A General Approach To High-vield Biosynthesis Of Chimeric RNAs Bearing Various Types Of Functional Small RNAs For Broad Applications

Small RNAs are a category of non-coding RNA(ncRNAs)that usually regulate target gene expression via RNA-RNA interactions.The most important and well studied function for small RNA is RNA interference(RNAi),which is a gene knockdown process mainly mediated by microRNAs(miRNAs)and siRNAs.miRNAs are genomically encoded ncRNAs around 22 nt and are highly conservative.They bind to the target 3'UTR and regulate target gene expression via translational repression and/or mRNA degradation.siRNAs are a class of small RNAs which are usually exogenous,?22 nt in length and follow the same mechanisms as miRNAs where siRNAs may bind to any target regions of mRNA and mainly cause mRNA cleavage.microRNAs and siRNAs are indispensible tools for gene function study,and may also serve as therapeutic agents.Indeed there are a number of RNAi-based therapies under clinical trials and pre-clinical development.RNA aptamers are small RNAs which can bind with target molecules with high affinity and specificity.RNA aptsamers may be derived by systematic evolution of ligands by exponential enrichment(SELEX).Because of its selectivity,RNA aptamers are widely used for basic research and has promising applications to disease treatment,e.g.,Pegaptanib approved by the United States Food and Drug Administration for the treatment of age-related macular degeneration.So far,the production of those RNA reagents mainly relies on chemical synthesis or in vitro transcription.In this project,we developed a new optimal noncoding RNA scaffold(OnRS)for high-level expression of chimeric ncRNAs in E.coli that carry functional miRNAs,siRNAs and RNA aptamers,which offers an easy and cost-effective way to produce target RNA agents for broad applications.1.Development of an OnRS to achieving high-yield production of recombinant RNAi agentsProduction of RNA in E.coli using tRNA scaffold has been reported.To employ this strategy to biosynthesize different microRNA precursors,we constructed a series of plasmids harboring target microRNA precursors.MicroRNA precursors were amplified by PCR using specific primers and human genomic DNA template,and the precursors were inserted into target vector through seamless cloning.After confirmed by Sanger sequencing,plasmids were used to transform HST08 competent cells.After overnight culture,total RNAs were isolated and analyzed by denaturing urea polyacrylamide gel electrophoresis.To our surprise,besides tRNA/mir-1291,tRNA/mir-125b-1 and tRNA/mir-34a,all other 8 target pre-miRNAs were not expressed or at a neglectable level.The results indicate that high-yield production of RNAi agents using tRNA scaffold remains a challenge.By replacing the mature miR-34a in its precursor with target microRNAs and substituting their complementary sequences accordingly,we hypothesized that tRNA/mir-34a showing a consistent high level expression in bacteria may be used as a more reliable carrier namely OnRS to accommodate target microRNAs.2.Utility of OnRS for high-level production of active microRNA chimeras in bacteriaTaking hsa-miR-124 as a starting point,we replaced the 22 nt miR-34a sequence in its precursor with the 20 nt miR-124 sequence(OnRS/miR-124).The target OnRS/miR-124 was successfully expressed in E.coli at high levels.A negative control(OnRS/Neg)was also constructed by inserting a 22 nt scramble RNA into the OnRS,which was highly expressed in E.coli.Recombinant ncRNAs were purified to a high degree of homogeneity by fast performance liquid chromatography(FPLC)method.Further deep sequencing study indicated that OnRS/miR-124 was selectively processed to large numbers of 20 nt miR-124 in A549 cells,while the tRNA scaffold was degraded to tRFs.Stem-loop RT-qPCR also comfirmed three orders of magnitude increase in miR-124 level in the cells.Compared to OnRS/Neg,OnRS/miR-124 was able to suppress the protein levels of STAT3,a well-defined target of miR-124.Consequently,OnRS/miR-124 inhibited the growth of human carcinoma cells and enhanced apoptosis.These results demonstrate that chimeric OnRS/miR-124 is biologically/pharmacological active in regulating miR-124 target gene expression and controlling cancer cell growth after being processed to mature miR-124 in the cells.3.Utility of OnRS for high-level production of active siRNA in bacteriaWe further evaluated whether OnRS could be employed to produce siRNA agents in the same manner.A 22 nt GFP-siRNA was chosen as a model siRNA and inserted into the OnRS.Target OnRS/GFP-siRNA showed high-level expression in bacteria and...