The Optimization Of RNA In Vitro Transcription

In recent decades, more and more studies have found that non-coding RNA fragment have the potential commercial value of applied in the field of diagnosis and treat critical disease. Non-coding RNA fragment includes non-coding RNA(ncRNA)and untranslated region(UTR) on mRNA. All non-protein-coding RNA is referred to ncRNA, represent by the small RNA(microRNA, miRNA) and long non-coding RNA(lncRNA). Riboswitch is the representative of non-coding segments of mRNA.MiRNAs are a class of non-coding RNA which length between 18~25 nucleotides, studies had shown that approximately 30% of human encoding protein gene are regulated by miRNAs. They are involved in the regulation of gene expression and RNA processing and protein translation. They also affect the growth and development of creature. LncRNA is a type of noncoding RNA which length greater than two hundred nucleotides, they participate in the regulation of gene expression, RNA processing and editing protein translational, imprinting control and telomere system regulation. At the same time, they can form their own to ribozyme or riboswitches.Riboswitches are a class of mRNA who through binding to small molecule metabolites caused a conformational changed thus control the expression of the downstream gene. Riboswitches have the most widely distributed in almost all bacteria, while control many important gene expression, regulating basic life metabolism and signal transduction.Usually, the study of structure and function of biological macromolecules(including proteins and nucleic acids) use the Structural Biology reached our main research target. Using RNA as an example include the following steps: obtain the desired RNA and purification, RNA crystallization, collection and correction the data of diffraction and phase, finally describing the structure and function. As we can see, how to get a amount of high purity RNA is the basis of all the structural and functional analysis, the first step is extremely important.Currently, there are some reports about RNA in vitro transcribed by RNA polymerase. However, the researches about in vitro transcription for RNA crystal are rarely reported, especially about crystal structure optimization experiments in vitro transcription had not been reported.Purposive point and the innovation of this paper is select SAM-V riboswitches crystal structure template as a representation, according to Doudna reported in vitro transcription reaction system as a standard control system and optimize the system to increasing the efficiency of in vitro transcribed RNA.We optimized the reaction conditions, including reaction time, NTPs addition concentration and magnesium ions addition concentration. According to standard control system, every time only change one factor in the preliminary design of the experimental conditions, and the experimental results detected by polyacrylamide gel, then take gel photographed and analyze data by Bio-rad instrument. In the same background estimates the product concentration. Then calculated the target RNA percentage, concentration trend was painted.The outcome of the reaction time shows that the relative percentage of desired RNA increased as the growing reaction time, reaching the highest value at 8h, the target RNA yield increased by 50% than standard control system; the results of NTPs addition concentration showed that the relative percentage of desired RNA increased as the growing concentration of NTPs, achieving the highest value at the 4 mM/L, increased by 50% than the standard control system; the results of magnesium ions addition concentration showed that the relative percentage of desired RNA increased as the growing concentration of Mg2+, achieving the highest value at the 85 mM/L, increased almost by 80% than the standard control system; Finally, the above optimum conditions combined into a optimization system for RNA in vitro transcribed, compare the generated percentages of desired RNA by two in vitro transcription systems. The results showed that the percentage of the target RNA transcribed by the optimization system than the standard system increased nearly 100 percent.