Detection Of MiRNA Related To Tongue Squamous Cell Carcinoma By Nanopore Sensing Based On DNA Origami Technology

Objective:To design a set of detection system,and then use this detection system to detect tongue squamous cell carcinoma-related miRNA in nanopore sensing detectionMethods:In the first stage,tongue squamous cell carcinoma data sets were screened from GEO database,and then miRNA expression differential analysis was performed on the downloaded data sets.According to the established difference criteria,miRNAs with significant expression differences in two or more different data sets were identified as key differentially expressed miRNAs,which were used as the target miRNAs for subsequent studies.In the second stage,the DNA stem-loop structure was designed based on the DNA origami principle,which was used as tag molecules and signal amplifiers in the nanopore sensing detection system.The optimal assembly conditions were explored by changing the salt ion concentration of the assembly buffer.Then,the target miRNA sequence was found,and the miRNA double-ended base assignment strategy（sandwich method）was used to capture the target miRNA.The effectiveness and specificity of the miRNA detection strategy was verified by the gel imaging system,and the detection sensitivity of the gel imaging method was explored.In the third stage,nanopore sensing detection technology is introduced to make up for the low sensitivity of the gel imaging method.Firstly,M13mp18molecule is used to characterize the electrochemical signal characteristics of a single molecule passing through the silicon nitride nanopore,and then the samples of the designed detection system are successively passed through the nanopore in the absence or presence of target mirnas to capture and record the current trajectory.Clampfit software and Matlab software are used to fit the current trajectory,and the fitting data are statistically analyzed with Origin software,and signals are distinguished from the two aspects of the dwell time and current blockage.Results:（1）four key differentially expressed miRNA in tongue carcinoma were identified by bioinformatics method,which were has-mi R-370,has-mi R-204-5p,has-mi R-149-5p and has-mi R-99a-5p respectively.（2）the best buffer for stem-loop structure assembly is the mixture of 10 m M Tris-HCl（p H 8.0）and 20 m M Mg Cl₂,the sandwich method can specifically recognize the target miRNA--mi R-370,and the sensitivity of this method is 1n M under gel imaging.（3）When the M13mp18molecules pass through the nanopore,both the dwell time and the current blockage are voltage-dependent.The general trend is that with the increase of the voltage,the dwell time decreases and the current blockage amplitude increases.The dwell time and current blockage also showed the voltage dependence of the same trend as M13mp18,and the detection system that detected mi R-370 had a larger current blockade amplitude at the same voltage.Conclusion:The stem-loop structure designed in this study can be used as a molecular label for nanopore sensing detection,and can also successfully amplify the miRNA signal.The designed detection system can specifically recognize the target miRNA,and has higher detection sensitivity than the gel imaging method.