The Research Of Fluorogenic DNAzyme Probe Based On Specfic Recognition Of MDA-MB-231 Cancer Cells

Breast cancer,one of the most frequently diagnosed cancers,is the second leading cause of death by cancer in women.Early detection has long been considered to be a vital strategy in the control of the diseases’ development,which can lead to timely care of patients and prevent potential tumor metastasis.However,due to the lack of effective breast cancer markers,there are still some weakness in early detection of breast cancer.RNA-cleaving Fluorogenic DNAzyme(RFD)is a labeled DNA molecule,which can recognize specific target and binding to achieve RNA cleavage.In a previous study,we obtained a selective RNA-cleaving Fluorogenic DNAzyme(RFD)probe against MDA-MB-231 cells,typical triple negative breast cancer cells,through the systematic evolution of ligands by exponential process(SELEX)named AAI2-5.AAI2-5 is the first RFD probe to recognize breast cancer in all the world and successfully used for clinical test.In order to make AAI2-5 into further practical application,we have studied the properties of RFD probe AAI2-5,and optimized the reaction conditions and structure of molecular probe in order to improve the sensitivity and practicality of the probe.First,we made a series of experiments to optimize the reaction conditions of the RFD probe AAI2-5,including the reaction time,the pH of the reaction buffer,the type and the concentration of cofactor ions.And we determined the optimal reaction condition of the RFD probe AAI2-5.In the next step,we minimized the length of the probe and simulated its secondary structure.The length of the active domain of the probe reduced to 25 nt from 40 nt after optimization,which was synthesized more easily and economically.Therefore,the RFD probe AAI2-5 is more likely to be widely used in clinical detection.And through the software simulation,we determined the secondary structure of the two probe before and after optimization.By comparing the secondary structure of the two probes,we found that the secondary structure of the optimized probe is more simple on the basis of the retention of the main structure.Finally,we tested the specificity and sensitivity of the optimizedprobe.The results showed that the optimized probe showed specificity as the original probe in specificity.But,in terms of sensitivity,we found that the optimized The detection limit of the RFD probe AAI2-5 was reduced from 5000cells/mL(almost 0.5 μg/mL of MDA-MB-231 cell protein)to 2000 cells/mL(almost 0.2 μg/mL MDA-MB-231 cell protein)through a series of optimization experiments.The probe after a series optimization with a shorter sequence,lower detection limits and good specificity,which makes the RFD probe can be applied to the clinical detection of which provides a strong support.At the same time in this study,we established an optimization system for future reference to the optimization of DNAzyme for other targets.