Structure of highly effective siRNA. See the text for details.

Generation of the non-redundant sequence set of genes from alternative splice variants located on the same locus.

(A) The vertical axis is the number of 19 nt sequences of the both-strand mismatch tolerance shown in the horizontal axis. The solid line is the distribution for the non-redundant sequence set. For comparison, the dotted line shows the distribution when all the RefSeq and Unique UniGene sequences are used without removing any duplicates. Observe the dramatic reduction of redundant sequences of mismatch tolerance zero and the increases in the number of sequences of mismatch tolerance one or two. (B) The statistical chart (A) is recomputed for plus-strand mismatch tolerance. Note that the number of 19 nt sequences of mismatch tolerance three increases twofold. (C) The horizontal axis shows the requirement for the minimum number of effective, both/plus-strand specific siRNA candidates on a single mRNA. The vertical axis is the fraction of qualified genes in RefSeq that fulfill the constraint in the horizontal axis. Note that the fraction of qualified genes decreases severely when more both-strand specific siRNA sequences are designed, while the fraction decreases much more slowly when plus-strand specific siRNA are designed. This indicates the usefulness of the plus-strand specificity for designing multiple effective siRNA sequences on a single mRNA sequence. (D) The statistical chart in (A) is restricted to redundant 19 nt sequences and recalculated. This demonstrates that the non-redundant sequence set is indispensable for evaluating mismatch tolerances of redundant sequences correctly.

Flowchart of siRNA sequence selection by siDirect. (A) An arbitrary mRNA sequence is input. (B) Both/plus-strand specific precomputed siRNA sequences are presented in front. Both-strand specific (plus-strand specific, respectively) sequences are colored blue (light blue) and are placed under the mRNA sequence. Other siRNA sequences that meet the four guidelines of effective sequences are colored white. Clicking on an siRNA sequence displays the complete list of off-target candidates. (C) The alignment between each off-target candidate and the siRNA sequence clarifies the locations of mismatches.