A weapon that can distinguish between nearly identical targets sounds more like a military device than a molecular biology tool. Schwarz et al. report the design specifications for small interfering RNA (siRNA) that can differentiate two genes that vary by just a single nucleotide in a recent article in PLoS Genetics.
A guanine-to-cytosine point mutation at nucleotide 323 causes a glycine-to-arginine substitution at amino acid 85 of superoxide dimutase 1 (SOD1) that is associated with a familial form of amyotrophic lateral sclerosis. SOD1 prevents oxidative damage, but the disease-associated mutation makes SOD1 toxic. Therefore, the authors aimed to create siRNA that would reduce the expression of disease-associated SOD1 without affecting wild-type SOD1.
siRNA destroys target RNA by inducing an endogenous RNA interference pathway. Many siRNAs target RNA sequences with less than 100% sequence complementarity. Therefore, the authors determined which nucleotide of the 21-nucleotide siRNA should be complementary to the point mutation to best distinguish between disease-associated and wild-type SOD1. Because the final two nucleotides are not important to siRNA function, the authors designed 19 siRNAs with the point mutation tiled to each of the first 19 positions.
All 19 siRNAs reduced disease-associated SOD1 mRNA expression by at least 60%, but only 10 of the siRNAs were specific enough to reduce wild-type SOD1 mRNA expression by less than 40%. Only P12, which carried the point mutation at position 12, and P16 failed to cleave wild-type SOD1 in a cell-free assay.
When the authors inserted other nucleotides at position 323 of SOD1, few siRNAs with mismatches in positions 2–7 (the 'seed' region important for target mRNA recognition) discriminated target from mismatched mRNA. In contrast, discrimination was better for siRNAs with mismatches 3' to the seed region, including P8, P11, P13, P14 and P16. According to the authors, mismatches in this region of the siRNA disturb RNA helix formation that is necessary for the cleavage of target mRNA.
Using microarrays and RT-PCR, the authors found that P16 best distinguished its target, disease-associated SOD1, from all other genes, including wild-type SOD1. They also found that siRNA with purines mismatched to purines in the wild-type mRNA (purine:purine) distinguished between matched mutant and mismatched wild-type mRNA better than purine:pyrimidine or pyrimidine:pyrimidine mismatches.
The authors designed siRNA with a purine:purine mismatch at P16 that targeted a Huntington disease-associated single nucleotide polymorphism without altering wild-type mRNA. Therefore siRNAs with purine:purine mismatches at P16 might successfully target alleles with point mutations associated with other dominantly inherited diseases, such as sickle cell anemia.