The present invention provides improved hairpin and fractured hairpin
constructs for use in gene silencing through the RNA interference
pathway. An exemplary short hairpin polynucleotide for use in gene
silencing can include a polynucleotide having from about 42 nucleotides
to about 106 nucleotides configured for being processed by Dicer. The
polynucleotide can include a first region having from about 19 to about
35 nucleotides, a loop region coupled to the first region, the loop
region having from about 4 to about 30 nucleotides, and a second region
having from about 19 to about 35 nucleotides and having at least about
80% complementarity to the first region. Optionally, one of the first
region or second region can have an overhang having less than about 6
nucleotides. Also, the short hairpin can be formed of a plurality of
polynucleotides that cooperate to form a hairpin structure.