Abstract
In this study, we formulate a computational reaction model following a chemical kinetic theory approach to predict the binding rate constant for the siRNA-RISC complex formation reaction. The model allowed us to study the potency difference between 2-nt 3' overhangs against blunt-ended siRNA molecules in an RNA interference (RNAi) system. The rate constant predicted by this model was fed into a stochastic simulation of the RNAi system (using the Gillespie stochastic simulator) to study the overall potency effect. We observed that the stochasticity in the transcription/translation machinery has no observable effects in the RNAi pathway. Sustained gene silencing using siRNAs can be achieved only if there is a way to replenish the dsRNA molecules in the cell. Initial findings show about 1.5 times more blunt-ended molecules will be required to keep the mRNA at the same reduced level compared to the 2-nt overhang siRNAs. However, the mRNA levels jump back to saturation after a longer time when blunt-ended siRNAs are used. We found that the siRNA-RISC complex formation reaction rate was 2 times slower when blunt-ended molecules were used pointing to the fact that the presence of the 2-nt overhangs has a greater effect on the reaction in which the bound RISC complex cleaves the mRNA.
Highlights
RNA interference (RNAi) refers to a post-transcriptional gene silencing mechanism with potential therapeutic application for the treatment of various diseases including cancer, viral infections, and neurodegenerative disorders [1]
We identified that the small interfering RNAs (siRNAs)-RNA-induced silencing complexes (RISCs) complex formation reaction rate was altered due to the different siRNA molecular structures
Let us consider a simple RNAi system from Table 1. mRNA is transcribed according to reaction 4 and degraded according to reaction 6. dsRNA is synthesized from mRNA by RNAdependent RNA polymerase (RDR) and is cleaved into 10 siRNAs according to reactions 3 and 2 respectively. siRNA can associate with mRNA according to reaction 1 to cleave the mRNA and produce garbage RNA
Summary
RNA interference (RNAi) refers to a post-transcriptional gene silencing mechanism with potential therapeutic application for the treatment of various diseases including cancer, viral infections, and neurodegenerative disorders [1]. RNAi is being extensively used to study the functions of individual genes based on its intrinsic property of regulating the expression of a distinct mRNA species in mammalian cells [3,4] as well as offering additional improvements over alternative technologies including knock-out by homologous recombination and antisense. [5], the authors performed a comparative analysis of the suppressive effects of three knockdown methods, namely, methods based on RNA interference (RNAi), antisense ODNs, and ribozymes, using a luciferase reporter system. Their doseresponse experiments revealed that the IC50 value for the siRNA was about 100-fold lower than that of the antisense ODN besides providing useful information about the positional effects in RNAi
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