RNAi sequence-independent modification formats, and stabilized forms thereof
Modification formats having modified nucleotides are provided for siRNA. Short interfering RNA having modification formats and modified nucleotides provided herein reduce off-target effects in RNA interference of endogenous genes. Further modification formatted siRNAs are demonstrated to be stabilized to nuclease-rich environments. Unexpectedly, increasing or maintaining strand bias, while necessary to maintain potency for endogenous RNA interference, is not sufficient for reducing off-target effects in cell biology assays.
1. A RNA, comprising:
a sense oligonucleotide having a length of 17 to 30 nucleotides and comprising:
sequence-independent modification:
5′ m p -N x -m-m-N q -n r 3′ wherein p is 2, x is 10; r is 0, 1, or 2; and q is an integer representing the length of the sense oligonucleotide minus (p+x+r+2);
and
an antisense oligonucleotide having 17 to 30 nucleotides, a region of continuous complementarity to the sense oligonucleotide of at least 12 nucleotides; the antisense oligonucleotide further having complementarity to at least a portion of a target mRNA;
wherein
each m is independently a 2′-modified nucleotide wherein the 2′ modification is OR
wherein R is alkyl, and where the alkyl portion is C1-C6;
each n is independently a deoxynucleotide, modified nucleotide, or ribonucleotide; and
each N is independently a nucleotide other than the 2′-modified nucleotide,
wherein the RNA does not have Format G:
Passenger (Sense) Strand Guide (Antisense) Strand
2. The RNA of claim 1 wherein
each n is an overhanging nucleotide.
3. The RNA of claim 2 wherein the 2′ modification is OR wherein R is methyl.
4. The RNA of claim 3 wherein r is 2.
5. The RNA of claim 1 wherein a 5′-end is further derivatized with a phosphate, C 1-12 -alkyl, C 1-12 -alkylamine, C 1-12 -alkenyl, C 1-12 -alkynyl, C 1-12 -cycloalkyl, C 1-12 -aralkyl, aryl, acyl, or silyl substituent.
6. The RNA of claim 1 wherein at least one internucleoside linkage is other than a phosphodiester internucleoside linkage.
7. The RNA of claim 1 wherein the RNA is further associated with a cell-targeting ligand.