Sirna sequence-independent modification formats for reducing off-target phenotypic effects in rnai, and stabilized forms thereof

A method of minimizing cleavage by a passenger strand in inhibition of expression of an exogenously provided target gene by RNA interference, comprising:

• contacting a cell containing the target gene with a chemically synthesized short interfering RNA in an amount sufficient to minimize cleavage by a passenger strand while maintaining potency of a guide strand, the short interfering RNA comprising:
• a passenger (sense) oligonucleotide having a length of 15 to 30 nucleotides, and comprising one of sequence-independent modification format (1), sequence-independent modification format (2), and sequence-independent modification format (3) as follows:
• (1) 5′ Np-m-m-Nx-m-m-Nq-nr 3′, wherein p is 0 or 1;
• x is 7, 8, 9, 10, or 11;
• r is 0, 1, or 2;
• and q is an integer representing the length of the passenger strand minus (p+x+r+4);
• (2) 5′ Np-m-m-Ny-m-Nz-m-Nq-nr 3′, wherein p is 0 or 1;
• y is 7, 8, or 9 and z is 1;
• or y is 7 and z is 2 or 3;
• r is 0, 1, or 2;
• and q is an integer representing the length of the passenger strand minus (p+y+z+r+4);
• (3) 5′m-N1-m-Nx-m-m-Nq-nr 3′, wherein x is 8, 9, or 10;
• r is 0, 1, or 2;
• and q is an integer representing the length of the passenger strand minus (x+r+5);
• wherein
• each m is independently a 2′-modified nucleotide;
• each n is independently a deoxynucleotide, modified nucleotide, or ribonucleotide;
• each N is independently an unmodified nucleotide;
• and
• a guide (antisense) oligonucleotide having 15 to 30 nucleotides, a region of continuous complementarity to the passenger (sense) oligonucleotide of at least 12 nucleotides;
• the guide strand further having complementarity to at least a portion of a target mRNA.

A short interfering RNA, comprising:

• a passenger (sense) oligonucleotide having a length of 17 to 30 nucleotides and comprising:
• one of sequence-independent modification format (4), format (5), and format (6):
• (4) 5′ mp-Nx-m-m-Nq-nr 3′ wherein when p is 0, x is 12;
• when p is 1, x is 11;
• when p is 2, x is 10;
• when p is 3, x is 9, when p is 4, x is 8;
• r is 0, 1, or 2;
• and q is an integer representing the length of the passenger strand minus (p+x+r+2);
• (5) 5′ m-m-Nx-m-Nq-nr 3′ wherein x is 11;
• r is 0, 1, or 2;
• and q is an integer representing the length of the passenger strand minus (x+r+3);
• (6) 5′ mp-Ny-m-Nz-m-Nq-nr 3′, wherein p is 3;
• y is 9 and z is 1;
• r is 0, 1, or 2;
• and q is an integer representing the length of the passenger strand minus (p+y+z+r+2);
• and
• a guide (antisense) oligonucleotide having 17 to 30 nucleotides, a region of continuous complementarity to the passenger (sense) oligonucleotide of at least 12 nucleotides;
• the guide strand further having complementarity to at least a portion of target mRNA;
• wherein
• each m is independently a bicyclonucleotide, a tricyclonucleotide, or a 2′-modified nucleotide;
• each n is independently a deoxynucleotide, modified nucleotide, or ribonucleotide and is an overhanging nucleotide;
• when m is a bicyclonucleotide, each N is independently a nucleotide other than a bicyclonucleotide,
• when m is a tricyclonucleotide, each N is independently a nucleotide other than a tricyclonucleotide, and
• when m is a 2′-modified nucleotide, each N is independently a nucleotide other than a 2′-modified nucleotide.