Study On Binding Mechanism And Detection Application Of Kanamycin Aptamer

Aptamers are single-stranded nucleotide chains that can specifically recognize the target obtained by SELEX(systematic evolution of ligands by exponential enrichment)technology,which have the advantages of high stability,easy modification and low immunogenicity.Therefore,they play an important role in chemical analysis,medical diagnosis,and drug delivery.Although sensors using aptamers as recognition elements for target detection have been developed with high sensitivity,traditional aptasensors still have two major difficulties: first,the specific recognition mechanism of most aptamers and targets was still a black box problem;second,aptamers are susceptible to degradation by various enzymes present in the human body and difficult to exist stably in biological samples,thus having limitations in physiological applications.Therefore,in this study,we investigated the mechanism of aptamer-target identification by fluorescence polarization(FP),isothermal calorimetric titration(ITC),and circular dichroism(CD)and developed a fluorescent method for the detection of aptamer-target in real food samples based on the theoretical study.In addition,this study introduced aptamer cyclization to broaden the application of aptamer in biofluid samples and developed a corresponding application aptasensor based on the binding mechanism of aptamer and target,using the circular aptamer as the recognition element.This topic focuses on kanamycin aptamer,and the specific research work was carried out in the following three aspects:First,exploration of the binding mechanism of aptamer and target and expansion of practical applications.Based on the reported linear aptamer of kanamycin,the secondary structure of the aptamer was fitted and confirmed as a stem-loop structure.Firstly,the possible binding region of the target was initially confirmed near the T8 base with the help of fluorescence polarization.Secondly,after narrowing down the binding region,several variants were obtained by targeted mutation of specific bases,and the dissociation constants(Kd)of the mutants and the original aptamer were measured and compared by ITC to confirm that the key binding bases of the aptamer are T7,T8,C13 and A15.Again,the thermodynamic parameters of ITC and the spinodal analysis of CD confirmed that the binding of the aptamer to the target is mainly enthalpy-driven.The binding of the aptamer to the target is mainly enthalpy-driven,and the binding leads to changes in hydrogen bonding and π-π stacking forces.Finally,based on the fluorescence resonance energy transfer principle(FRET),a fluorescence sensor was constructed for the detection of Kana using the reported aptamer(Kd = 6.91 μmol/L)and the better affinity Kmut3(Kd = 2.18 μmol/L)obtained during the mutation process as the recognition primitive,respectively.Distance between FAM at the 5’ end and BHQ1 at the 3’ end,which restores the originally quenched fluorescence signal.The detection limits of the constructed fluorescence detection technique were 59 nmol/L and 148 nmol/L,respectively,and the recoveries in actual milk samples were good,which are of good practical application.Secondly,the cycling process of linear aptamers was investigated.In the presence of T4 DNA ligase,the original aptamer and Kmut3 were utilized as recognition elements and cyclized to produce bivalent aptamers.After gel electrophoresis analysis,it was determined that the survival time of the cyclic bivalent aptamer in serum was twice as long as that of the original aptamer and Kmut3,from 12 h to 24 h,with increased stability in biological samples.The thermal stability of the bivalent cyclic aptamer was improved,and the temperature of unchaining was raised by 10°C.Consequently,the bivalent cyclic aptamer was more effective.Last,using a bivalent circular aptamer as the recognition element and replacing the Cy7 dye monomer complexed on the aptamer with kanamycin,which will revert to the multimeric form in the detection buffer of the aqueous phase,resulting in a signal change in the absorbance value,a colorimetric sensing detector for the detection of Kana in real samples was constructed.The constructed sensor is user-friendly and can complete all detection processes within one hour.The detection threshold was 129 nmol/L,and the linear range was between 0.2 μmol/L and 2.5 μmol/L.The method was applied to milk and serum samples containing 93%-105% with excellent recoveries and detection stability.