Designing highly active siRNAs for therapeutic applications

Abstract

The discovery of RNA interference (RNAi) generated considerable interest in developing short interfering RNAs (siRNAs) for understanding basic biology and as the active agents in a new variety of therapeutics. Early studies showed that selecting an active siRNA was not as straightforward as simply picking a sequence on the target mRNA and synthesizing the siRNA complementary to that sequence. As interest in applying RNAi has increased, the methods for identifying active siRNA sequences have evolved from focusing on the simplicity of synthesis and purification, to identifying preferred target sequences and secondary structures, to predicting the thermodynamic stability of the siRNA. As more specific details of the RNAi mechanism have been defined, these have been incorporated into more complex siRNA selection algorithms, increasing the reliability of selecting active siRNAs against a single target. Ultimately, design of the best siRNA therapeutics will require design of the siRNA itself, in addition to design of the vehicle and other components necessary for it to function in vivo. In this minireview, we summarize the evolution of siRNA selection techniques with a particular focus on one issue of current importance to the field, how best to identify those siRNA sequences likely to have high activity. Approaches to designing active siRNAs through chemical and structural modifications will also be highlighted. As the understanding of how to control the activity and specificity of siRNAs improves, the potential utility of siRNAs as human therapeutics will concomitantly grow.

Figure 1. Key design options for siRNAs

siRNAs can be modified on either their guide (red) or passenger (blue) strands. Multiple chemical modifications can be made along the length of the siRNA or at the termini as discussed elsewhere [50, 51], including modifying the backbone, base, and terminal chemistry, especially of the passenger strand. Formation of intramolecular secondary structure by the guide strand after separation from the passenger strand has also been examined as a design criterion [–34]. Although positional base preferences have been suggested for multiple locations along the siRNA (e.g. [28]), this review focuses primarily on the characteristics of the siRNA termini, hybridization asymmetry and sequence (i.e., the 5′-nucleotide on each strand), for use in the design of highly active siRNAs.

Figure 2. Sorting of siRNA Activity Results (from Shabalina, et. al, [30])

The activities of siRNAs were plotted according to the 5′ nucleotides on their antisense and sense strands (horizontal axis) and the ΔΔG calculated using 3 nearest neighbors (vertical axis). The data points were then interpolated to simplify visualization of data trends. The scale is red (least active siRNAs; highest mRNA concentration) to violet (most active siRNAs; lowest mRNA level). The figure is divided into the four quadrants where check marks (✓) indicate the approach would identify the correct guide strand, and x’s (✘) indicate the approach would identify the incorrect guide strand. Therefore, sequences in the upper left quadrant are those that would be predicted by both methods to prefer the proper guide strand. Plots using the Novartis data [31] with calculations using 1–4 nearest neighbors are available in the supporting information (Figures S1–S8) but are visually similar to this plot.