Single stranded extended dicer substrate agents and methods for the specific inhibition of gene expression

A method for reducing expression of a target gene in an animal, comprising:

• treating an animal with an isolated double stranded nucleic acid in an amount effective to reduce expression of a target gene in a cell of the animal, wherein said double stranded nucleic acid comprises a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, wherein each said 5′ terminus comprises a 5′ terminal nucleotide and each said 3′ terminus comprises a 3′ terminal nucleotide, wherein:
• said first strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1to 23 of said first strand comprise at least 8 ribonucleotides;
• said second strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of said first strand to form a duplex;
• wherein at least the 3′ terminal nucleotide of said second strand is unpaired with said first strand, and UP to 6 consecutive 3′ terminal nucleotides are unpaired with said first strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides;
• wherein the 5′ terminus of said second strand comprises from 10-30 consecutive nucleotides which are unpaired with said first strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang;
• wherein at least the first strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of said second strand when said first and second strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between said first and second strands;
• and
• said second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of said second strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell.

A method for reducing expression of a target gene in a cell, comprising:

• contacting a cell with an isolated multi-stranded nucleic acid in an amount effective to reduce expression of a target gene in a cell, wherein said multi-stranded nucleic acid comprises a first oligonucleotide strand having a 5′ terminus and a 3′ terminus, a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, and a third oligonucleotide strand having a 5′ terminus and a 3′ terminus, wherein each said 5′terminus comprises a 5′ terminal nucleotide and each said 3′ terminus comprises a 3′ terminal nucleotide, wherein:
• said first strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of said first strand comprise at least 8 ribonucleotides;
• said second strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of said first strand to form a duplex;
• said third strand is 10-30 nucleotide residues in length;
• wherein at least the 3′ terminal nucleotide of said second strand is unpaired with said first strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with said first strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides;
• wherein the 5′ terminus of said second strand comprises from 10-30 consecutive nucleotides which are unpaired with said first strand and wherein at least 10 consecutive nucleotides and at most 30 consecutive nucleotides of said third strand are sufficiently complementary to said second strand to hybridize with said second strand at said 10-30 consecutive nucleotides of said second strand which are unpaired with said first strand;
• wherein at least the first strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of said second strand when said first and second strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between said first and second strands;
• and
• said second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of said second strand length to reduce target gene expression when said multi-stranded nucleic acid is introduced into a mammalian cell.

A method for reducing expression of a target gene in an animal, comprising:

• treating an animal with an isolated multi-stranded nucleic acid in an amount effective to reduce expression of a target gene in a cell of the animal, wherein said multi-stranded nucleic acid comprises a first oligonucleotide strand having a 5′ terminus and a 3′ terminus, a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, and a third oligonucleotide strand having a 5′ terminus and a 3′ terminus, wherein each said 5′ terminus comprises a 5′ terminal nucleotide and each said 3′ terminus comprises a 3′ terminal nucleotide, wherein:
• said first strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of said first strand comprise at least 8 ribonucleotides;
• said second strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of said first strand to form a duplex;
• said third strand is 10-30 nucleotide residues in length;
• wherein at least the 3′ terminal nucleotide of said second strand is unpaired with said first strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with said first strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides;
• wherein the 5′ terminus of said second strand comprises from 10-30 consecutive nucleotides which are unpaired with said first strand and wherein at least 10 consecutive nucleotides and at most 30 consecutive nucleotides of said third strand are sufficiently complementary to said second strand to hybridize with said second strand at said 10-30 consecutive nucleotides of said second strand which are unpaired with said first strand;
• wherein at least the first strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of said second strand when said first and second strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between said first and second strands;
• and
• said second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of said second strand length to reduce target gene expression when said multi-stranded nucleic acid is introduced into a mammalian cell.