In Vitro Selection Of Circular DNAzymes For Biosensing Applications

With the advancement of science and technology and the rapid development of economy and society,more and more harmful pollutants are released into the environment,which has a serious impact on the quality of the environment.Therefore,there is an urgent need for low-cost,rapid and portable new detection tools to detect pollutants in the environment.DNAzyme biosensors have the characteristics of strong specificity and sensitive response,and have been applied in the fields of human health and environmental analysis widely.DNAzymes are generally linear and are easily degraded in a complex matrix environment,which affects the catalytic reaction and affects the sensing effect.Circular DNAzymes is a closed-loop structure,which has the characteristics of enhanced biological stability and thermodynamic stability against exonuclease degradation.To this end,in this paper,the circular DNAzymes was obtained by in vitro selection technology,and after characterizing its performance,a circular aptazyme was constructed on this basis,and successfully responded to ATP in cells.The details are as follows:（1）In vitro selection strategy for circular DNAzymes was designed.A total of 12 rounds of in vitro selection were performed.The linear library of catalytic cracking was removed by reverse selection and the circular library of catalytic cracking was obtained by positive selection.After the SELEX,the products were subjected to high-throughput sequencing,and the top five sequences in the sequencing results were synthesized and the catalytic activity was verified.The first two sequences have the characteristics of high catalytic activity in circular form and low catalytic activity in linear form,and the corresponding circular DNAzymes CD1 and CD2are the target sequences that meet the screening requirements.（2）The relative properties of circular DNAzymes were characterized.Since the catalytic activity of CD2 is better than that of CD1,CD2 is selected as the characterization object,and its optimum reaction temperature,optimum p H,optimum Mn²⁺concentration and divalent metal ion specificity were studied.Through in vitro stability characterization experiments,it was verified that the circular sequence was more stable than the linear sequence in serum and cell lysates.The circular DNAzyme sequence CD2N4 with fewer bases was further obtained by sequence truncation experiment,and its apparent catalytic rate could reach 0.012 min^-1.It was further found that the circular DNAzyme no longer had catalytic activity after the modification group on the substrate was removed,which confirmed that the modification group on the substrate had a great influence on the catalysis.（3）On the basis of CD2,the ATP aptamer was incorporated to construct the ATP-responsive circular aptazyme CD2-ATPapt.In vitro feasibility assay confirmed that ATP can regulate the activity of CD2-ATPapt,and the ATP aptazyme still maintains its selection specificity.Further,the circular aptazyme was transfected into A549 cells.Through confocal microscopy imaging and flow cytometry analysis,the fluorescence release of the cells transfected into the circular aptazyme was much higher than that of the cells transfected into the mutant sequence and of cells was inhibited by intracellular ATP,which confirmed that the ATP aptazyme can effectively bind to intracellular ATP and can successfully respond to intracellular ATP.In this paper,the circular DNAzymes stably existed in complex matrix were obtained for the first time by in vitro selection technology,and the sensing application was successfully carried out in cells.We envisage that the circular DNAzymes screened in this paper will be widely used in the fields of environmental analysis and biomedicine.