1305] 1B 3B

[1306] To a solution of PCk (35 g.25486 mmol) in THF (1000 mL) was added TEA (51.58 g. 509.71 mmol, 70.95 mL) and 3-hydroxypropanenitrile (36.23 g, 509.71 mmol, 34.67 L). The mixture was stirred at 25°C for 2 hr. TLC indicated compound IB was consumed completely and one new spot formed The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Compound 3B (44 g, 83.58% yield) was obtained as a colorless oil

[1307] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf= 0 12

[1308] 2. Preparation of compound 4B:

[1309] Cl— i\

[1310]

[1311] THF, 0-25 °C, 3h O'"\

[1312] 3B 4B

[1313] To a solution of compound 3B (10 g, 48.41 mmol) in THF (100 mL) was added bromo(ethynyl)niagnesium (0.5 M. 116.19 mL) at 0 °C. The mixture was stirred at 0-25°C for 5 hr. TLC indicated compound 3B was consumed completely and two new spots formed. The reaction mixture was quenched by addition NILC1 50 mL at 0°C, and then diluted with water 100 mL and extracted with EtOAc ( 100 ml., *3). The combined organic layers were dried over NazSCL, filtered, and concentrated under reduced pressure to give a residue. Compound 4B (6 g, 63.19% yield) was obtained as a yellow oil.

[1314] 'HNMR (400 MHz, CHLOROFORM-d) 6 = 4.17 - 4.09 (m, 4H).3.19 (d, J = 2.1 Hz, 1 H), 2.67 (t, J = 6.1 Hz. 4H)

[1315] 31P NMR (162 MHz, CHLOROFORM-d) 5 = 132.04 (s, IP)

[1316] For the scale up batch:

[1317] To a solution of compound 3B (44 g, 213.01 mmol) in THF (1000 mL) was added bromo(ethynyl)magnesium (0.5 M, 511.22 mL) at 0°C. The mixture was stirred at 0-25°C for 3hr. TLC indicated compound 3B was consumed completely and two new spots formed. The reaction mixture was quenched by sat. NFLCl (200 mL) at 0°C, then extracted with DCM (500 mL*3). The combined organic layers were dried over Na2SCL, filtered to get the crude No purification. Compound 4B (40 g, crude) was obtained as a yellow oil.

[1318] TLC: petroleum ether: ethyl acetate = 1:1, Rf = 0.393. Preparation of compound 5B:

[1319] mCPBA O

[1320] ==-- p'

[1321]

[1322] O-'X.^N DCM

[1323] 48 5®

[1324] To a solution of compound 4B (40 g. 203.93 mmol) in DCM (1000 mL) was added mCPBA (62.10 g, 305.90 mmol, 85% purity)- The mixture was stirred at 0-25°C for 2 hr. TLC indicated compound 4B was consumed completely and one new spot formed. The reaction mixture was quenched by sat, NajSOs (2000 mL) and NaHCO? (2000ml.,), then extracted with DCM (1000 ml, *2). The combined organic layers were washed with brine (500ml), dried over Na2SC>4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCty petroleum ether: ethyl acetate = 10:1 to 0:1). Compound 5 B (13 g, 30.05% yield) was obtained as a yellow oil.

[1325] ’H NMR (400 MHz. CHLOROFORM-d) 5 = 4.37 - 4.29 (m. 4H), 3.16 (dd..1 = 1.3. 139 Hz, HI). 2.81 (t. J - 6.1 Hz, 4H)

[1326] 31P NMR (162 MHz, CHLOROFORM-d) S - -8.61 (s. IP)

[1327] TLC: Petroleum ether: Ethyl acetate = 1:1, Rf = 0.23

[1328] 4. Preparation of compound 2:

[1329] O

[1330] l2, PPh3, imidazole

[1331] THF

[1332] 0-25 °C, 12 h OH CMa HO OMe

[1333]

[1334] 1

[1335] To a solution of compound 1 (50 g, 193.63 mmol) in THF (700 ml,) was added imidazole (34.27 g, 503.43 mmol). I? (78.63 g. 309.80 mmol) and PPh? (81.26 g, 309.80 mmol) at 0°C. The mixture was stirred at 25°C for 12 hr. LCMS showed compound 1 was consumed completely and the desired mass was detected. The reaction was quenched by 10% aqueous sodium thiosulfate solution (500 ml). After removing the solvent and volatiles under reduced pressure, the residue was extracted into EtOAc (200 mL*3) and washed with saturated aqueous NaHCO, solution. The organic layer was separated, dried over anhydrous NacSOi, filtered and concentrated. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 1:0 to 0:1). Compound 2 (270 g, 94.73% yield, together with four batches) was obtained as a white solid.

[1336] ’H NMR (400 MHz, DMSO-d6) 5 - 11.42 (s, 1H), 7.68 (d, J - 8.0 Hz. 1H), 5.86 (d, J - 5.4 Hz, 1H). 5.69 (d, J = 8.0 Hz, 1H). 5.45 (d, J = 6.0 Hz, 1H). 4.06 - 4.01 (m, 1H), 4.00 - 3.96 (m, 1H), 3.85 (Id, J - 5.0, 6.2 Hz, 1H), 3.55 (dd, J - 5.4. 10.6 Hz, 1H), 3.40 (dd, J - 6.9, 10.6 Hz, 1H), 3.34 (s, 3H)LCMS (M+H+): 368.9

[1337] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf = 0.5

[1338] 5. Preparation of compound 3:

[1339] Q O

[1340] NaN3

[1341] DMF

[1342] 0-50 °C, 3 h

[1343] HO OMe HO OMe

[1344]

[1345] 2

[1346] To a solution of compound 2 (10 g, 27.16 mmol) in DMF (100 mL) was added NaNs (1.86 g, 28.66 mmol) at 0°C. The mixture was stirred at 0-50°C for 3 hr. LCMS showed compound 2 was consumed completely and the desired mass was detected. The reaction was quenched by H2O (1500 mL), and extracted with Ethyl acetate (500 mL*3). The combined organic layers were washed with saturated aqueous NaCl 100 mL, dried over Na-SCL, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO?„ petroleum ether: ethyl acetate = 1:0 to 0: 1). The etude product was purified by re-crystallization from DCM (200 mL) at 25°C. Compound 3 (57 g, 93.44% yield) was obtained as a white solid.

[1347] 'H NMR (400 MHz, DMSO-d6) 6 = 11.41 (br s. 1H). 7.70 (d. J = 8.0 Hz. 1H), 5.83 (d, J = 4.9 Hz, HI). 5.68 (d, J == 8.0 Hz, 1H), 5.35 (br d, J - 6.0 Hz. HI), 4.07 (q, J - 5.3 Hz. HI), 3.95 - 3.89 (m, 2H). 3.61 (d. J - 4.9 Hz. 2H). 3.36 (s. 3H)

[1348] LCMS: (M+H-r): 284.0, LCMS purity: 100%

[1349] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf 0.45

[1350] 6. Preparation of compound WV-NU-336:

[1351] O

[1352] 9

[1353] sodium ascorbate (1.2 eq.) CUSO45H2O (1.2 eq.)

[1354]

[1355] HO OMe THF / H? O(1:1), 65 °C, 10 h OH OMe

[1356] 3 WV-NU-336

[1357] To a solution of compound 3 (5 g, 17.65 mmol) and compound 5B (4.49 g, 21.18 mmol) in THF (10 mL) and FLO (10 mL) was degassed and purged with N? for 3 times, then CuSCL. SHjO (5.29 g. 21.18 mmol), sodium ascorbate (4.20 g, 21.18 mmol) was added. The mixture was stirred at 65°C for 10 hr under N2atmosphere. LCMS sho ed compound 3 was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate =10:1 to 0:1, ethyl acetate MeCN = 8:1 to 0:1). Compound WV-NU-336 (5.3 g, 60.61% yield) was obtained as a white solid.

[1358] 'HNMR (400 MHz, DMSO-d6) 5 = 11.40 (d, = 1.6 Hz, 1H), 8.72 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 5.79 (d. J = 4.8 Hz, 1H), 5.66 (dd, J = 2.1, 8.0 Hz, 1H). 5.50 (d, J = 6.1 Hz, 1H). 4.87 - 4.73 (m, 2H), 4.32 - 4.17 (m, 5H). 4.14 (q, J = 5.5 Hz, 1H), 3.93 (t, J = 5.0 Hz, 1H), 3.36 (s, 3H), 2.95 (t, J = 5.9 Hz, 4H)

[1359] 31P NMR (162 MHz, DMSO-d6) 5 = 7.72 (s, IP)

[1360] I. CMS (M+H+): 496.1, purity: 93.49%

[1361] TLC: di chloromethane: methanol = 8:1. Rf = 0.13

[1362] EXAMPLE 6. Synthesis of WV-NU-347

[1363] o

[1364]

[1365] OH F

[1366] WV-NU-347

[1367] 2'-Fluoro-5'-PO(POM)2-Triazolyl Phosphorate Uridine (WV-NU-347) (((1-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2 / - / )-yi)~4-fluoro-3- hydroxytetrahydrofuran-2-yl)methyl)-1 H-1,2,3-triazol-4- yl)phosphoryl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoate)

[1368] General Scheme:

[1369] 0:— MgBr 0 POM-I (4eq.), 4A MS p X. p' a ' 'L THF, 0-25 °C, 2 h 1- ACN, 82 °C, 15 h O o 1C 2C 5A 0 l2, PPti3, imidazole NBN3THF DME: H2O = 5:1 0-25 °C, 4 h 0-90 °C, 12 h HO F

[1370]

[1371] 3A-, OPOM

[1372] / p\

[1373] OPOMSA

[1374] sodium ascorbate (1.2 eq.)

[1375] CuSO45H2O (1.2 eq.)

[1376] THF / H2O(1:1), 65 °C, 3 h OH F

[1377]

[1378] WV-NU-347

[1379] 2'-Fluoro-5'-PO(POM)2-Triazolyl Phosphorate Uridine (WV-NU-347)

[1380] Experimental Procedure:

[1381] 1. Preparation of compound 2C:

[1382] °Co. _ °Co.

[1383] c

[1384]

[1385] r 'A o THF, 0-25 °C, 2 h A o

[1386] 1C 2C

[1387] To a solution of compound 1C (36 g, 249.15 mmol) in THF (400 L) was added bromo(ethynyl)niagnesium (0.5 M, 498.29 mL) under N. The mixture was stirred at 0-25°C for 2 hr. TLC indicated compound 1C was consumed completely and one new spot formed. Each reaction mixture was quenched by addition NH4C1 lOOmL at 0°C. and then diluted with water 300 mL and extracted with EtOAc (100 mL*3). The combined organic layers were dried over NazSO.-i, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.-. petroleum ether: ethyl acetate = 10:1 to 0:1). Compound 2C (28.5 g, 28.44% yield) was obtained as a yellow oil.

[1388] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf = 035

[1389] 2. Preparation of compound 5 / 4:

[1390] 6 > POM-I (4eq.), 4A MS, — 0x'

[1391] ACN, 82 °C, 15 h XY n

[1392] % O i or

[1393]

[1394] 2C 5A

[1395] To a solution of compound 2C (9.5 g. 70.86 mmol) in ACN (500 mL) was added 4A MS (3 g, 70.86 mmol), iodomethyl 2,2-dimethylpropanoate (68.61 g, 283.43 mmol). The mixture was stirred at 82°C for 15 hr. TLC indicated Reactant 1 was consumed completely and t o new spots formed The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO?. petroleum ether: ethyl acetate = 10:1 tol: 1). Compound 5A (45 g, 63.32% yield) was obtained as a colorless oil.

[1396] *H NMR (400 MHz. CHLOROFORM-d) 8 - 5.72 (d. J = 1.2 Hz. 2H), 5.68 (s, 2H), 3 04 (d, J= 14.3 Hz, 1H), 1.22 (s, 18H)

[1397] 3 IP NMR (162 MHz. CHLOROFORM -d) 0 = -10.36 (s, IP)

[1398] TLC: Petroleum ether: Ethyl acetate = 3:1, Rf = 0.38

[1399] 3. Preparation of compound 2:

[1400]

[1401] 1 2

[1402] To a solution of compound 1 (15 g, 60.93 mmol) in THF (210 mL) was added imidazole (10.78 g, 158.41 mmol), h (24.74 g, 97.48 mmol) and PPM (25.57 g, 97.48 mmol) at 0°C. The mixture was stirred at 25°C for 4 hr. LCMS showed compound 1 was consumed completely and the desired mass was detected. The reaction was quenched by 10% aqueous sodium thiosulfate solution (100 ml). After removing the solvent and volatiles under reduced pressure, the residue was extracted into EtOAc (50 mL*5) and washed with saturated aqueous NaHCOi solution. The organic layer was separated, dried over anhydrous NacSCL, filtered and concentrated. The residue was purified by column chromatography (SiCL, petroleum ether: ethyl acetate = 1:0 to 0:1 to Dichloromethane: Methanol = 1:0 to 3:1). The crude product was purified by re-crystallization from Ethyl acetate (100 mL) and Methanol (10 mL) at 15°C. Compound 2 (43 g (total three batches), 66.15% yield,) was obtained as a yellow solid.

[1403] 'H NMR (400 MHz, DMSO-d6) 5 = 11.46 (br s. 1H), 7.66 (d, J = 8.0 Hz. 1H), 5.94 - 5.79 (m.

[1404] 2H), 5.66 (d. J - 8.0 Hz. 1H), 5.33 - 5.13 (m. 1H), 4.10 - 3.98 (m. 1H). 3.77 (dt, J - 3.4, 7.2 Hz. 1H).

[1405] 3.60 (dd, J = 3 6. 11.0 Hz, 1H). 3.41 (dd, J = 6.8, 11.0 Hz, 1H)

[1406] 19F NMR (376 MHz. DMSO-d6) 5 - -199.11 (s. IF)

[1407] LCMS (M+H+): 356.9. purity: 95.16%

[1408] TLC: Dichloromethane: Methanol = 10:1 Rf =0.45

[1409] 4. Preparation of compound 3:

[1410]

[1411] To a solution of compound 2 (10 g, 28.08 mmol) in 1,2-dimethoxyethane (100 mL) and H2O (20 mL) was added NaNa ( 1.83 g. 28.08 mmol) at 0°C under N2. The mixture was stirred at 90°C for12 hr. LCMS showed compound 2 was remained and tire desired mass was detected. The reaction was quenched by H2O (50 mL), and extracted with ethyl acetate (100 mL*3). The combined organic layers were washed with saturated aqueous NaCl 50 mL, dried over Na2SO4, filtered and concentrated trader reduced pressure to give a residue. The residue was purified by column chromatography (SiO-. petroleum ether: ethyl acetate = 1:0 to 0: 1). The crude product was purified by re-crystallization from ethyl acetate (100 mL) al 20°C. Compound 3 (28 g, 85.63% yield) was obtained as a white solid.

[1412] ’H NMR (400 MHz, DMSO-d6) 6 = 11.45 (br s. 1H), 7.67 (d, J = 8.0 Hz, 1H), 5.90 - 5.81 (m, 1H), 5.75 (br s, 1H), 5.66 (d. J = 8.0 Hz, 1H). 5.29 - 5.10 (m, 1H). 4.31 - 4.17 (m, 1H). 3.94-3.93 (m, 1H). 3.79 - 3.48 (m, 2H)

[1413] l9FNMR (376 MHz. DMSO-d6) 6 = -198.74 (s. IF)

[1414] LCMS: (M+H-F): 272.0, purity: 92.29%

[1415] TLC: Dichloromethane: Methanol = 10:1. Rf = 0.45

[1416] 5. Preparation of WV-NU-347:

[1417] 9- OPOM 'Z' ' OPOM5Asodium ascorbate (1.2 eq.) CuSO45H2O (1.2 eq.) HO F THF / H20(1:1), 65 °C, 3 h

[1418]

[1419] 2'-Fiuoro-5’-(POM)2-Triazoiyl Phosphonate Uridine (WV-NU-347)

[1420] To a solution of compound 3 (5 g, 18.44 mmol) and compound 5 A (7.40 g, 22.12 mmol) in HzO (25 ml) and THF (25 ml) was degassed and purged with N2for 3 times. CuSO4.5H2O (5.52 g, 22.12 mmol), sodium ascorbate (4.38 g, 22.12 mmol) was added. The mixture was stirred at 65°C for 3 hr under N2 atmosphere. LCMS showed compound 3 was consumed completely and the desired mass was detected. The reaction mixture was extracted with ethyl acetate (30mL*3). The combined organic layers were washed with saturated aqueous NaCl 20 mL, dried over Na2SC)4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate=l: O to 0:1 to ethyl acetate: methanol=l:0 to 2: 1) Compound WV-NU- 347 (14.5 g, 65.91% yield) was obtained as a yellow solid.

[1421] ’H NMR (400 MHz, DMSO-d6) 6 - 11.42 (d, J - 1.6 Hz. 1H). 8.71 (s. 1H). 7.56 (d, J - 8.2 Hz, 1H). 5.91 (d. J = 6.0 Hz. 1H), 5.87 - 5.78 (m. 1H). 5.69 (d. J = 13.8 Hz. 4H), 5.62 (dd. J = 2.0. 8.1 Hz, HI), 5.31 - 5.09 (m, 1H), 4.91 - 4.71 (m, 2H). 4.30 - 4.16 (m, 2H), 1.08 (s, 18H)

[1422] 31P NMR (162 MHz. DMSO-d6) 5 = 7.04 (s. IP)

[1423] 19F NMR (376 MHz, DMSO-d6) 8 = -199 16 (s, I F)

[1424] LCMS (M-i-H-F): 606.2. purity: 98.32%TLC: Petroleum ether: Ethyl acetate = 0:1, Rf 0.55

[1425] EXAMPLE 7. Synthesis of WV-NU-348

[1426]

[1427] WV-NU-348

[1428] 2'-OMOE-5’-PO(POM)2-Triazolyl Phosphonate Uridine (WV-NU-348) (((l-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3-hydroxy-4-(2- metboxyethoxy)tetrahydrofuran-2-yl)metbyl)-lH-l,2,3-triazol-4- yl)phosphoryl)bis(oxy))bis(meihylene) bis(2,2-dimethylpropanoate) General Scheme;

[1429] 5A sodium ascorbate (1.2 eq.) CUSO4.5H2O (1.2 eq.) THF / H2O(1:1), 65RC,6 h

[1430]

[1431] WV-NU-348

[1432] Experimental Procedure:

[1433] 1. Preparation of compound 2C:

[1434] p'x->.

[1435] C

[1436]

[1437] K 'L THF, 0-25 °C, 2 h 'L

[1438] 0 o

[1439] 1C 2C

[1440] To a solution of compound 1C (36 g, 249.15 mmol, 26.87 mL) in THF (400 mL) was added bromo(cthynyl)magncsium (0.5 M, 498.29 mL) under N2. Tbc mixture was stirred at 0-25°C for 2 h.TLC indicated compound 1C was consumed completely and one new spot formed. The each reaction mixture was quenched by addition NH.; C1 100 mL at 0°C, and then diluted with water 300 ml, and extracted with EtOAc (100 mL*3). The combined organic layers were dried over NacSCL, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography t Si() petroleum ether: ethyl acetate = 10:1 to 0:1). Compound 2C (23 g, 17.21% yield) was obtained as a yellow oil.

[1441] ’H NMR (400 MHz, CHLOROFORM-d) 3 = 3.82 (s. 3H), 3.80 (s. 3H), 2.96 (d, J = 13.4 Hz, 1H)

[1442] 31P NMR (162 MHz. CHLOROFORM-d) 8 - -5.07 ■ -5.22 (m, IP)

[1443] TLC: Petroleum ether: Ethyl acetate = 0: 1, Rf = 0.31

[1444] 2. Preparation of compound SA:

[1445] O \ „ POM-! (4eq.), 4A MS / — Ox|

[1446] ACN, 82 °C, 15 h 4 XL ^O.

[1447] A O V o

[1448]

[1449] 2C 5A

[1450] To a solution of compound 2C (11.5 g, 85.78 mmol) in ACN (500 mL) was added 4A MS (5 g. 85.78 mmol), iodomethyl 2,2-dimcthylpropanoatc (83.05 g, 343.10 mmol). The mixture was stirred at 82°C for 15h. TLC indicated compound 2C was consumed completely and two new spots formed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography iSiO-. petroleum ether: ethyl acetate = 10:1 to 0:1). Compound 5 A (33 g, 57.89% yield) was obtained as a colorless liquid.

[1451] ’H NMR (400 MHz, CHLOROFORM-d) o = 5.70 (d, J = 1.5 Hz, 2H). 5.66 (d. J = 0.8 Hz, 2H), 3.06 (d, J = 14.3 Hz, 1H). 1.20 (s, 18H)

[1452] 31P NMR (162 MHz. CHLOROFORM-d) 3 - -10.31 (s, IP)

[1453] TLC: Petroleum ether: Ethyl acetate = 3: 1, Rf = 038

[1454] 3. Preparation of compound 2:

[1455] l2, PPh3, imidazole

[1456] - THF

[1457]

[1458] 1 2

[1459] To a solution of compound 2 (17.5 g, 57.89 mmol) in THF (400 L) was added imidazole (10.25 g, 150.52 mmol), h (23.51 g, 92.63 mmol. 18.66 mL) and PPhj (24.30 g, 92.63 mmol) at 0°C.The mixture was stirred at 20°C for 12 h. LCMS showed compound 2 was consumed completely and one main peak with desired mass was detected. Two reactions were combined for work up. The reaction mixture was quenched by addition 10% Na2S2O3aq. 100 mb at 25°C, and then concentrated under reduced pressure to remove THF, diluted with H2O (700 mL) and extracted with EtOAc (500 mL*2). The combined organic layers were washed with brine (600 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product w as used directly for the next step w ithout purification. Compound 2 (48 g, crude) was obtained as a yellow oil.

[1460] LCMS: (M+H'): 412.9

[1461] 4. Preparation of compound 3:

[1462] OMe OMe

[1463]

[1464] 2 3

[1465] To a solution of compound 2 (22 g. 53.38 mmol) in DMF (250 mi.) was added NaN3(3.09 g, 47.53 mmol) at 0°C under N2. The mixture was stirred at 90°C for 12 h. LCMS showed compound 2 was consumed completely and desired mass was detected. The reaction was quenched by H2O (500 mb), and extracted with Ethyl acetate (500 mL*3). The combined organic dried over N2SO4. filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 0:1). Compound 3 (34 g, crude) was obtained as a yellow oil.

[1466] !HNMR (400 MHz, DMSO-d6) 6 = 11.40 (s, 1H), 7.70(d, J = 8.2 Hz. 1H). 5.82 (d. J = 4.5 Hz, 1H). 5.68 (d. J - 7.8 Hz, 1H), 5.26 (d, J - 5.6 Hz, 1H), 4.10 - 4.02 (m. 2H), 3.92 (q, J - 4.8 Hz. 1H), 3.74 - 3.67 (m. 1H). 3.66 - 3.57 (tn, 3H), 3.47 - 3.42 (m, 2H). 3.25 - 3.20 (m, 3H)

[1467] LCMS: (M+IP): 328.2

[1468] TI C. Petroleum ether: Ethyl acetate - 0: 1, Rf = 0.34

[1469] 5. Preparation of compound WV-NU-348:

[1470] Ap-OPOM '3? OPOM 5A sodium ascorbate (1.2 eq.) CUSO4.5H2O (1.2 eq.)

[1471]

[1472] THF / H2O(1:1), 65 °C, 6 h 3 WV-NU-348

[1473] 2’-OMOE-5'-PO(POM)2-Triazolyl Phosphonate Uridine (WV-NU-348)To a solution of compound 3 (11.5 g, 35.14 mmol) and compound 5A (15.27 g, 45.68 mmol) in THF (50 mL) and H? O (50 mL) was added sodium ascorbate (8.35 g, 42,16 mmol) and CuSCL. SHzO (10.53 g, 42.16 mmol). The mixture was stirred at 65°C for 6 h. LCMS showed compound 3 was consumed completely and desired mass was detected. The mixture was concentrated, the reaction mixture was extracted with EtOAc (50 mL*3) and HzO 50 mL. Then the combined organic layers were dried oxer Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiOa. petroleum ether: ethyl acetate = 1:0 to 0:1). Compound WV-NU-348 (10.7 g, 45.48% yield, 97.77zo purity') was obtained as a yellow solid.

[1474] ’HNMR (400 MHz, DMSO-d6) 5 - 11.45 - 11.36 (m. 1H), 8.70 (s, 1H). 7.62 (d. J - 8.2 Hz, 1H), 5.80 (d, J = 4.6 Hz, 1H), 5.70 (s, 2H), 5.68 - 5.64 (m, 3H), 5.40 (d, J = 5.6 Hz, 1H), 4.85 - 4.71 (m, 2H), 4.21 - 4.17 (m, 1H), 4.14 - 4.08 (m, 2H). 3.75 - 3.67 (m, 1H), 3.67 - 3.59 (m, 1H), 3.46 (t, J - 4.8 Hz. 2H). 3.22 - 3.22 (m, 1H). 322 (s. 2H). 1.08 (s, 18H)

[1475] 3)PNMR (162 MHz, DMSO-d6) 5 - 7.08 (s. IP)

[1476] LCMS: (M+H% 662.2, LCMS purity: 97.77%

[1477] TLC: Dicbloromethane: Methanol = 10: 1, Rf = 0.32

[1478] EXAMPLE 8. Synthesis of WV-NU-349

[1479]

[1480] WV-NU-349

[1481] 2'-LNA-5'-PO(POM)2-Triazolyl Phosphonate Uridine (WV-NU-349) (((l-(((lS,3R,4R>7S)-3-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-7-hydroxy-2,5- dioxabicyclo[2.2.1]heptan-l-yl)methyl)-lH-1.2.3-triazol-4-yl)phosphoiyl)bis(oxy))bis(methylene) bis(2,2-dimethylpropanoate)

[1482] General Scheme:

[1483]

[1484] R, OPOM OPOM

[1485] sodium ascorbate CUSO4.5H2O THF / H20(1:1)

[1486]

[1487] 8

[1488] WV-NU-349 Experimental Procedure:

[1489] 1. Preparation of compound 2C:

[1490] O / ::::::: - MgBf

[1491] C

[1492]

[1493] l'' A O THF

[1494] 1C 2C

[1495] To a solution of compound 1C (36 g, 249.15 mmol) in THF (400 mL) was added bromo (ethynyl) magnesium (0.5 M. 498.29 mL) under N2. The mixture was stirred at 0-25°C for 2 hr. TLC indicated compound 1C was consumed completely and one new spot formed. The each reaction mixture was quenched by addition NILiCl lOOmL at 0°C. And then diluted with water 300 mL and extracted with EtOAc (100 mL*3). The combined organic layers were dried over NacSO^ filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO-. petroleum ether: ethyl acetate - 10:1 to 0:1). Compound 2C (23 g, 17.21% yield) was obtained as a yellow oil,

[1496] ’H NMR (400 MHz, CHLOROFORM -d) 5 = 3.82 (s, 3H), 3.79 (s, 3H), 2.97 (d, J -13.4 Hz, 1H)

[1497] 3IP NMR (162 MHz, CHLOROFORM-d) 5 = -5.07 - -5.22 (m, IP)

[1498] TLC: Petroleum ether: Ethyl acetate - 0:1, Rf 0.31

[1499] 2. Preparation of compound 5A:

[1500] O > POM-I (4eq.), 4A MS / — O w

[1501] p" o' I -0ACN / ■<

[1502] Y

[1503]

[1504] 2C 5A

[1505] To a solution of compound 2C (11.5 g, 85.78 mmol) in ACN (500 mL) as added 4A MS (5 g, 85.78 mmol), iodomethyl 2,2-dimethylpropanoate (83.05 g, 343.10 mmol). The mixture was stirredat 82°C for 15 hr. TLC indicated compound 2C as consumed completely and two new;spots formed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue 'as purified by column chromatography (SiOr, petroleum ether: ethyl acetate = 10:1 to 0:1). Compound 5A (33 g, 57.89% yield) was obtained as a colorless.

[1506] ’H NMR (400 MHz, CHLOROFORM-d) 5 = 5.70 (d, J = 1.5 Hz, 2H). 5.66 (d, J = 0.8Hz, 2H), 3.06 (d, J - 14.3 Hz, 1H), 1.20 (s, 18H)

[1507] 31P NMR (162 MHz, CHLOROFORM-d) 5 = -10.31 (s, IP)

[1508] TLC: petroleum ether: ethyl acetate = 3:1. Rf = 0.38

[1509] 3. Preparation of compound 2:

[1510] MsCl, pyridine

[1511]

[1512] To a solution of compound 1 (50 g, 161.11 mmol) its DCM (800 mL; was added pyridine (82.84 g. 1.05 mol) and the mixture was cooled to 0°C. then MsCl (46.18 g. 403.14 mmol) was slowly added the mixture. The mixture was stirred at 0-20°C for 12 hr. LCMS showed compound 1 was consumed completely and desired mass was detected. The mixture was cooled to 0°C. the mixture of 100 ml, ice water and aqueous NaHCCh 100 mL was dropped to the mixture under N2. and stirred for 5 min. The organic layer wns separated and washed with saturated aqueous NaHCO? (300 ml*2) and with water. The combined organic phases were dried over MgSC>4 and the solvent was removed under reduced pressure. The crude product was used into the next step without further purification. Compound 2 (150 g, crude) was obtained as a yellow’ oil.

[1513] T-INMR (400 MHz, CHLOROFORM-d) 5 - 7.41 - 7.35 (m. 5H). 5.79 (d, J - 3.8 Hz, 1H). 4.88 (d. J = 12.0 Hz, lH).4.77 (d. J = 11.6 Hz, 1H). 4.68 - 4.63 (m, 1H). 4.57 (d. J = 11.6 Hz. 1H), 4.41 (d, J = 12.0 Hz, 1H), 4.32 (d. J = 11.0 Hz, 1H), 4.22 - 4.12 (m, 2H), 3.08 (s, 3H), 2.98 (s, 3H), 1.68 (s. 3H), 1.34 (s.3H)

[1514] LCMS (M+Na+): 489

[1515] 4. Preparation of compound 3:

[1516] 80% TFA

[1517]

[1518] To a solution of compound 2 (50 g, 107.18 mmol) was added TFA (250 mL) and ILO (62.5 mL). The mixture was stirred at 20°C for 12 hr. I., CMS showed compound 2 was consumed completely and desired mass was detected. The reaction mixture was concentrated under reduced pressure toremove TFA. The residue was diluted with NaHCOj (500 mL) and extracted with DCM (300 mL*2). The combined organic layers were washed with brine 500 mL, dried over Nap. SO.i, filtered and concentrated under reduced pressure to give a residue. The crude product was used into the next step without further purification. Compound 3 (137 g, crude) w as obtained as a yellow oil.

[1519] ’H NMR (400 MHz, CHLOROFORM-d) <5 = 8.64 (br d,, T = 4.6 Hz, 1H), 7.48 - 7.45 (m, 1H), 7.41 - 7.30 (m, 5H), 5.39 - 5.34 (m, 1H), 4.66 - 4.59 (m, 2H), 4.35 -4.33 (m, 1H), 4.31 - 4.20 (m, 1H), 4,19 - 4,14 (m, 1H). 4.11 (d, J = 5.0 Hz, 1H), 3.05 - 2.97 (m, 6H)

[1520] LCMS (M+Na+): 449.1

[1521] 5. Preparation of compound 4:

[1522] MsCX

[1523] AC2O

[1524] ^-°'yOAc

[1525] pyridine

[1526]

[1527] MsC^nO OAc

[1528] 3 4

[1529] To a solution of compound 3 (65 g, 152.42 mmol) in pyridine (600 mL) was added AC2O (57.57 g. 563,95 mmol). The mixture was stirred at 20°C for 12 hr. LCMS showed compound 3 was consumed completely and desired mass was detected. The reaction mixture was diluted with NallCOs (1000 mL) and extracted with EtOAc (500 mL*2). The combined organic layers were washed with brine 800 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, petroleum ether: ethyl acetate = 0:1 to 1:1). Compound 4 (155 g, crude) was obtained as a yellow oil,

[1530] !H NMR (400 MHz, CHLOROFORM-d) 5 = 7.42 - 7.33 (m, 5 H ) 6.18 (s. 1H), 5.38 (d, J = 4.8 Hz. 1H), 4.65 - 4.60 (m, 1H), 4.55 - 4.48 (m, 2H). 4.43 id. J - 4.8 Hz, 1H), 4.31 - 4.28 (m, 1H), 4.23 - 4.17 (m. 2H), 3.02 (d, J = 2.0 Hz, 6H), 2.16 (s, 3H), 2.11 (s, 3H)

[1531] LCMS (M+Na+): 533.1

[1532] TI C Petroleum ether: Ethyl acetate = 1:1. Rf = 0.5

[1533] 6. Preparation of compound 5:

[1534] uraciS, BSA. TMSOTf

[1535] ACN

[1536]

[1537] 5

[1538] To a solution of compound 4 (50 g, 97.94 mmol) and uracil (21 95 g, 195.87 mmol) in ACN (700 mL) was added BSA (69.73 g, 342.78 mmol) at 80 °C for 1 hr. Then TMSOTf (5442 g, 244.84 mmol) was added to the mixture. The mixture was stirred at 60°C for 12 hr. LCMS showed compound 4 was consumed completely and desired mass was detected. The reaction mixture as cooled to 0°C,and added to NallCCh (800 inL). The residue was extracted with EtOAc (500 mL*3). The combined orgastic layers were washed with brine 500 L, dried over Na. SO-, filtered asid concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 1:0 to 0:1). Compound 5 (78 g, 47.27% yield) and 78 g (crude) w as obtained as a while solid.

[1539] ’H NMR (400 MHz, DMSO-d6) 5 - 11.46 (br s. HI), 7.71 (d, J - 8.0 Hz, HI), 7.39 - 7.29 (m, 5H), 5.98 (d, J = 4.6 Hz, IH), 5.68 (d, J = 80 Hz, IH). 5.55 - 5.50 (tn, 1H), 4.65 - 453 (m. 3H), 4.45 -4.37 (m, 3H), 4.36 - 4.30 (m. 1H), 3.26 (s, 3H), 3.19 (s, 3H), 2.05 (s, 3H)

[1540] LCMS (M-t-Na+): 585.1, purity: 92.5%

[1541] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf = 0.5

[1542] 7. Preparation of compound 6:

[1543]

[1544] To a solution of compound 5 (41.5 g, 73 77 mmol) in dioxane (100 ml.,) and H2O (100 tnL) was added NaOH (2 M, 221.31 mL). The mixture was stirred at 20°C for 12 hr. LCMS showed compound 5 was consumed completely and one main peak with desired mass was detected. The reaction mixture was w ashed with 10% AcOH (300 mL). The residue was diluted with H2O (500 mL) and extracted with DCM (300 mL*3). The combined organic layers were washed with NaHCCL (200 ml.,) and brine (200 mL), dried over NazSO-r, filtered and concentrated under reduced pressure to give a residue. The crude product as triturated with EtOAc at 20°C for 5 min. The mixture was filtered and the cake was concentrated under reduced pressure to give product. Compound 6 (50 g, 79.85% yield) was obtained as a white solid,

[1545] ‘H NMR (400 MHz, DMSO-d6) 5 - 11.42 (br s. HI), 7.62 (d, J - 8.0 Hz. IH), 7.37 - 7.25 (m, 5H), 5.60 (d, J = 8.0 Hz. IH), 5.56 (s, 1H), 4.83 (d. J = 12.0 Hz. 1H), 4.63 (d, J = 3.0 Hz, 2H), 4.61 -4.56 (m, 2H), 4.03 - 3.97 (ro. 2H), 3.84 (d. J = 8.0 Hz, IH). 3.27 (s. 3H)

[1546] LCMS (M-i-H-t-): 425.0, purity: 92.4%

[1547] TLC: Petroleum ether: Ethyl acetate = 0:1 Rf = 0.4

[1548] 8. Preparation of compound 7:

[1549]

[1550] 6 7

[1551] To a solution of compound 6 (20 g. 47.12 mmol) in DMF (200 mL) was added NaNs (3.17 g„ 48.76 mmol). The mixture was stirred at 60°C for 12 hr. LCMS showed compound 6 was consumed completely and desired mass was detected. The reaction was quenched by H2O (TOO mL). and extracted with Ethyl acetate (200 mL*3). The combined organic was dried over Na? SO i. filtered and concentrated under reduced pressure to give a residue. The crude product was triturated with petroleum ether: ethyl acetate = 10: 1. at 20°C for 5 min. Then the mixture was filtered, the cake was washed with Petroleum ether, concentrated under reduced pressure to give product. Compound 7 (15.5 g, 88.58% yield) was obtained as a white solid.

[1552] 'H NMR (400 MHz, DMSO-d6) 8 = 11.42 (s. 1H), 7.60 (d, J - 8.2 Hz, 1H). 7.37 - 7.27 (m, 5H), 5.65 (d. J - 8.2 Hz, 1H). 5 54 (s, 1H), 4.66 - 4.57 (m, 2H). 4.54 (s, 1H), 4.01 (d, J - 14.0 Hz, 1H), 3.94 - 3.89 (m, 2H), 3.87 - 3.77 (m, 2H)

[1553] LCMS (M+H+): 372.0

[1554] , OPOM

[1555] , PS

[1556] OPOM 5A

[1557] sodium ascorbate CuSO4r5H2O

[1558] THF / H2O(1:1)

[1559]

[1560] To a solution of compound 7 (10 g, 26.93 mmol) and compound 5A (10.80 g, 32.31 mmol) in THF (100 mL) and H2O (100 mL) was added copper; sulfate; pentahydrate (807 g. 32. 1 mmol) and sodium; (2R)-2-[(lS)-l,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-olate (6.40 g, 32.31 mmol). The mixture was stirred at 65°C for 6 hr. TLC indicated compound 7 was consumed completely and one new spot formed. The reaction mixture was concentrated under reduced pressure to remove ’THF. The residue was diluted with H2O (50 mL) and extracted with EtOAc (100 mL*2). The combined organic layers were dried over Na2SCh, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO-. petroleum ether: ethyl acetate = 1:0 to 0:1). Compound 8 (18 g, 94.72% yield) was obtained as a white solid.

[1561] ’H NMR (400 MHz, DMSO-d6) 8 =11.39 (s, 1H), 8.78 (s, 1H), 7.36 (d, J = 4.2 Hz, 4H), 7.33 -7.26 (m, 2H), 5.73 (s, 2H), 5.69 (s, 2H), 5.52 (br d, J = 8.0 Hz, 1H), 5.47 (s, 1H), 5.28 (br d. J = 15.0Hz, 1H), 5.02 (br d, J = 15.0 Hz, 1H). 4.66 (s, 2H), 4.57 (s, 1H), 4.11 (d, J = 8.0 Hz, 1 H), 3.58 (br d, J = 8.2 Hz, 2H), 1.10 (s, 18H)

[1562] 3,P NMR (162 MHz, DMSO-d6) 8 = 6.73 (s, IP)

[1563] LCMS (M+H+); 706.3, purity: 94.7%

[1564] TLC: Petroleum ether: Ethyl acetate = 0:1, Rf = 0.3

[1565] 10. Preparation of WV-NU-349:

[1566] Pd / C (15Psi), Hz

[1567] AcOH

[1568]

[1569] 8 WV-NU-349

[1570] 2MNA-5'-PO(POM)2-Triazolyi Phosphonate Uridine (WV-NU-349) To a solution of compound 8 (9 g, 12.75 mmol) in AcOH (100 mL) was added Pd / C (1.36 g, 1.28 mmol, 10% purity). The mixture was stirred at 30°C for 12 hr under H2 (15 Psi). LCMS showed compound 8 was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 0:1, ethyl acetate: methanol = 1:0 to 10: 1). Compound WV-NU-349 (7.49 g, 4993% yield) was obtained as a white solid.

[1571] ’H NMR (400 MHz, DMSO-d6) 5 - 11.38 (s, 1H), 8.78 (s. 1H), 7.29 (d, J = 8.0 Hz, 1H), 6.11 (br d, J = 4.0 Hz, 1H), 5.72 (br d. J = 13.8 Hz. 3H), 5.51 (d, J = 8.0 Hz, 1H). 5.39 (s, 1H), 5.22 (d. J = 15.0 Hz, 1H), 4.96 (d, J = 15.0 Hz, 1H). 4.25 (s, 1H). 4.07 (d. J = 8.0 Hz, 1H). 3.86 (d, J = 3.6 Hz, 1H), 3.50 (d. J - 8.0 Hz. 1H). 1.11 (s, 18H)

[1572] 3,P NMR (162 MHz. DMSO-dg) 8 = 6.75 (s. IP)

[1573] LCMS (M-H+): 616.2, purity: 98.88%

[1574] TLC: Ethyl acetate: Methanol = 10:1, Rf = 0.3

[1575] EXAMPLE 9. Synthesis of WV-NU-350

[1576]

[1577] OH OMe

[1578] WV-NU-350

[1579] 2'-OMe-5'-PS(OEt)2-Triazolyl Thlophosphonate Uridine (WV-NU-350) 0,0-diethy 1 (1 -(((2R,3R,4R.5R)-5 -(2.4-dioxo-3,4-dihy dropyrimidin- 1 (2H)-y l)-3 -hydroxy -4-mcthoxytctrahydrofuran-2-yl)mcthyl)-lH-l,2,3-triazoI-4-yl)phosphonothioatc General Scheme:

[1580] EtO MgBr sulfur EtO,, $ P-CI P-, EK) THF, 0-15 °C, 2 h DOM, 2 h Etd 1A 2A 3A

[1581]

[1582] WV-NU-350

[1583] Experimental Procedure:

[1584] 1. Preparation of compound 2A:

[1585] =

[1586] P-CI - »•, P.

[1587] EtO THF, 0-15 °C, 2 hEt0'

[1588]

[1589] 1A 2A

[1590] To a solution of compound 1A (9.3 g, 59.41 mmol) in THF (100 mL) was added bromo (ethynyl) magnesium (0.5 M, 120.00 L) al 0°C under N2. The resulting mixture was stirred at 0-15°C for 2 hr, TL. C indicated compound 1 A was consumed completely and two new spots formed. The reaction was clean according to TLC. The mixture was quenched by addition sat. NILC1 (aq. 150 mL) at 0 °C, then diluted with H2O (100 mL) and extracted with DCM (200 mL*3). The combined organic layers were dried over Na2SO4, filtered to get the crude. Without purification. Compound 2A (26 g, crude) was obtained as a brown oil.

[1591] TLC: petroleum ether: ethyl acetate = 1:1. Rf = 0,95

[1592] 2. Preparation of compound 5 / 1OEt sulfurEtQ, S

[1593] p - Px.

[1594] EtO'' DCM, 2 h EtQ wy

[1595]

[1596] 2A 3A

[1597] To a solution of compound 2A (13 g, 88.97 mmol) in DCM (150 mL) was added S (5.40 g, 168.41 mmol). The mixture was stirred at 15°C for 2 hr. TLC indicated compound 2A was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction mixture was cooled to 0c,C and quenched by addition H2O 80 mL, and then diluted with H? O 50 mL and extracted with DCM 150 mL (50mL*3). The combined organic layers were dried over Na2SCL. filtered and concentrated under reduced pressure to give a residue. 'The residue was purified by column chromatography (SiO-. petroleum ether: ethyl acetate = 1:0 to 0:1). Compound 3 A (14 g.44.16% yield) was obtained as a yellow oil.

[1598] T-I NMR (400 MHz, CHLOROFORM-d) 5 - 4.16 (qd. J - 7.2. 10.3 Hz, 4H), 3.12 (d, J = 12.4 Hz. 1H). 1.34 (t. J - 7.2 Hz, 6H)

[1599] 31P NMR (162 MHz. CHLOROFORM-d) <5 = 51.30 (s. 1 P)

[1600] LCMS (M+H+): 179.1

[1601] TLC: Petroleum ether: Ethyl acetate = 5:1. Rf = 0.7

[1602] 5. Preparation of compound 2:

[1603] p q

[1604] L imidazole li

[1605] f ri r

[1606] HO. % - »- k % A.

[1607] LQJ ° THF, 0-25°C '°

[1608] OH OMe HO OMe

[1609]

[1610] 1 2

[1611] To a solution of compound 1 (30 g. 116.18 mmol) in THF (420 L) was added imidazole (20.56 g, 302.06 mmol), I2(47.18 g. 185.88 mmol) and PPh2(48.75 g. 185.88 mmol) at 0°C. The mixture was stirred at 25°C for 6 hr. TLC indicated compound 1 w-as consumed completely and two new spots formed The reaction was clean according to TLC. The reaction was quenched by710% aqueous sodium thiosulfate solution (800 ml). After removing the solvent and volatiles under reduced pressure, the residue was extracted into Dichloromethane (200 mL*3) and washed with saturated aqueous NaHCOs solution. The organic layer was separated, dried over anhydrous Na2SCL, filtered and concentrated. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 0: 1 to dichloromethane: methanol = 1:0 to 3:1). The crude product was purified by recrystallization from ethyl acetate (100 mL), dichloromethane (300 mL) and methanol (50 mL) at 15 °C. Compound 2 (200 g, 93.55% yield) was obtained as a purple solid.

[1612] Tl NMR (400 MHz, DMSO-d6) 5 = 11.43 (s, 1H), 7.68 (d, J = 8.2 Hz, 1H), 5.87...

Claims

1. CLAIMSWhat is claimed is:

1. A double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: the guide strand is complementary' or substantially complementary to a target RNA sequence: the guide strand comprises a seed region at its 5 ’-end region that is capable of mediating the initial recognition of the target RNA sequence; andthe passenger strand comprises a PN linkage or a backbone phosphoryl guanidine chiral center between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide.

2. The dsRNAi agent of claim 1, wherein the guide strand comprises a PN linkage or a backbone phosphoryl guanidine chiral center in the seed region.

3. The dsRNAi agent of claim 1 or 2. wherein the PN linkage or the backbone phosphoryl guanidine chiral center between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide of the passenger strand is in the A’p configuration.

4. The dsRNAi agent of any of claims 1-3. wherein the passenger strand further comprises a PN linkage or a backbone phosphory l guanidine chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide.

5. The dsRNAi agent of claim 4, wherein the PN linkage or the backbone phosphoryl guanidine chiral center between the 3‘ terminal (N) nucleotide and the penultimate (N-l) nucleotide of the passenger strand is in the Rp configuration.

6. The dsRNAi agent of any of claims 1-5, wherein the PN linkage or the backbone phosphoryl —N?I N P O'~N Oguanidine chiral center comprises the structure of 'f(nOOl).

7. The dsRNAi agent of any of claims 1-6, wherein the PN linkage or backbone phosphoryl guanidine chiral center in the seed region is between the +3 nucleotide and the immediately downstream (+4) nucleotide, relati ve to the 5’ terminal nucleotide of the guide strand.

8. The dsRNAi agent of claim 7, wherein the PN linkage or the backbone phosphoryl guanidine chiral center in the seed region of the guide strand is in the. Sb configuration.

9. The dsRNAi agent of claim 8, wherein the nucleoside 3’ to the PN linkage or the backbonephosphory l guanidine chiral center in the seed region comprises a 2’-F ribose modification.

10. The dsRNAi agent of any of claims 1-9, wherein the guide strand further comprises a PN linkage or a backbone phosphoryl guanidine chiral center between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5’ terminal nucleotide of the guide strand.

11. The dsRNAi agent of claim 10, wherein the nucleoside 3’ to the PN linkage or the backbone phosphoryl guanidine chiral center between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5' terminal nucleotide of the guide strand, comprises a 2'-F ribose modification.

12. The dsRNAi agent of claim 11. wherein the PN linkage or the backbone phosphoryl guanidine chiral center between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5’ terminal nucleotide of the guide strand, is in the Rp configuration.

13. The dsRNAi agent of any of claims 1-12. wherein the guide strand comprises a 5’ phosphate modification.

14. The dsRNAi agent of claim 13. wherein the 5’ phosphate modification is a 5’ phosphate mimic modification.

15. The dsRNAi agent of claim 14, wherein the 5’ phosphate mimic modification is5, wherein:the base is N3U, or is selected from A. C, G, T. U, abasic. and modified nucleobases other than N3U; andR1is selected from H, OH. O-alkyl, O-methyl (O-Me), F, O-methoxy ethyl (MOE), and 2’-0.4’C-methylene-bridged or locked nucleic acid (2’,4’-BNA or LNA).

16. The dsRNAi agent of claim 15, wherein R1is O-methyl (O-Me).

17. The dsRNAi agent of any of claims 1-16, wherein the guide strand further comprises a PN linkage or a backbone phosphoryl guanidine chiral center between the 3’ terminal < TN) nucleotide and the penultimate (N-1) nucleotide, and / or a PN linkage or a backbone phosphoryl guanidine chiral center between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide.

18. The dsRNAi agent of claim 17. wherein the PN linkage or the backbone phosphoryl guanidine chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide, and / or the PN linkage or the backbone phosphoryl guanidine chiral center between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, is / are in the Rp configuration.

19. The dsRNAi agent of claim 18, wherein the guide strand does not comprise a PN linkage or a backbone phosphory l guanidine chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide.

20. The dsRNAi agent of claim 19, wherein the guide strand comprises a phosphorothioate (PS) chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide.

21. The dsRNAi agent of claim 20, wherein the phosphorothioate (PS) chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide of the guide strand is in the Sp configuration.

22. The dsRNAi agent of any of claims 1-21, wherein the guide strand comprises a phosphoryl guanidine cap at its 5’-end (5’-end PN cap).

23. The dsRNAi agent of claim 22, wherein the 5’-end PN cap is selected fromthe base is N3U, or is selected from A. C, G, T, U, abasic. and modified nucleobases other than N3U; andR1is selected from H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxy ethyl (MOE), and 2’- 0, 4 Y -methylene-bridged or locked nucleic acid (2’,4’-BNA or LNA).

24. The dsRNAi agent of claim 23, wherein R‘ is O-methyl (O-Me).

25. The dsRNAi agent of any of claims 1-24, wherein the guide strand comprises a natural phosphate linkage (PO) between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and / or a natural phosphate linkage (PO) between the +2 nucleotide and the +3 nucleotide, relative to the 5' terminal nucleotide.

26. A double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: the guide strand is complementary or substantially complementary' to a target RNA sequence; the guide strand comprises a seed region at its 5‘-end region that is capable of mediating the initial recognition of the target RNA sequence;andthe guide strand comprises a phosphoryl guanidine cap at its 5’-end (5’-end PN cap).

27. The dsRNAi of claim 26, wherein the guide strand comprises a backbone phosphoryl guanidine chiral center in the seed region.

28. The dsRNAi agent of claims 26 or 27, wherein the 5 ’-end PN cap is selected fromthe base is N3U, or is selected from A, C, G, T, U, abasic, and modified nucleobases other than N3U; andR1is selected from H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2’- 0,4’C-methylene-bridged or locked nucleic acid (2',4‘-BNA or LNA).

29. The dsRNAi agent of claim 28, wherein R1is O-methyl (O-Me).

30. A double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: the guide strand is complementary or substantially complementary to a target RNA sequence; the guide strand comprises a seed region at its 5 ’-end region that is capable of mediating the initial recognition of the target RNA sequence;the guide strand comprises a PN linkage or a backbone phosphoryl guanidine chiral center in the seed region; andthe guide strand comprises a natural phosphate linkage (PO) between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and / or a natural phosphate linkage (PO) between the +2 nucleotide and the +3 nucleotide, relative to the 5’ terminal nucleotide.

31. The dsRNAi agent of claim 30. wherein the guide strand comprises a 5’ phosphatemodification.

32. The dsRNAi agent of claim 31. wherein the 5’ phosphate modification is a 5’ phosphate mimic modification.

33. The dsRNAi agent of claim 32, wherein the 5’ phosphate mimic modification is’. wherein:the base is N3U, or is selected from A. C, G, T. U, abasic. and modified nucleobases other than N3U; andR1is selected from H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxy ethyl (MOE), and 2’-0.4’C-methylene-bridged or locked nucleic acid (2’,4’-BNA or LNA).

34. The dsRNAi agent of claim 33, wherein R‘ is O-methyl (O-Me).

35. A double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein: the guide strand is complementary or substantially complementary to a target RNA sequence; the guide strand comprises a seed region at its 5 ’-end region that is capable of mediating the initial recognition of the target RNA sequence; andthe guide strand comprises a PN linkage or a backbone phosphoryl guanidine chiral center in the seed region,wherein the nucleoside 3 ’ to the PN linkage or the backbone phosphoryl guanidine chiral center in the seed region comprises a 2’-F ribose modification or a 2’-OMe ribose modification.

36. The dsRNAi agent of claim 35, wherein the PN linkage or the backbone phosphoryl guanidine chiral center in the seed region is between the +3 nucleotide and the immediately downstream (+4) nucleotide, relative to the 5’ terminal nucleotide of the guide strand.

37. The dsRNAi agent of claim 35 or 36, wherein the PN linkage or the backbone phosphoryl guanidine chiral center in the seed region of the guide strand is in the Sp configuration.

38. The dsRNAi agent of any of claims 35-37, wherein the guide strand further comprises a PN linkage or a backbone phosphoryl guanidine chiral center between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5’ terminal nucleotide of the guide strand, wherein the nucleoside 3 ’ to the PN linkage or the backbone phosphoryl guanidine chiral center comprises a 2’-F ribose modification.

39. The dsRNAi agent of claim 38. wherein the PN linkage or the backbone phosphoryl guanidine chiral center between the +10 nucleotide and the immediately downstream (+11) nucleotide, relative to the 5’ terminal nucleotide of the guide strand, is in the Rp configuration.

40. The dsRNAi agent of any of claims 35-39, wherein the guide strand further comprises a PN linkage or a backbone phosphoryl guanidine chiral center between the 3' terminal (N) nucleotide and the penultimate (N-l ) nucleotide, and / or a PN linkage or a backbone phosphoryl guanidine chiral center between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide.

41. The dsRNAi agent of claim 40, wherein the PN linkage or the backbone phosphoryl guanidine chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide, and / or the PN linkage or the backbone phosphory l guanidine chiral center between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide, is / are in the Rp configuration.

42. The dsRNAi agent of claim 41, wherein the guide strand does not comprise a PN linkage or a backbone phosphoryl guanidine chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide43. The dsRNAi agent of claim 42. wherein the guide strand does not comprise a PN linkage or a backbone phosphoryl guanidine chiral center between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide.

44. The dsRNAi agent of claim 43, wherein the guide strand comprises a phosphorothioatc (PS) chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide.

45. The dsRNAi agent of claim 44, wherein the phosphorothioatc (PS) chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide of the guide strand is in the Sp configuration.

46. The dsRNAi agent of any of claims 35-45, wherein the guide strand comprises a 5’ phosphate modification.

47. The dsRNAi agent of claim 46, wherein the 5 ’ phosphate modification is a 5’ phosphate mimic modification.

48. The dsRNAi agent of claim 47, wherein the 5’ phosphate mimic modification is’. wherein:the base is N3U, or is selected from A. C, G, T, U, abasic. and modified nucleobases other than N3U; andR1is selected from H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2’-0,4’C-methylene-bridged or locked nucleic acid (2’.4’-BNA or LNA).

49. The dsRNAi agent of claim 48, wherein R1is O-methyl (O-Me).

50. The dsRNAi agent of any of claims 35-49, wherein the nucleoside 3’ to the backbone phosphoryl guanidine chiral center in the seed region comprises a 2'-OMe ribose modification, and wherein the nucleoside 5’ to the backbone phosphoryl guanidine chiral center in the seed region comprises a 2'-F ribose modification.

51. The dsRNAi agent of any of claims 35-50, wherein the backbone linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide of the guide strand is an unmodified phosphodi ester (PO) linkage.

50. A double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein:the guide strand is complementary or substantially complementary to a target RNA sequence; the guide strand comprises a seed region at its 5 ’-end region that is capable of mediating the initial recognition of the target RNA sequence; andthe guide strand comprises a contiguous stretch of five backbone phosphoro thioate (PS) chiral centers between the +13 nucleotide and the +18 nucleotide, relative to the 5’ terminal nucleotide, and / or a contiguous stretch of four backbone phosphorothioate (PS) chiral centers between the +19 nucleotide and the 3’ terminal (N) nucleotide.

51. The dsRNAi agent of claim 50, wherein the guide strand comprises a PN linkage or a backbone phosphoryl guanidine chiral center in the seed region.

52. The dsRNAi agent of claim 50 or 51, wherein the seed region comprises nucleotides at positions 2- 6. at positions 2-7, or at positions 2-8. relative to the 5 ’-end of the guide strand.

53. The dsRNAi agent of any of claims 50-52. wherein the PN linkage or the backbone phosphorylguanidine chiral center in the seed region is between the +3 nucleotide and the immediately downstream (+4) nucleotide, relative to the 5’ terminal nucleotide of the guide strand.

54. The dsRNAi agent of any of claims 50-53, wherein the PN linkage or the backbone phosphoryl guanidine chiral center in the seed region of the guide strand is in the Sp configuration.

55. The dsRNAi agent of any of claims 50-54, wherein the PN linkage or the backbone phosphoryl Ir-N?I )c=N— P=O6 £guanidine chiral center comprises the structureof1(nOOl).

56. The dsRNAi agent of any of claims 50-55, wherein the contiguous stretch of five backbone phosphorothioate (PS) chiral centers between the + 13 nucleotide and the -M8 nucleotide, relative to the 5’ terminal nucleotide, and / or the contiguous stretch of four backbone phosphorothioate (PS) chiral centers between the +19 nucleotide and the 3’ terminal (N) nucleotide, of the guide strand, are in the Sp configuration.

57. The dsRNAi agent of any of claims 50-56, wherein the passenger strand comprises a backbone phosphorothioate (PS) chiral center between the 5’ terminal (+1) nucleotide and the immediately downstream ( +2) nucleotide, and / or a backbone phosphorothioate (PS) chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l) nucleotide.

58. The dsRNAi agent of claim 57, wherein the backbone phosphorothioate (PS) chiral center between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and''or the backbone phosphorothioate (PS) chiral center between the 3’ terminal (N) nucleotide and the penultimate (N-l), of the passenger strand, is / are in the Sp configuration.

59. The dsRNAi agent of any of claims 50-58, wherein the guide strand further comprises a PN linkage or a backbone phosphoryl guanidine chiral center between the +7 nucleotide and the +8 nucleotide, and / or a PN linkage or a backbone phosphoryl guanidine chiral center between the +18 nucleotide and the +19 nucleotide thereof.

60. The dsRNAi agent of claim 50-59, wherein the PN linkage or the backbone phosphoryl guanidine chiral center between the +7 nucleotide and the +8 nucleotide, and / or the PN linkage or the backbone phosphoryl guanidine chiral center between the +18 nucleotide and the +19 nucleotide, of the guide strand, is / are in the Sp configuration.

61. The dsRNAi agent of claim 59 or 60, wherein the PN linkage or the backbone phosphory l guanidine chiral center between the +7 nucleotide and the +8 nucleotide, and / or the PN linkage or the backbone phosphory l guanidine chiral center between the +18 nucleotide and the +19 nucleotidethereof, comprises the structureof ' (nOOl).

62. The dsRNAi agent of any of claims 50-61, wherein the passenger strand comprises a PN linkage or a backbone phosphory l guanidine chiral center between the +7 nucleotide and the immediately downstream (+8) nucleotide, and / or a PN linkage or a backbone phosphory l guanidine chiral center between the +15 nucleotide and the immediately downstream (+16) nucleotide.

63. The dsRNAi agent of claim 62, wherein the PN linkage or the backbone phosphoryl guanidine chiral center between the +7 nucleotide and the immediately downstream (+8) nucleotide, and / or the PN linkage or the backbone phosphoryl guanidine chiral center between the +15 nucleotide and the immediately downstream (+16) nucleotide, of the passenger strand, is / are in the Rp configuration.

64. The dsRNAi agent of claim 62 or 63, w herein the PN linkage or the backbone phosphory l guanidine I I iIP— o“N 6chiral center comprises the structure of ' * (nOOl).

65. A double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein:the guide strand is complementary or substantially complementary’ to a target RN A sequence; the guide strand comprises a seed region at its 5 ’ -end region that is capable of mediating the initial recognition of the target RNA sequence; andthe guide strand comprises one or more N-3-uridine base modifications (N3U) having the structure0fA N A D66. The dsRNAi agent of claim 65, wherein the seed region comprises nucleotides at positions 2-6, at positions 2-7, or at positions 2-8, relative to the 5 ‘-end of the guide strand.

67. The dsRNAi agent of claim 65 or 66, wherein the guide strand comprises a PN linkage or abackbone phosphory l guanidine chiral center.

68. The dsRNAi agent of claim 67, wherein the PN linkage or the backbone phosphoryl guanidine chiral center is in the seed region.

69. The dsRNAi agent of claim 67 and 68, wherein the PN linkage or the backbone phosphory l guanidine chiral center is in the Sp configuration.

70. The dsRNAi agent of any of claims 65-69. wherein the guide strand comprises a contiguous stretch of four backbone phosphoro thioate (PS) chiral centers between the -t-19 nucleotide and the 3’ terminal (N) nucleotide.

71. The dsRNAi agent of arty of claims 65-70, wherein the guide strand comprises an N3U base modification at the 5’ terminal (+1) nucleotide.

72. The dsRNAi agent of any of claims 65-70, wherein the guide strand comprises an N3U base modification at the 3’ terminal nucleotide, the penultimate (N-l) nucleotide, or at both the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

73. The dsRNAi agent of claim 72, wherein the guide strand comprises an N3U base modification at the penultimate (N-l) nucleotide.

74. The dsRNAi agent of any of claims 65-73, further comprising a 5’ phosphate modification.

75. The dsRNAi agent of claim 74. wherein the 5’ phosphate modification is a 5’ phosphate mimic modification.

76. The dsRNAi agent of claim 75, wherein the 5' phosphate mimic modification is selected from o~O~P=Oo R1-o-p=o, R2Base Base<s7O R1wherein:the base is N3U, or is selected from A, C, G, T, U, abasic, and modified nucleobases other than N3U;R1is selected from H, OH, O-alkyl, O-methyl (O-Me), F, O-methoxyethyl (MOE), and 2’- 0,4’C-methylene-bridged or locked nucleic acid (2’,4‘-BNA or LNA); andR is selected from alkyl, methyl, ethyl, isopropyl, propyl, cyclohexyl, benzyl, phenyl, tolyl, xylyl, aryl, and arene group.

77. The dsRNAi agent of claim 76, wherein the 5’ phosphate mimic modification is78. The dsRNM agent of claim 76 or 77, wherein R1is O-Me.

79. The dsRNAi agent of claim 76, wherein R2inoris methyl.

80. The dsRNAi agent of any of claims 65-79, wherein the guide strand further comprises a backbone phosphorothioate (PS) chiral center in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and a backbone PS chiral center in the < S'p configuration between the +2 nucleotide and immediately downstream (+3)nucleotide.

81. The dsRNAi agent of any of claims 65-79, wherein the guide strand further comprises a backbone phosphorothioate (PS) chiral center in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and wherein the backbone linkage between the +2 nucleotide and the immediately downstream (+3) nucleotide of the guide strand is an unmodified phosphodiester (PO) linkage,82. A double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein the guide strand is complementary or substantially complementary to a target RNA sequence; wherein the guide strand comprises a 5’ phosphate modification; and wherein the guide strand further comprises a backbone phosphorothioate (PS) chiral center in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and a backbone PS chiral center in the Sp or ftp configuration between the +2 nucleotide and immediately downstream (+3) nucleotide.

83. A double-stranded RNAi (dsRNAi) agent capable of directing target-specific RNA interference, the dsRNAi agent comprising a guide strand and a passenger strand, wherein the guide strand is complementary’ or substantially complementary to a target RNA sequence; wherein the guide strand comprises a 5’ phosphate modification; wherein the guide strand further comprises a backbone phosphorothioate (PS) chiral center in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream (+2) nucleotide; and wherein the backbone linkage between the + 2 nucleotide and the immediately downstream (+3) nucleotide of the guide strand is an unmodified phosphodiester (PO) linkage.

84. The dsRNAi agent of claim 82, wherein the backbone PS chiral center between the +2 nucleotide and immediately downstream (+3) nucleotide is in the Sp configuration.

85. The dsRNAi agent of claim 82, -wherein the backbone PS chiral center between the +2 nucleotide and immediately downstream (+3) nucleotide is in the ftp configuration86. The dsRNAi agent of claims 82-85, wherein the 5' phosphate modification is a 5’ phosphate mimic modification.

87. The dsRNAi agent of claim 86. wherein the 5’ phosphate mimic modification is selected from:the base is N3U, or is selected from A. C, G, T. U, abasic. and modified nucleobases other thanN3U;R1is selected from H, OH, O-alkyl, O-raethyl (O-Me), F, O-methoxyethyl (MOE), and 2‘- O,4’C-methylene-bridged or locked nucleic acid (2’,4‘-BNA or LNA); andR2is selected from alkyl, methyl, ethyl, isopropyl, propyl, cyclohex 1, benzyl, phenyl, tolyl, xylyl, aryl, and arene group.

88. The dsRNAi agent of claim 87. wherein the 5’ phosphate mimic modification is189. The dsRNAi agent of claim 87 or 88, wherein the R' is LNA bridge to the 4' position.

90. The dsR Ai agent of claim 87 or 88, wherein the R1is MOE.

91. The dsRNAi agent of claim 87 or 88. wherein the R1is F.

92. The dsRNAi agent of claim 87. wherein R2oris methyl. The dsRNAi agent of claim 87, wherein the 5" phosphate mimic modification is, wherein the base is U or abasic, R1is H or O-alkyl.

94. The dsRNAi agent of claim 93. wherein R1is O-C1.5 alkyl.

95. The dsRNAi agent of claims 93-94, wherein R6is H.

96. The dsRNAi agent of claim 87, wherein the 5‘ phosphate mimic modification is, wherein R1is O-Me and R6is H.

98. The dsRNAi agent of claim 87, wherein the 5’ phosphate mimic modification isY"NO=P— O. Baseo~wherein the base is abasic and R!is H.

99. The dsRNAi agent of claim 87, wherein the 5’ phosphate mimic modification is O N^NR6O -S N Base0VYO R1wherein the R1is O-Me and R6is II.

100. The dsRNAi agent of any of the preceding claims, wherein the guide strand further comprises a PN linkage or a backbone phosphoryl guanidine chiral center in the Sp configuration between the +3 nucleotide and the immediately downstream (+4) nucleotide,101. The dsRNAi agent of any of the preceding claims, wherein the guide strand further comprises backbone PS chiral centers in 5p configuration between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide and between the penultimate (N-l) nucleotide and the immediately upstream (N-2) nucleotide.

102. The dsRNAi agent of any of the preceding claims, wherein the guide strand comprises an Rp.Sp, or stereorandom non-negatively charged internucleotidic linkage between the tenth (+10) and eleventh (+11) nucleotides, relative to the 5' terminal nucleotide.

103. The dsRNAi agent of claim 102, wherein the guide strand further comprises a PN linkage or a backbone phosphoryl guanidine chiral center between the +10 and the +11 nucleotides in the Rp configuration.

104. The dsRNAi agent of any of the preceding claims, wherein the guide strand comprises one or more Rp, Sp. or stereorandom non-negatively charged internucleotidic linkages 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- i ) nucleotide of the guide strand, where N is the 3‘ terminal nucleotide.

105. The dsRNAi agent of any of the preceding claims, wherein the guide strand comprises a 2’ modification, of the 3’ nucleotide of a nucleotide pair linked by an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage.

106. 'The dsRNAi agent of any of the preceding claims, wherein the guide strand comprises an Rp, Sp, or stereorandom non-negatively charged internucleotidic linkage between the seventh (+7) and eighth (+8) nucleotides, relative to the 5’ terminal nucleotide.

107. The dsRNAi agent of any of the preceding claims, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration where linkages occur between any two adjacent nucleotides between the penultimate 3’ nucleotide of the guide strand, where N is the 3’ terminal nucleotide, and the upstream N-10 nucleotide.

108. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages occur upstream, i.e., in the 5’ direction, relative to the central nucleotide of the passenger strand.

109. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises one or more Rp, Sp, or stereorandom non-negatively charged internucleotidic linkages occur downstream, i.e., in the 3' direction, relative to the central nucleotide of the passenger strand.

110. The dsRNAi agent of any of the precedin g claims, wherein the passenger strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkages occur upstream, i.e., in the 5’ direction, relative to the central nucleotide of thepassenger strand.

111. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration internucleotidic linkage internucleotidic linkages occur downstream, i.e., in the 3’ direction, relative to the central nucleotide of the passenger strand.1 12. The dsR Ai agent of any of the preceding claims, wherein the passenger strand in combination with one or more of the aforementioned guide strands, comprises one or more modified sugars between the 5’ terminal (+1) nucleotide and the penultimate (IN- 1) nucleotide.

113. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises one or more of:a. 0-n Rp, Sp. or stereorandom non-negatively charged internucleotidic linkages, where n is about 1 to 49;b. one or more backbone chiral centers in Rp or Sp configuration:c. one or more backbone phosphoryl guanidine chiral centers in the Rp configuration between the +7 nucleotide and the immediately' downstream (+8) nucleotide, i.e., in the 3' direction;d. one or more backbone phosphor l guanidine chiral centers in the Rp configuration between the +15 nucleotide and the immediately downstream (+16) nucleotide, i.e., in the 3’ direction; and / ore. backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+1) nucleotide and the immediately downstream, i.e., in the 3' direction, (+2) nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

114. The dsRNAi agent of any of the preceding claims, wherein each strand of the dsRNAi agent independently has a length of about 15 to about 49 nucleotides.

115. The dsRNAi agent of any of the preceding claims, wherein the Rp, Sp. or stereorandom non- negatively charged backbone internucleotidic linkages have neutral charge.

116. The dsRNAi agent of claim 115, wherein the neutral backbone internucleotidic linkage is[CH2]nCH3„O>=N— P<'N OO>[CH2]mCH3wherein n is about 0 to 49 and m is about 0 to 49.

117. The dsRNAi agent of claim 116, wherein the guide strand comprises a linkage having thefollowing structurebetween the third (+3) and fourth (+4) nucleotides of the guide strand, between the tenth (+10) and eleventh (+11) nucleotides of the guide strand, or both.

118. The dsRNAi agent of claim 116. wherein the guide strand comprises a linkage having the following structurebetween the third (+3) and fourth (+4) nucleotides of the guide strand, between the seventh (+7) and eighth (+8) nucleotides of the guide strand, between the tenth (+10) and eleventh (+11) nucleotides of the guide strand, between the eighteenth (+18) and nineteenth ( H 9) nucleotides of the guide strand, or combinations thereof.

119. The dsRNAi agent of claim 116, wherein the passenger strand comprises a linkage having the [CH2]nCH3following structure1 2Jm 3, where n is about 0 to 49 and m is about 0 to 49. at 5’ to the central nucleotide of the passenger strand, 3 ’ to the central nucleotide of the passenger strand, or both.120 The dsRNAi agent of claim 116, w’herein the passenger strand comprises a linkage having the;[CH2]nCH3■Np..Ofollowing structure[CH2]mCH3wherein n is about 11 to 49 and m is 0.

121. The dsRNAi agent of claim 120, wherein n is 11 or 15.

122. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises:i. a non-negatively charged backbone internucleotidic linkage in the Rp configuration between the +7 nucleotide and the +8 nucleotide;ii. a non-negatively charged backbone internucleotidic linkage in the Rp configuration between the +15 nucleotide and the +16 nucleotide; and / oriii. backbone phosphorothioate chiral centers in the Sp configuration between the 5’ terminal (+1) nucleotide and the +2 nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

123. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises:in the Rp configuration between the +7 nucleotide and the +8 nucleotide;+°> PTOx / Nii.\!in the Rp configuration between the +15 nucleotide and the +16 nucleotide; andiii. backbone phosphorothioate chiral centers in the Sp configuration between the +1 nucleotide and the +2 nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

124. The dsRNAi agent of any of the preceding claims, wherein the passenger strand comprises:+°, / fx / P-0N / in the Rp configuration between the +7 nucleotide and the +8 nucleotide; andbackbone phosphorothioate chiral centers in the Sp configuration between the +1 nucleotide and the +2 nucleotide and between the 3’ terminal nucleotide and the penultimate (N-l) nucleotide.

125. A method for reducing level and / or activity of a transcript or a protein encoded thereby comprising administering to a cell expressing the transcript a dsRNAi agent of any of the preceding claims, wherein the guide strand of the dsRNAi agent comprises a target-binding sequence that is completely complementary to a target sequence in the transcript.

126. The method of claim 125, wherein the cell is an immune cell, a blood cell, a cardiac cell, a lung cell, an optic cell, a muscle cell, a liver cell, a kidney cell, a cell of the central nervous system, or a cell of the peripheral nervous system.

127. The method of claim 126, wherein the cell of the central nervous system is a brain cell.

128. The method of any of claims 125-127. wherein when the dsRNAi agent is contacted with a cell comprising transcripts of both the target allele and another allele of the same nucleic acid sequence, it shows suppression of transcripts of the particular allele at a level that is:a. greater than when the dsRNAi agent is absent;b greater than a level of suppression observed for another allele of the same nucleic acid sequence; orc. both greater than when the dsRNAi agent is absent, and greater than a level of suppression observed for another allele of the same nucleic acid sequence.d. of suppression observed for another allele of the same nucleic acid sequence.

129. An oligonucleotide, wherein the oligonucleotide has the structure of a guide strand described in any one of the preceding claims.

130. An oligonucleotide, wherein the oligonucleotide has the structure of a passenger strand described in any one of the preceding claims.

131. A method, comprising:assessing potency and / or durability of a first dsRNAi agent, wherein the PN linkage between the first nucleoside and the second nucleoside from the 5 ‘-end of the sense strand of the first dsRNAi agent is a PN linkage; andassessing potency and / or durability of a second dsRNAi agent, wherein the PN linkage between the first nucleoside and the second nucleoside from the 5 ’ -end of the sense strand of the second dsRNAi agent is not a PN linkage.

132. The method of claim 131, wherein the PN linkage between the first nucleoside and the second nucleoside from the 5’-end of the sense strand of the second dsRNAi agent is a phosphorothioateinternucleotidic linkage.

133. The agent, composition, or method of any one of the preceding claims, wherein diastereopurity of a chiral linkage phosphorus is about or at least about 90%.

134. The agent, composition, or method of any one of the preceding claims, wherein diastereopurity of a chiral linkage phosphorus is about or at least about 40%-60%.

135. The agent, composition, or method of any one of claims 1-133, wherein diastereopurity of each chiral linkage phosphorus is about or at least about 90%.

136. The agent, composition, or method of any one of claims 1-133, wherein diastereopurity’ of each chiral linkage phosphorus is about or at least about 95%.137, The agent, composition, or method of any one of claims 1 -136. wherein diastereopurity of the oligonucleotide is about or at least about (DS)nc, wherein DS is about 90%-100%, and nc is the number of chiral linkage phosphorus in the oligonucleotide.