Ribonuclease Detection And Cell Imaging Based On DNA Tetrahedral Nanoprobes

As a classic polyhedron,the tetrahedral DNA nanostructure(TDN)is a rigid,simple,stable,and highly modified nano-scale material generated by the complementary pairing of four single DNA strands.Due to its good biocompatibility and cell permeability,it provides a universal and promising platform for constructing a series of biosensors and drug delivery systems for living cell research.In addition,the high degree of programmability of the DNA tetrahedron determines that it can be ingeniously designed and combined with other materials.With the rapid development of DNA tetrahedrons and DNA nanotechnology,new approaches and opportunities have been opened up in the fields of analytical chemistry,molecular biology and medicine.Based on previous research findings,we use TDN-based fluorescent probes for ribonuclease(RNase)detection,which is closely related to the occurrence of diseases,and successfully constructed a natural compound screening platform and achieved in-situ imaging in living cells.The specific contents are as follows:1.Research on natural compound screening platform targeting ribonuclease H(RNase H)based on radar-like DNA tetrahedral nanoprobes.Based on the specific cleavage of RNA in the DNA/RNA hybrid chain by RNase H,the fluorescent-labeled DNA/RNA hybrid chain is designed at the protruding end of the TDN to construct a radar-like DNA monitor(D-TDN)for RNase Highly sensitive and specific analysis of H activity.In addition,a natural compound screening platform targeting RNase H has been further constructed.The experimental results show that the optimal detection conditions of the RNase H activity analysis system are 20 m M Mg²⁺,D-TDN/L6 concentration ratio of 1.5:1,reaction temperature of37?,and reaction time of 30 minutes;Under the optimal detection conditions,the linear detection of RNase H concentration 0?0.5 U/m L can be achieved,and the detection limit is 0.01 U/m L;Specificity experiments show that the probe has good selectivity to RNase H,which can resist DNase I cleavage at high concentration.This method was used to screen natural compounds targeting RNase H activity,and three potential inhibitors were obtained.The half-inhibitory concentrations were 5.27±2.03?M,9.06±4.21?M,and 18.21±1.11?M,respectively.Cell imaging results also confirmed the inhibitory effect.This radar-like DNA monitoring system with strong specificity and high sensitivity has broad prospects in the fields of activity detection,natural compound screening and living cells.2.Research on ribonuclease A(RNase A)detection system based on natural compound assisted TDN probeAccurate monitoring of RNase A level is helpful for molecular mechanism research,drug screening,clinical diagnosis and prognosis evaluation of tumor pathogenesis.Based on the spatial structure of TDN,the close-distance quenching effect of double fluorophore,the characteristics of natural compounds to activate RNase A and the characteristics of RNase A to specifically degrade RNA base U,a natural compound assisted TDN probe system was successfully constructed to realize ultra-sensitivity RNase A detection.The experimental results show that the optimal detection system for the TDN nanoprobe to detect RNase A is a reaction temperature of 37?,a reaction time of 60 minutes,an in vitro detection linear range of 0.05 pg/?L?10 pg/?L,and a detection limit of 0.09 pg/?L.In the following drug screening experiments,we obtained 4 natural compounds,which can activate RNase A activity.By applying natural compound G10,an activator,as a content of reactive buffer,it was found that the detection limit of RNase A was as low as 0.005 pg/?L,and the detection limit increased about one order of magnitude.Finally,the G10-assisted RNase A detection method was successfully used for visual dynamic monitoring of RNase A in living cells.