Selecting effective siRNAs based on guide RNA structure

In RNA interference, guide RNAs direct RNA-induced silencing complexes to mRNA targets, mediating cleavage and ultimately leading to gene silencing. We have observed that unstructured guide strands, which either completely lack complementary bases or in which internal base pairing is thermodynamically unlikely, confer strongest silencing, whereas structures with base-paired ends are inactive. Thus, the structure of the guide strand represents a major determinant of small interfering RNA activity. Here we describe a detailed computational protocol for identification of unstructured guide strands for a given mRNA target sequence. Sequentially, all guide sequences with target complementarity are simulated, their corresponding structures are folded and unstructured guide strands are selected and rated according to thermodynamic parameters. Although this procedure is new and remains to be validated by the community, it allows reliable identification of highly active siRNAs that can be used for functional target validation or drug development.