Design Of FRET-based DNA Fluorescent Probes And Their Application In Biosensing

Since the concept of DNA nanotechnology was proposed in the 1980s,DNA nanotechnology has received extensive attention in the fields of chemistry,biology,materials,and nanotechnology.Scientists have successfully constructed a variety of DNA nanostructures and DNA nanomachines with desired functions,and have achieved great progresses in self-recognition and programmability of DNA molecules.Hybrid assembly of DNA nanostructures with one or more DNA structures with recognition capabilities,such as aptamers,molecular beacons,i-motifs,etc.,can produce multifunctional DNA biosensors or nanomachines.Tetrahedron DNA nanostructure is one kind of DNA nanostructure,which has the characteristics of high yield and controllable size.In addition to being used as a drug carrier,tetrahedron DNA nanostructure can also be used to construct biological probes.The fluorescence analysis method has the advantages of good bioimaging,simple operation and high sensitivity,and has become an important analysis tool in the field of biomedical molecular detection.Due to the superior cell penetration ability,excellent biocompatibility and stable structures,DNA nanoprobes that use fluorescence as the detection output signal have become a research hotspot in the field of in-situ detection of intracellular markers.DNA nanotechnology is changing the field of live cell imaging,providing a simple and versatile method for real-time monitoring of important analytes in cells.Proteins,nucleic acids,and small biological molecules in cells play an important role in complex biological processes,some diseases often cause changes in the activity or content of specific biological molecules.Therefore,they can be used as biomarkers to diagnose diseases such as malignant tumors and metabolic diseases.In addition,the stability of ion concentration and physiochemical index in cells is the basis for maintaining normal life activities.Therefore,the development of biosensors capable of sensing related disease biomarkers and intracellular physiochemical index is of great significance.In this study,DNA self-assembly nanotechnology is used to functionalize the classic tetrahedron DNA nanostructure(TDN).A variety of FRET based DNA biosensors with the advantages of high sensitivity,time-saving and improved specificity have been constructed,and they are finally used in the sensing of telomerase,p H,ATP.The specific work is described as follows:First,to deal with the problems of poor cellular uptake,poor stability,and false-positive signal faced by conventional DNA probes for cell imaging,a DNA biosensor TDNp based on fluorescence resonance energy transfer(FRET)was developed for telomerase activity imaging in cells.Through the introduction of DNA tetrahedral nanostructures,TDNp exhibits superior cell penetration and good biological stability.Meanwhile,the ratiometric"OFF-ON"characteristic of FRET makes TDNp can effectively avoid the false positive signals when imaging of cells.With the absence of telomerase,the FRET signal of the system is in the OFF state;when telomerase presence,the telomerase primer in TDNp would be extended,and the molecular beacon MB is replaced and released to form a hairpin structure,showing a FRET ON signal.Electrophoresis characterization results show that TDNp is efficiently prepared.The stability experiments results show that TDNp has a stable structure that can resist the degradation of DNase I and Exonuclease III in the environment.In the presence of fetal bovine serum(FBS)or p H changes,the FRET signal does not change,indicating that TDNp can effectively avoid false positive signals caused by the interference.Fluorescence measurement results show that the modified quencher BHQ2 can reduce the background signal of TDNp.When used for detecting of telomerase extract activity,the detection limit for the telomerase activity test is as low as 35 He La cells,with good linearity in the range of 50-2000 cells(R~2=0.992).Cell imaging experiments results show that TDNp can be effectively taken up by a variety of cells,and exhibits a telomerase activity-dependent FRET signal in the cells,which has the potential for drug screening of telomerase inhibitors.On the basis of the work above,the design of the TDN bisosensor was promoted,to adjust the sensing range.Firstly,using i-motif as the recognition element,FRET-based p H biosensors was estabilished accordingly.p H-i,p H-s and p H-ss are constructed by"splitting"i-motif and adjusting the distance between the two parts of the sequence after disconnection,the p H transition midpoint(p H_t)of the three probes realized the regulation from 6.81 to 5.71.In addition,this strategy can optimize the signal-to-noise ratio(S/N)of the FRET probe,and the S/N of the probe p H-s+1 is as high as 22.38,which is much higher than that of the conventional FRET probe.Combining the"splitting"i-motif with the TDN,the estabilished biosensor p H-Td not only has the advantages of the adjustable response range,but also benefit to the signal-to-noise ratio high biostability and strong cell penetration.Cell imaging analysis showed that p H-Td mainly distributed in lysosomes,which can be used to trace lysosomes,and finally used to image lysosomal p H fluctuations under drug stimulation.When the i-motif sequence was replaced with an ATP aptamer"splitting"and anchored on the vertices of the TDN,a sensitive FRET DNA bisensor was then developed for ATP,which proved the general applicability of the proposed strategy.In this work,FRET biosensors that sensitive to telomerase,p H,and ATP were successfully constructed,and the possibility of DNA biosensors for intracellular analyte imaging and related drug screening were investigated.The DNA biosensor provides an important example for the development of biosensor for other intracellular analytes.Because the DNA nanoprobe can be precisely assembled,it is expected to develop universal platform for other biomarker detection and drug screening by replacing the recognition units.