6×His RNA Aptamer Were Screened By SELEX

SELEX(Systematic Evolution of Ligands by Exponential Enrichment)was developed by Ellington and Szostake labo ratories in 1990,after decades of continuous development,SELEX technology has been greatly in proved and developed towards automation and simplicity.The development of this technology has changed from the original2?3 months to 3?4 weeks to screen the desired adaptor,greatly shortening the time of screen adaptor and reducing the consumption of human and material resources.Present,SELEX and its derivative technique has been widely used in the research,diagnosis and treatment,the adaptation of the body is obtained by screening for the target molecule is wide and can be aimed at different targets such as protein,cell and microorganism,compound molecules,has a very high affinity and stability,to specific target molecules,It has great potential in the production of new antibodies,cancer treatment and biomedical research.The basic steps of using artificial construct a random oligonucleotide library can be a single stranded DNA or RNA,ideally library can almost cover the nature of all kinds of target molecules,mix between library and molecular target,under the condition of changing different makes library within different conformational changes of oligonucleotides,From the library-target complex,which can be separated from other unbound oligonucleotides through different elution conditions,so as to obtain nucleic acid molecules specifically bound to the target molecule,and then PCR amplification to obtain a new library for the next round of screening.By repeating the above steps,after 15?20 rounds,less with the target molecules olignucleotide screening will be removed,eventually get fit body is the strongest nucleic acid molecules,and molecular target affinity repeating this process can achieve the effect of the enrichment of aptamer.The aptamers with high specific binding to target molecules are obtained by SELEX screening,separation and enrichment.SELEX method is used to screen 6×His aptamer,which is the exponential enrichment systematic evolution method of SELEX.Through repeated screening,a fragment of oligonucleotide,single strand DNA or RNA that can bind with small peptides with His tag with high specificity and high affinity is finally obtained.The results of this study are as follows: 1)Aptamer templates were designed and RNA fold prediction was used to obtain the template that the variable stem-loop structure was most likely to bind to 6×His;2)High concentration and high purity DNA library was obtained by optimizing error-prone PCR conditions;3)The DNA library with the maximum random mutation rate was obtained by continuous dilution and multiplication,which was amplified by dilution using the optimal conditions of error-prone PCR to obtain the maximum mutation library;4)The DNA fragments amplified by Taq were inserted into recipient cells through T vector,and the DNA library size obtained by sequencing;5)By optimizing the condition of in vitro transcription to get high quality,high concentration of RNA;6)Purified RNA libraries through with His column,the gravity of the tags by changing the solution concentration makes RNA present different conformation,some RNA can combine with His will stay on the pillar,washing column will combined with weak or combination of RNA to remove,finally by changing the salt concentration makes RNA transform conformation from gravity elution column,In this way,the single-stranded oligonucleotide specifically binding to His can be obtained.Repeat the above steps to obtain adaptor with the strongest specific binding ability to His through continuous screening and amplification.