Prediction Of Secondary Structure Of SAM-V Riboswitch By SHAPE Chemical Detection

In recent decades,more and more studies have shown that RNA's non-coding genes can regulate the expression of protein-coding genes at the transcriptional and post-transcriptional levels,and have potential applications in the diagnosis and treatment of diseases and other aspects.The non-coding genes of RNA comprise non-coding RNA and untranslated regions(UTR)of the mRNA that encoding the protein.For the study of mRNA that encoding proteins,people prefer to put more attention to the open reading frame(ORF),but often overlooked non-coding region of the mRNA.But in 2002,the Breaker team discovered and confirmed that certain structural elements located in the mRNA non-coding region can also regulate gene expression,and this structure named "riboswitch".Riboswitch is located in the non-coding region(UTR)of the mRNA.It is widely distributed in most bacteria,fungi and plants.It is a kind of mRNA molecule that can regulate the expression of downstream genes by binding to metabolic small molecules.There are many uses of riboswitch have been reported,such as riboswitch to participate in the study of new genes and gene expression regulation and riboswitch for the new molecular sensor and the development of new antibiotics to provide new ideas.Due to the many applications of riboswitch,researchers have increasingly focused on riboswitch research in recent decades.At present,more than 20 kinds of riboswitches have been discovered,among which S-adenosylmethionine(SAM)riboswitch family has attracted much attention because of its most members and the most diversified regulatory mechanism.The five members of the SAM ribo switch family,named SAM-I~SAM-V riboswitch because of their binding to the same ligand S-adenosylmethionine.The structures and functions of SAM-I~SAM-III riboswitches have been reported by many biologists.However,the structure and function of SAM-IV and SAM-V riboswitches are less reported.The structure of the SAM-V riboswitch is only the prediction of its secondary structure.Due to the conserved sequence of the SAM-V riboswitch and SAM-II ribos witch are similarity,its secondary structure is also based on SAM-II riboswitch predicted.Studies on the structure and function of riboswitch are usually performed by X-ray crystallography and multidimensional NMR techniques.Although the three-dimensional structureof the riboswitch and the binding site of the ligand can be seen intuitively by these techniques,the structural changes of the riboswitch before and after the binding of the ligand can not be detected.The conception and innovation of this paper is to study the secondary structure of the SAM-V riboswitch RNA sequence before and after the binding SAM by SHAPE chemistry method,which can be used for the further study of its crystal structure.SHAPE chemical method is currently used in the analysis and prediction of RNA secondary structure of the most simple and most practical chemical method,its basic principle is the use of an electrophilic reagent and RNA react to form a stable adduct,thus blocking The subsequent primer extension reaction is broken down and the reaction site of the RNA is determined by the band on the sequencing gel.Because of according to the SHAPE protocol by the paper‘ Selective 2?-hydroxyl acylation analyzed by primer extension(SHAPE): quantitative RNA structure analysis at single nucleotide resolution ' by Kevin A Wilkinson,Edward J Merino & Kevin Meeks report at NATURE 2006,complete the SAM-V riobswitch SHAPE experiment found that a series of problems,such as SHAPE experimental results,the disappearance of the full-length RNA,modified signal is low and so on.Therefore,the first step of this study is to optimize the SHAPE chemistry to obtain a better SHAPE protocol for the SAM-V riboswitch,so as to lay a solid foundation that predict the SAM-V riboswitch RNA conserved sequence for secondary structure.The steps of SHAPE chemistry application in establishing secondary structure include: RNA design,RNA purification,RNA folding,RNA modification,RNA primer extension,and final lateral sequence analysis.It can be seen how to get the pure RNA is a crucial step,in this study though in vitro transcription to obtain the target RNA,in vitro transcription requires the target gene and T7 RNA polymerase,obtain the desired gene need Taq DNA polymerase.Therefore,the first step of this study is the introduction of Taq and T7 plasmid into BL21 competent cells for expression and purification to obtain the necessary tools for the experiment enzyme.The template of the target gene SAM-V riboswitch was obtained by PCR amplification,then the target RNA was obtained by in vitro transcription,and purified RNA was obtained by running gel,concentration and other steps.The final SHAPE test was found to be a failure when the initial concentration of the target RNA was selected to be 0.1?M and the concentration of the modified NMIA was 13 m M to complete the SHAPE experiment according to the protocol of the SHAPE chemistry experiment reported in the literature.According to the steps of SHAPE chemical experiment,the initial concentration of RNA was optimized.After optimizing the initial concentration of RNA,the concentration of NMIA was optimized.The experimental results were obtained by using polyacrylamide Ammonia gel detection simultaneously fluorescence detection analysis.The results of SAM-V RNA initial concentration showed that when the reaction system was 10?l,the reverse transcriptase was 1?l,the initial RNA concentration was 0.3?M and 1?M,the final SHAPE full length were lower,when the initial RNA concentration was between 2 and 6?M,the SHAPE results showed that the full length were thicker and had no significant difference.Therefore,SHAPE was performed at the initial concentration of 2?M in the subsequent SHAPE Therefore,SHAPE was performed at the initial concentration of 2?M in the subsequent SHAPE experiment.The NMIA optimization results showed that with the increase of NMIA concentration,the full-length c DNA of SHAPE was decreased gradually,and the signal of NMIA was decreased with the increase of NMIA concentration.When the concentration of NMIA was 13 m M,the full length and the sequencing signal of the SHAPE experiment result is lower,and the result of this experiment was not good for the final analysis.When the concentration of NMIA was 1.3m M,the full length and the sequencing signal of the SHAPE experiment result is higher.The experimental results are favorable for the final analysis and can be used for the analysis and prediction of the final RNA secondary structure.At last,the secondary structure of SAM-V riboswitch was predicted by the optimized SHAPE data.The results showed that there were several differences between the SAM-V riboswitch secondary structure and the reported results,but the final result was studies confirm.