Immobilization-free Selection And Characterization Of Aptamers Target To C-reactive Protein

C-reactive protein (CRP) is a major acute-phase reactant protein, and is the mostpowerful indicator and risk factor for inflammatory and cardiovascular disease(CVD). The most commonly used diagnostic methods are the antibody-based assays.Compared with antibodies, aptamers exhibit advantageous features: comparableaffinity, easy in vitro synthesis, better stability and easy tagging with otherfunctionalities. Therefore, some researchers try to isolate aptamers and develop theaptamer-based CRP sensors for diagnostics. But it was rarely reported that theanalysis and detection of CRP were achieved by using the aptamer-based sensors.Conventional methods for aptamer selection mainly rely on immobilizing targetsor ssDNA library on solid supports to isolate target-binding aptamers from therandom library. However, the immobilization of ssDNA library may cause sterichindrance to affect the binding to targets, and immobilization of targets may inducetheir conformational change. It was reported that the three aptamers were selected byimmobilizing CRP on the microbead, plate or magnetic bead. However, it waspreviously reported that the isoform conversion of CRP was resulted due to theimmobilization of CRP, and a RNA aptamer and a DNA aptamer specifically boundto monomeric but not pentameric form of CRP. Hence, it is necessary to isolateaptamers specific to CRP by adopting immobilization-free selection methods, so as toprovide probe with good performance.However, the developed immobilization-free selection methods such asmembrane filtration and capillary electrophoresis still had some problems. Filtrationbased methods suffer strong nonspecific binding toward targets and oligonucleicacids, and are limited to large target molecules, while capillary electrophoresis basedmethods usually need specialized instruments. Since graphene oxide (GO) couldstrongly adsorb of ssDNA, with the weak absorption to dsDNA or aptamer bindingthe target, GO was applied to separate unbound DNA from the bound DNA in theprocess of selection. An immobilization-free selection method assisted by GO(GO-SELEX) had been developed, which overcame the above described drawbacks ofmembrane filtration and capillary electrophoresis.In this thesis, for solving the above problems and further improving the affinity of the selected aptamers, we adopted the modified immobilization-free GO-SELEXstrategy to isloate aptamers target to CRP, and characterized the properties of selectedaptamers. Research contents include:(1) modifying the immobilization-free GO-SELEX method and selecting theaptamers against CRPIn order to avoid the conformational change of CRP due to its immobilizationand improve the affinity of aptamers, some modifications were made for theimmobilization-free GO-SELEX method. The DNA library was firstly mixed with GO.This step was allowed the ssDNA was absorbed on the GO by-stackinginteraction. Then the CRP was added to the mixture. The strongly bound DNA withCRP was released from the GO due to the formation of CRP-DNA complex, while theunbound or weakly bound DNA was retained on GO. The bound DNA was collectedby centrifugation. With the increasing rounds of screening, the counter selectionagainst BSA, HSA and Myo was simultaneously conducted, and the molar ratiobetween library and CRP was gradually decreased. The modification for GO-SELEXmethod was aimed at improving the affinity of the selected aptamers, because theweakly bound DNA was firstly absorbed on GO and hard to form the DNA/CRPcomplex. Ten rounds screening were carried out, and the library of the7th round wassubjected to high-throughput sequencing. Finally, the twenty candidate sequenceswas obtained by the preliminary analysis for the result of sequencing.(2) the optimization and properties investigation for CRP specific aptamersBy analyzing for the secondary structures of the twenty candidate sequences,nine candidate aptamers was obtained and further characterized for their affinityusing SPR analysis. The four aptamers CRP-80-2, CRP-80-5, CRP-80-13, andCRP-80-17was obtained, and their dissociation constants (Kd) were1.3M,24.8nM,40.7nM and3.9nM, respectively. The aptamer CRP-80-17with the highest affinitywas further optimized by cutting bases of the two constant regions. Thus a shorteraptamer CRP-40-17with comparable affinity to CRP, containing only40nt, wasobtained. The specificity and ability of immobilized aptamer CRP-80-17andCRP-40-17to bind free CRP were demonstrated in the condition of immobilizatingaptamers on the SPR Au films. Moreover, the recognition sites between aptamer andantibody against CRP were different by preliminary investigation.