Abstract
Aim:
Our previous study demonstrated an siRNA-mediated, allele-specific silencing of mutant genes that cause amyotrophic lateral sclerosis. To improve siRNA design for better therapeutic use of RNA interference, we systematically tested the base-pairing mismatch strategy in the design of asymmetric siRNA.
Methods:
A naturally symmetric siRNA that targets the human Cu Zn superoxide dismutase G85R mutant allele was modified by placing either 1 or 2 mismatches at the end of the siRNA from position 1 to 4 at each time. The target preference and silencing efficacy of modified siRNA were measured using a modified dual luciferase system.
Results:
The modification of single base-pairing mismatch successfully achieved the conversion of the siRNA that was originally favored to the antisense of the mutant allele to the one that was favored to the sense strand of the gene. Compared to the single-mismatched siRNA, those with double-mismatch at one end demonstrated an increased asymmetry, and thus, an enhanced specificity and efficacy of gene silencing. In addition, the siRNA with double-mismatch at both ends remained in symmetry.
Conclusion:
Our results suggest the effectiveness of converting a symmetric siRNA to an asymmetric one by introducing mismatches into its structure, and the superiority of double-mismatched siRNA to single-mismatched siRNA in producing selective gene silencing resulting from the disruption of siRNA symmetry. The double-mismatch strategy is an improvement of the single-mismatch method and could be useful in the design of effective siRNAs for the treatment of diseases caused by dominant, gain-of-function gene mutations, such as ALS.
Similar content being viewed by others
Article PDF
References
Taylor J P, Hardy J, Fischbeck KH . Toxic proteins in neurodegenerative disease. Science 2002; 296: 1991–5.
Cleveland DW, Rothstein JD . From charcot to lou gehrig: Deciphering selective motor neuron death in ALS. Nat Rev Neurosci 2001; 2: 806–19.
Matzuk MM, Dionne L, Guo Q, Kumar TR, Lebovitz RM . Ovarian function in superoxide dismutase 1 and 2 knockout mice. Endocrinology 1998; 139: 4008–11.
Shefner JM, Reaume AG, Flood DG, Scott RW, Kowall NW, Ferrante RJ, et al. Mice lacking cytosolic copper/zinc superoxide dismutase display a distinctive motor axonopathy. Neurology 1999; 53: 1239–46.
McFadden SL, Ding D, Salvi R . Anatomical, metabolic and genetic aspects of age-related hearing loss in mice. Audiology 2001; 40: 313–21.
Flood DG, Reaume AG, Gruner JA, Hoffman EK, Hirsch JD, Lin YG, et al. Hindlimb motor neurons require Cu/Zn superoxide dismutase for maintenance of neuromuscular junctions. Am J Pathol 1999; 155: 663–72.
Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A . Rational siRNA design for RNA interference. Nat Biotechnol 2004; 22: 326–30.
Kawase M, Murakami K, Fujimura M, Morita-Fujimura Y, Gasche Y, Kondo T, et al. Exacerbation of delayed cell injury after transient global ischemia in mutant mice with CuZn superoxide dismutase deficiency. Stroke 1999; 30: 1962–8.
Kondo T, Reaume AG, Huang TT, Carlson E, Murakami K, Chen SF, et al. Reduction of CuZn-superoxide dismutase activity exacerbates neuronal cell injury and edema formation after transient focal cerebral ischemia. J Neurosci 1997; 17: 4180–9.
Matz PG, Copin JC, Chan PH . Cell death after exposure to subarachnoid hemolysate correlates inversely with expression of CuZn-superoxide dismutase. Stroke 2000; 31: 2450–9.
Behndig A, Karlsson K, Reaume AG, Sentman ML, Marklund SL . In vitro photochemical cataract in mice lacking copper-zinc superoxide dismutase. Free Radic Biol Med 2001; 31: 738–44.
Hutvagner G, Zamore PD . RNAi: Nature abhors a double-strand. Curr Opin Genet Dev 2002; 12: 225–32.
Hannon GJ . RNA interference. Nature 2002; 418: 244–51.
McManus MT, Sharp PA . Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002; 3: 737–47.
Zamore PD, Tuschl T, Sharp PA, Bartel DP . RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000; 101: 25–33.
Hamilton AJ, Baulcombe DC . A species of small anti-sense RNA in posttranscriptional gene silencing in plants. Science 1999; 286: 950–2.
Hammond SM, Bernstein E, Beach D, Hannon GJ . An RNA-directed nuclease mediates post-transcriptional gene silencing in drosophila cells. Nature 2000; 404: 293–6.
Elbashir SM, Lendeckel W, Tuschl T . RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 2001; 15: 188–200.
Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA . Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci USA 2001; 98: 9742–7.
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T . Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411: 494–8.
Ding H, Schwarz DS, Keene A, Affar el B, Fenton L, Xia X, et al. Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis. Aging Cell 2003; 2: 209–17.
Uprichard SL . The therapeutic potential of RNA interference. FEBS Lett 2005; 579: 5996–6007.
Heale BS, Soifer HS, Bowers C, Rossi JJ . siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Res 2005; 33: e30.
Brown KM, Chu CY, Rana TM . Target accessibility dictates the potency of human RISC. Nat Struct Mol Biol 2005; 12: 469–70.
Khvorova A, Reynolds A, Jayasena SD . Functional siRNAs and miRNAs exhibit strand bias. Cell 2003; 115: 209–16.
Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD . Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003; 115: 199–208.
Chalk AM, Warfinge RE, Georgii-Hemming P, Sonnhammer EL . siRNAdb: A database of siRNA sequences. Nucleic Acids Res 2005; 33: D131–4.
Schwarz DS, Ding H, Kennington L, Moore JT, Schelter J, Burchard J et al. Designing siRNA that distinguish between genes that differ by a single nucleotide. PLoS Genet 2006 Sep 8; 2: e140.
Author information
Authors and Affiliations
Corresponding author
Additional information
This work was supported by grants from the Amyotrophic Lateral Sclerosis (ALS) Association, the National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) No (R01NS048145).
Rights and permissions
About this article
Cite this article
Geng, Cm., Ding, Hl. Double-mismatched siRNAs enhance selective gene silencing of a mutant ALS-causing allele. Acta Pharmacol Sin 29, 211–216 (2008). https://doi.org/10.1111/j.1745-7254.2008.00740.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2008.00740.x
Keywords
This article is cited by
-
Silencing the FOP gene
Gene Therapy (2012)