The Study On SiRNA Delivery Of RNA Self-Assembled Nanostructures Based On Rolling Circle Transcription

RNA interference?RNAi?,as a post-transcriptional regulatory mechanism,can achieve efficient gene silencing by degradation of target mRNA.small interference RNA?siRNA?,a double strand RNA,is widely used in nucleic acid drug development due to its high efficiency and specificity based on RNAi mechanism.Currently,a variety of cationic carriers have been developed for siRNA delivery.However,due to the relatively strong rigid structure and low anionic charge density,it is difficult for siRNA to form a stable and compact complex with cationic carriers.Therefore,the application of siRNA still faces many challenges,such as inefficient cellular uptake,lack of specificity in cells and tissues,poor stability in delivery process,potential cytotoxicity and high initial immune response,etc.In recent years,nucleic acid self-assembled nanoparticles have attracted wide attentions due to their flexible structures and high negative charge density,which will be very useful to achieve efficient delivery and gene silencing of siRNA drugs.Here,we dedicated to develop a new transcription template to construct nucleic acid self-assembled nanospheres,and to study the structural characteristics,cellular uptake and gene regulation of the nanospheres.?1?In this study,dumbbell circular DNA template was designed and synthesized,and poly-RNA was synthesized based on rolling circle transcription.Poly-RNA was further self-assembled based on complementary pairing of bases and binded to magnesium pyrophosphate crystals to form spongy RNA nanoparticles?RNP?.The formation of poly-RNA was verified by agarose gel electrophoresis.?2?The composition,particle size,surface potential,structure and stability of RNP were characterized in this study.RNP was in the form of a sheet-like microporous structure.The particle size of the RNP rely on the Mg²⁺concentration in transcription system.When the concentration of Mg²⁺increased from 6 mM to 15 mM,the particle size of the RNP decreased from 2?m³.5?m to about 1?m.Zeta potential measurement showed that the surface potential of RNP was-12 mV^-14 mV.The serum stability test showed that the RNP had better stability than the native siRNA.With the prolongation of time,the native siRNA degraded to about 20%within 6 h and almost completely degraded within 24 h.The degradation rate of RNP was slower than that of siRNA,and 40%of them were still undegraded after 24 h.?3?Cellcular uptake and lysosome escape of siRNA are the key processes in gene silencing.Herein,the cellcular uptake and lysosome escape process of RNP was researched using HepG2 cells.The uptake efficiency of RNP was higher than that of the native siRNA,and RNP could escape from lysosomes after 12 hours transfection.Finally,we confirmed that the expression of green fluorescent protein gene was silenced by RNP.