The Study On Interactions Between RNA Aptamer And Small Metabolic Molecule

Nucleic acid aptamers are selected from single strand pools of random-sequenceoligonucleotides through SELEX (Systematic Evolution of Ligands by Exponential Enrichment).Nucleic acid aptamers are divided into DNA aptamer and RNA aptamer. RNA aptamers possessvarious secondary structures, which lead to high affinity and specificity for their wide range oftargets. Besides, they are easy to be synthesized and modified. Owing to the advantages listedbefore, RNA aptamers are the most promising aptamers in basic research, clinical diagnosis andtherapeutics. In this article, an arginine-binding RNA aptamer is obtained through SELEX and itsbiological activity is evaluated.We synthesize a100nucleotides random single strand DNA library, which is composed of37fixed nucleotides in both ends and63random nucleotides in the middle. The random RNAlibrary is built through PCR and in vitro transcription of the DNA pool, which demonstrateswell-proportioned bases and all the possibilities of random assortments.Arginine covalent with agarose bead is used as target to select RNA aptamers in ourexperiment. When we get the secondary pool after one cycle of bingding experiment, thebingding process is repeated through reverse transcription, PCR and transcription again until theproducts are proved with high affinity. We overcome the difficulties of PCR through regulate theamplification cycles and low transcription efficiency through adding regulation sequence. Finally,RNA sequence（named22-7）is obtained through22cycles of in vitro selection, which occurs6times in the100clones of the22nd cycle, as well as5times in the100clones of the20th cycle.Then, we use ITC to detect the affinity between22-7and free arginine.In order to study the internal function of22-7, we compare the similarity between sequence22-7and the RNA in Escherichia coli through BLAST, but no homology is found. Then we addthe transcription termination of sRNA to22-7and construct the over expression system inEscherichia coli DH5α. The two related enzymes, arginine succinyltransferase（astA）andsuccinylornithine transaminase（astC）in Arginine Succinyltransferase Pathway are choosed astargets to explore the function of22-7. The result proves that the expression level of astA and astC are obviously inhibited and regulated by22-7.Bacterial small RNA (sRNA) is a kind of newly discovered RNAs whose nucleotides aretypically range from tens to hundreds in length and functions by incomplete base pairing withtarget mRNA. We wonder whether the non-coding sRNA in bacteria possesses the same abilitiesas RNA aptamers and choose the sugar metabolism related sRNA—SgrS as the research object.SgrS is a glucose-related sRNA，which responds to the pressure of glucose phosphorylation andinhibits the transportation of glucose. We construct a sRNA over expression system and explorethe possibility of SgrS binding glucose. When we over express SgrS and stimulate the bacteriawith excessive glucose, the expression level of target mRNA remains the same as control. Weconclude that SgrS doesn’t interact with glucose when regulate the target gene ptsG.To sum up, we acquire an arginine-binding RNA aptamer through SELEX, construct theover expression system of small RNA and evaluate its biological function in Escherichia coli. Wealso explore the possibilities of small non-coding RNA-SgrS in bacteria function as RNA aptamer.Through the research of interactions between RNA aptamers and small metabolic molecules, weprovide new idea for the gene regulation by sRNA in prokaryote organism.