Kinetics Of RNA Tertiary Structure Folding And RNA Interference

RNA interference (RNAi) is a gene-silencing phenomenon that involves the double-stranded RNA (dsRNA) mediated cleavage of mRNA. It is triggered by double stranded RNA helices as small interfering (si)RNA or that have been produced endogenously from small non-coding RNA known as microRNAs (miRNAs). One strand of the interference RNA is incorporated into the RNA-induced silencing complex to guide the assocaitation of RISC with complementary target or substrate mRNAs, resulting in the cleavage or knock down of target mRNA. Since its discovery, RNAi has been adopted as a powerful research tool in the study of gene function and holds promise as a hopeful genetics therapy against diseases. However, although the mechanism of RNAi has been found, there are still challenges to meet these needs. One is that desighed siRNA should have predictable high accuracy and high efficiency, and another is to avoid the off-target effect.RNA must adopt various secondary and tertiary structures for its diverse biological functions. An RNA pseudoknot is minimally composed of two helical segments connected by single-strand regions or loops. RNA pseudoknots are functionally important in several known RNAs. Many ribozymes have pseudoknots to meet the needs of forming a well defined3D enzymatics shape. The HDV ribozyme, which is found in closely related genomic and antigenomic forms, undergoes rapid cleavage with the native structure containing two nested pseudoknots. RNA folding kinetics is directly tied to RNA biological functions. Developing methods to predict folding of RNA with pseudoknots can provide useful information for the research on ribozyme and therapy for HDV. Several tools have been developed to predict the folding of RNA, but most of them focus on the folding of RNA secondary structure without pseudoknot.The main research contents are as follows:(1) Kinetic analysis of the effects of target structure on siRNA efficiency. There are several factors that affect the efficiency of siRNA:the sequence of siRNA, the structure of siRNA, the interaction between protein and siRNA, the delivery of siRNA and the structure of target mRNA. Among these factors, how target structure affects siRNA efficiency is still under debate. Most of the existed works correlate the efficiency of RNAi with the thermodynamic features of the target site. Considering the RNA-induced silencing complex (RISC) as multiple turnover enzyme, we investigated the effect of target mRNA structure on siRNA efficiency with kinetic analysis.The4-step model was used to study the target cleavage kinetics process:(1) Hybridization nucleation at an accessible target site,(2) RISC-mRNA hybrid elongation along with mRNA target structure melting,(3) target cleavage, and (4) enzyme reactivation. At this model, the target accessibility, the seed and the nucleation site’s effects were all included. The results are in good agreements with that of experiments of different arguments about target’s effect on siRNA efficiency. It shows that the siRNA efficiency is influenced by the integrated factors of target accessibility, stability and the seed effects.(2) Controlling the off-target effects with siRNA concentration.It was found that some of the off-target effect can be eliminated by using lower concentration of siRNA, while others are preserved even when very low siRNA concentrations are employed. A simple model system of siRNA binding to two targets was proposed to study the off-target effect, and it was found that the off-target effect is also related to mRNA structure, whether it can be eliminated by using lower siRNA concentration depends on the target structure.(3) Developing method to predict the folding kinetics of RNA with pseudoknots and application to Hepatitis Delta Virus.RNA pseudoknots are functionally important in several known RNAs. RNA folding kinetics is directly tied to RNA biological functions. In our approach, the basic kinetics step is forming/disrupting/exchanging a helix. We identify the folding path by searching the net flux between the states from the native strate. We applied the model to predict the folding kinetics of HDV and mutated HDV sequences and the results show good agreements with that of experiments. We also indentified the folding path of the HDV and mutated HDV sequences and found that the different folding kinetics features of wild and mutated sequences arise from different pathways.For wild type HDV sequences, there are two main pathways:a slow pathway in which the population is trapped in an intermidate state I1, and the transition from I1to native structure takes30min; a fast pathway in which the population transits into native structure in less than10s. These two pathways folding kinetics explains the biphasic folding features of wild type HDV.The G11C mutation of the HDV sequence does not only change the slow folding pathway as the mutation breaks a helix in the intermediate state I1, which results the change of transition pathway from I1to the native structure and increase of transito rate, but also change the fast folding pathway as the mutation would reduce the stability of conformation in the fast pathway, which results the arise of an intermediate state that contains a pseudoknot and a decrease of the rate of the fast pathway. Due to the changes in both fast and slow pathway, the folding kinetics of G11C mutated HDV sequences become fast and single phasic.For the G11C/27D mutated sequence, several intermidate states that contain pseudoknot were found in its folding pathway. Since the stability of pseudoknot is strongly related to the ion environment, the folding pathway could be used to explain the different features of its folding kinetics under different ion conditions.We have also applied that model to predict the folding kintecs of HDV during transtcription. Our results show that, during transcription, the folding of wild type HDV can avoid the intermiediate state I1and quickly folds into native structure.