The Study Of Selection And Application Of Protein Aptamer

Since its first descripted in 1990, the SELEX technology was widely applied as an in vitro selection method to evolve nucleic acid ligands, called aptamers, with new functionalities. The term aptamer is derived from the Latin word''aptus''—which means fitting and the Greek word''meros''meaning particle. In fact, it is oligonucleotide that bind to targets with high affinity and specificity, contening RNA,modificatory RNA,ssDNA or dsDNA, which gaint from random oligonucleotide pool by SELEX(systemtic evolution of ligands by exponential enrichment).The aptamer has different classes of targets. aptamers can be developed for nucleic acid binding proteins like enzymes or regulatory proteins, but also for molecules by nature not associated with nucleic acids like growth factors and organic dyes or nucleic acids (nucleotides, cofactors), and even for molecules connected with whole cell and virus. Beside organic molecules, divalent metal ions were also used as target in SELEX experiments.Aptamers are short single-stranded nucleic acid oligomers (ssDNA or RNA) with a specific and complex three-dimensional shape characterized by stems, loops, bulges, hairpins, pseudoknots, triplexes or quadruplexes. Based on their three-dimensional structures, aptamers can well-fittingly bind to a wide variety of targets from singlemolecules to complex target mixtures or whole organisms. The binding of the aptamer to the target results from structure compatibility, stacking of aromatic rings, electrostaticand van hydrogen bondings or from a combination of these effects. In the presence of the target, aptamers can undergo adaptive conformational changes. The folding into defined three-dimensional structures like stem loop or G-quartet structure and by spermits makes the aptamers to completely interact with target. Proteins exhibit very large, multifunctional surfaces, which make them to be excellent aptamer targets, the broad area between targte and aptamer can form plentiful potential binding location, the advantage makes aptamer able to recognize a distinct tiny difference of a target molecule.By cycles of SELEX, many of the selected aptamers show affinities comparable to those observed for monoclonal antibodies. Aptamer can bind target high affinity, the Kd value is between pmol/L and nmol/L. Numerous variants of the original SELEX process were described to select aptamers with high affinities and specificities for their targets.In generl, the selection of SELEX teachonolyg needs 15-18 cycles, about 2-3 monthes to be completed. At present, the step of slection automatibly, saved labor, and shorter the selection time.The first aptamers developed consisted of RNA and modified RNA. Single-stranded DNA aptamers for different targets were described as well as aptamers containing chemically modified nucleotides. Chemical modification can introduce new features into the aptamers, improve their binding capabilities or enhance their stability. Denaturalized aptamer can renaturation in appriprite condition, by chemicl modified, aptamer can overtime the decompose of half life, stability and can be storaged and transported in room temperature, that was propitious to experiment dignose regent and clinic therapy. So based on the advantage of the aptamer,it was used in the field of analytical chemistry,gene therapy, proteome, and the exploitation of new medicament.Five protein as targets were conjugated on the wall of 96 well. First, a synthesized 80nt single stranded DNA(ssDNA) random library containing 40 ranodm sequences fianked by invariant primer was subjected to 5 rounds of selection againts five target protein, the affinity of ssDNA pool and targets was tested by RT-PCR. In this experiment, we builted the mothed of SELEX based on ELISA technology.In the application study of the aptamer, the method for analysis of the binding betweenα-human thrombin and its 35mer aptamer was developed based on capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The concentration of troombin was calculated through the area of the thrombin-aptamer complex peak, which was separated and detected by CE-LIF. Because of the binding favorable G-quartet conformation potentially involved in the specific aptamer, it was assumed that monovalent and bivalent cations promoting the formation of a stable G quadruplex conformation in the aptamer may enhance the binding of the aptamer and thrombin. Therefore, we investigated the effects of various metal cations on the binding of human thrombin and the aptamer. Our results show that cations like K+ and Mg2+ could not stabilize the affinity complex. After optimizing the method, we measured the linear range, detection limit and reproducibility. The linear range is 0.06-10 nmol/L (Y = 1.2×105x+5.33×104,r2 = 0.991), and the detection limit of thrombin in the method was3.56 pmol/L . Regarding the advantages of high efficient and rapid separation, low sample consumption, and high sensitivity, CE/LIF is a potential and powerful alternative to conventional immunoaffinity assays in clinical diagnostics.