title: Design of functional small interfering RNAs targeting amyotrophic lateral sclerosis-associated mutant alleles.
abstract: BACKGROUND
  RNA interference (RNAi) is a potential cure for amyotrophic lateral sclerosis (ALS) caused by dominant, gain-of-function superoxide dismutase 1 (SOD1) mutations. The success of such therapy relies on the functional small interfering RNAs (siRNAs) that can effectively deliver RNAi. This study aimed to design the functional siRNAs targeting ALS-associated mutant alleles.
  
  
  METHODS
  A modified dual luciferase system containing human SOD1 mRNA target was established to quantify siRNA efficacy. Coupled with validated siRNAs identified in the literature, we analyzed the rationale of siRNA design and subsequently developed an asymmetry rule-based strategy for designing siRNA. We then further tested the effectiveness of this design strategy in converting a naturally symmetric siRNA into functional siRNAs with favorable asymmetry for gene silencing of SOD1 alleles.
  
  
  RESULTS
  The efficacies of siRNAs could vary tremendously by one base-pair position change. Functional siRNAs could target the whole span of SOD1 mRNA coding sequence as well as non-coding region. While there is no distinguishable pattern of the distribution of nucleobases in these validated siRNAs, the high percent of GC count at the last two positions of siRNAs (P18 and P19) indicated a strong effect of asymmetry rule. Introducing a mismatch at position 1 of the 5' of antisense strand of siRNA successfully converted the inactive siRNA into functional siRNAs that silence SOD1 with desired efficacy.
  
  
  CONCLUSIONS
  Asymmetry rule-based strategy that incorporates a mismatch into siRNA most consistently enhances RNAi efficacy and guarantees producing functional siRNAs that successfully silence ALS-associated SOD1 mutant alleles regardless target positions. This strategy could also be useful to design siRNAs for silencing other disease-associated dominant, gain-of-function mutant genes.
authors.affiliation: Department of Neurology, Shanghai People's Liberation Army 85th Hospital, Shanghai 200052, China.
authors.forename: Chang-Ming
authors.initials: CM
authors.lastname: Geng
authors.forename: Hong-Liu
authors.initials: HL
authors.lastname: Ding
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics*; D002460:Cell Line; D006801:Humans; D034622:RNA Interference / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics* / Q000502:physiology; D013482:Superoxide Dismutase / Q000235:genetics*; D000072105:Superoxide Dismutase-1
chemical_list: D034741:RNA, Small Interfering; C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1