Buldging at the Seeds Abrogates Off-Targeting of siRNAs

Since the first descriptions of RNA interference in human cells a decade ago, the applications of small interfering RNAs (siRNAs) for studies of gene function as well as therapeutics have grown significantly. One of the major attractions of siRNAs is their sequence specificity as they guide base specific cleavage of target mRNAs. This notion was somewhat dispelled by microarray analyses of the transcriptome following siRNA applications which demonstrated that a single siRNA could affect the expression of dozens of non-targeted transcripts. It was quickly realized that this was due to the ability of siRNAs to mimic endogenous microRNAs, which bind to their targets with partial complementarity and only require what is called “seed sequence” base pairing between bases 2-7/8 of the guide strand with the target mRNA 3’UTR.

Since most siRNAs have the potential for off-target inhibition of gene expression, investigators have sought methods for preventing or minimizing this problem. The first approach used a 2’O Me backbone modification at position + 2 from the 5’end of the guide strand. For reasons which are not clear, this modification did a good job in minimizing off target effects. The importance of the +2 position in off target activity has recently been revisited in a September 2011 Molecular Therapy article by Dr. Pooja Dua and colleagues. These investigators determined that creating a buldged (non-base paired) nucleotide at this position in the siRNA resulted in greatly reduced off-target effects. In essence, they created an siRNA in which the guide strand is one nucleotide longer than the passenger strand, with the extra nucleotide being at position +2 from the 5’ end. They conjectured that this buldge makes the 5’ end thermodynamically less stable, favoring its incorporation into the active site of the RNAi effector protein argonaute 2. This eliminates the passenger strand incorporation, which reduces off targeting. More importantly, the buldge also prevents “seed sequence” miRNA type to off targets, but does not affect the interaction of the siRNA to the intended, otherwise full complementary, target.

Since the extra nucleotide can be incorporated in guide strand designs for promoter expressed short hairpin RNAs, it should be possible to eliminate off targeting by these as well. This simple adjustment in si/shRNA design should prove valuable in the use of siRNAs/shRNAs for therapeutics.