Constrution Of Nucleic Acid Nanosystem And Its Application In Biosensing

DNA nanomaterials have been widely used in biosensing,cell imaging,drug delivery,cancer treatment and other related fields due to their excellent properties such as good biocompatibility,programmable sequence,controllable structure,easy synthesis and adjustable function.In the process of tumor occurrence and development,the expression level or distribution state of tumor-related biomolecules(such as micro RNA)in cells will also change abnormally.Therefore,intracellular micro RNA(miRNA)monitoring is of great significance for early medical diagnosis and precise treatment of cancer.However,intracellular miRNA detection still faces many challenges.First,intracellular miRNA expression level is low and has a high degree of sequence homology.In order to achieve the purpose of early assessment,it is necessary to introduce some signal amplification methods to achieve highly sensitive detection of miRNA.Secondly,the traditional fluorescence sensing system is affected by the unstable signal output and low probe delivery efficiency,which can rarely achieve intracellular quantitative detection.In view of these difficulties,this study combined DNA nanoprobes with fluorescence imaging technology to develop a series of DNA self-assembled fluorescent nanoprobes for miRNA detection and real-time in-situ imaging in living cells,and realized accurate identification of cancer cells.The details are as follows:(1)A self-calibrating DNA self-assembly nanosystem has been developed for in situ imaging and quantitative detection of miRNA in cells.The system consists of five self-assembled strands of DNA with cholesterol-labeled ends that effectively anchor cell membranes and deliver them to various cells.In addition,multiple light-controlled sites are designed in the DNA nanosystem,which can be "fixed and timed" to regulate the probe structure and fluorescence signal in vivo by remote light control.The system innovatively proposed a "double reference ratio" strategy,using the detection signal of endogenous genes and the maximum fluorescence signal of the probe as the reference to achieve the calibration and normalization of signal output intensity,effectively avoiding the influence of external environment or operating conditions,with excellent stability and self-calibration performance.Finally,in situ imaging and direct quantification of miRNA in living cells were achieved.(2)The entropy-driven amplification cycle reaction(EDC)was combined with AND logic gate to construct a stimulus-responsive DNA tetrahedron nanoprobe that was simultaneously activated by miRNA-21 and APE1.DNA tetrahedron has good biocompatibility and is the carrier and functional modification interface of probe.The system introduced EDC signal amplification response and APE1 enzyme trigger response sensing strategy,showed excellent sensitivity and specificity,and realized in situ fluorescence imaging of mi R-21 in cells.When the two highly expressed targets of APE1 enzyme AND miRNA-21 in cancer cells coexist,namely,in the AND situation,the logic gate system will activate AND output fluorescence signals,so as to achieve fluorescence imaging AND accurate identification of cancer cells.This strategy is expected to provide an important adjunct method for in situ monitoring of intracellular miRNAs and tumor diagnosis.