Figure 1.

Core placement and the surrounding nucleotides. The shRNAs in this study were built around a designed 19 nucleotide siRNA core. We positioned the core at the base terminus of the shRNA (the open end), and extended the stem as necessary at the loop terminus (to 20 or 21 bp). The lower strand, or anti-sense strand region, was designed to be the intended siRNA guide strand which was therefore complementary to the target. Our shRNAs were designed so that the position of the first nucleotide of the primary core (p0) corresponds with the first base pair at the base terminus. The positions of the first and last nucleotides of the flanking p-2, p-1, p+1 and p+2 positions in the expected guide strand are shown (- positions in blue, + positions in purple). We considered the sequence of these flanking bases when selecting targets and estimating core conservations as it is presently unclear if these positions are incorporated in the processed siRNA product(s). The example shown is for a 20 bp stem, so that the last nucleotide of the p+2 position is also the last nucleotide of the loop. Likewise, shRNAs with 21 bp stems, the last nucleotide of the p+2 position is the last nucleotide of the stem.