Study On Structure And Activity Relationship Between RNA Secondary Structure And RNAi Efficiency

RNA interference (RNAi) was considered as one of the most important and highly conserved gene regulation pathway in most eukaryotic cells.Double-stranded small interfering RNA (siRNA) directed RISC to perform the endonuclease activity towards target RNAs to eliminate theirbiological functions. Since endogenous RNAi pathway was found in human, RNAi technology fordrug discovery has become the current research focus. Highly potent andhighly specific are key characters of siRNA for its application in drug discovery. Compared with the traditional small molecular and antibody drug candidates, target-universality and the rapid one-dimensionaldrug design are great superiority of siRNA drug application.Such an ideal drug technology is still facing serious actuality:poor stability in serum, hard to permeable into cell membrane, off-target effect and immunogenicity, which are great challenges in siRNA drugdiscovery. Improvements on RNAi efficiency (including highly potent and highly selective) is the key strategy to overcome such flaws. On the other hand, development of small molecular enhancer of RNAi activity can also reduce off-target effect. Our work systematically investigated the structure and activity relationship between RNA secondary structural characters and their RNAi efficiency. Based on chemical modified siRNAs, we achieved significantly improved guide strand RNAi potency and double-strand selectivity through changing RNA topological structures. Also, chemical modifications gave better serum stability, which increased the effective siRNA concentration. We indeed found small molecular enhancer for RNAi activity, which promoted us to deeply understand RNAi mechanism and protein function at molecular level.Our detailed discoveries shown as follow:1. Design and characterization of highly potent and specific short hairpin-shaped RNAs (shRNAs)Stem-loop structures are commonly found in native pre-microRNAs and virus RNAs, which inspired us about the particular biological functions of such loop structures. shRNAs with loop sequences are proven to have RNAi potency, but how detailed structural elements influence RNAi efficiency has still no answers. After careful investigation, we found that:(1) shRNAs with3’-overhang structure showed increased3’-arm strand RNAi activity while significantly reduced5’-arm strand RNAi potency, which resulted in the strand selection index up to1000compared to double-stranded siRNA sequence. which is the first discovery that loop structure of shRNA can significantly ameliorate double-strand off target effect in RNAi; and also a structural basis for understanding the function specificity of native RNAs.(2) shRNAs with different end structures, such as blunt,5’-overhang or3’-overhang have different RNAi potency towards sense and antisense dual-targets; we found3’-overhang and loopstructure can co-enhance RNAi activity of3’-arm strand together, which inspired us that key structural characters for improving RNA efficiency can not be unitary, multi-factors may participate in this regulation pathway. Elucidation of such regulation mode can be very important.(3) When stem region reached to25bp, shRNAs with3’-overhang structure, their RNAi activity of3’-arm strand have close relationship with shRNA thermodynamic property. Which is to say, RNAi activity of3’-arm strand is related to shRNA loop size. Using such loop and stem optimization strategies, we discovered an extremely potent shRNA sequence with its gene silencing IC508pM; also remarkably reduced passenger strand RNAi activity. The discovery of such highly potent and selective shRNAs also inspired us the current RNAi mechanism including Dicer recognition and cleavage mode may not be suitable for explaining such phenomenon.Our work based on native shRNA design and characterize built up the structure and activity relationship between RNA secondary structure and their RNAi efficiency. which promoted us the further investigation on whether chemical modification strategies can be applied in such manner to improve RNAi efficiency.2. Design, synthesis and characterization of highly potent and stable capped siRNA modifications.Using biocompatible Thiol-Michaeladdition reaction, end-thiol modified siRNA sense and antisense strand can easily be cross-linked, resulting in three different siRNA modifications withsingle-or double-end cross-linked features. We systematically evaluated the serum stability, Dicer cleavage activity and RNAi efficiency of each modification, and found:(1) Single-end cross-linked sequences LhpRNA and RhpRNA exhibited improved serum stability while double-end cross-linked sequence dbRNA gave the significantly improved serum stability, whose degradation t1/2reached to more than48h in50%normal human serum. which exhibited that simple man-made loop structure can significantly increase serum stability.(2) All the three modifications can be cleaved by recombinant human Dicerto release21-25nt small interfering RNAs for further RNAi efficiency. Which indicated that small molecular constituted loop structure cannot interfere Dicer recognition and processing procedure.(3) RhpRNA gave the most excellent antisense RNAi efficiency with its IC506.38pM, which is the most potent siRNA reported so far;LhpRNA and RhpRNA exhibited significantly different RNAi efficiency towards either sense target or antisense target. The cross-linked loop and selective3’-overhang structure can co-determine which strand can be incorporated into RISC for gene silencing effect.3. Mechanism study of enoxacin’s enhancement on RNAi activity.Our lab discovered that small molecular enoxacin can enhance RNAi efficiency by a dose-dependent manner. Based on its anti-bacterialactivity targeting bacterial DNA Gyrase, we raised the hypothesis that enoxacin may function through interaction with human RNA helicase A (RHA), another RISC loading factor in RNAi pathway. We performed the enoxacin’s enhancement experiments in normal HEK293cells, RHA overexpressed HEK293cells and RHA silenced HEK293cells to discover their regulation on siRNA guided RNAi activity:(1) Enoxacin can both enhance RNAi activity of either strand in siRNA.(2) When RHA was silenced, enoxacin’s enhancement effect reduced or barely detected, which suggested an RHA-dependent enhancement caused by enoxacin.4. Using ratiometric H2S fluorescentprobe SR400to investigate the generation and inhibition process of endogenous H2S in HEK293cells. Due to the significant function of endogenous H2S on signal transduction and maintaining cellular redox environment, there is absolutely every reason to study such important molecular maker in cellular condition.Endogenous H2S generation and inhibition process need to be verify in situ and quantification. We developed a FRET-based ratio matric fluorescent probe SR400to monitor cellular H2S generation and inhibition. We found:(1) D-cysteine can induce endogenous H2S generation more effectively than L-cysteine.(2) Universal D/L-PPG inhibitor can both inhibit the generation of endogenous H2S induced by D-cysteine and L-cysteine.(3) Chirality-dependent inhibitorD-PPG and L-PPG exhibitedchirality-dependent inhibition activity on the generation of endogenous H2S induced by either D-or L-cysteine. Which inspired us there are probablydifferent pathways for D-or L-cysteine induction of endogenous H2S.The above results will facilitate us to deeply investigate the core protein effectors involved in endogenous H2S generation and inhibition using highly potent and specific siRNA sequence as probes.In conclusion, thispaper was mainly focusedon how to improve RNAi efficiency, and systematically investigated the structure and activity relationship between. RNA secondary structures and their RNAi efficiency. Through chemical modification to change RNA topological structures, we achieved significantly improved guide strand RNAi potency (IC50value<10pM) and double-strand selectivity (more than1000times) through changing RNA topological structures. We also found small molecular enoxacin can enhance RNAi activity with a RHA protein expression-dependent manner. We developed a new class of FRET-based fluorescent probe to detect endogenous H2S with sensitivity, specificity and quantification, which also revealed that endogenous H2S generation and inhibition process are chirality cysteine dependent.