Design Of Molecular Beacon Based On G-triple And Its Sensing Performance

Molecular Beacon(MB)is a ssDNA labeled with fluorescence and quenching groups at the two ends.In the solution,MB spontaneously folds to form a stem loop structure,and the fluorescence is quenched due to the close contact between the fluorescence groups and the quenching groups.After binding with the target,the MB opens,the fluorescent group is far away from the quenching group,and the system presents fluorescence.Based on this "on off" signaling mechanism,MB has been used in the analysis of a variety of molecules.However,double labeling leads to the time-consuming and expensive synthesis of MB,which limits its widespread use.G-triplex(G3)is a special nucleic acid secondary structure formed by guanine(G)rich DNA sequence.In this paper,G3 was introduced into the stem of MB to obtain a label-free MB,and the sensing performance of this G3-based MB was studied.The details are described as following:1.Comparison of G3-based MB and G4-based MB for sensing performanceG-triplex(G3)is a nucleic acid secondary structure similar to G-quadruplex(G4),which can combine with thioflavin T(ThT)and light up the fluorescence of ThT.Both G4/ThT and G3/ThT can be used as fluorescent indicators to construct label-free molecular beacons.In this work,the sensing performance of G3-based MB and G4-based MB are compared.For 0.45 ?M target DNA,the signal-to-noise ratio of G3-based MB was 11.4,while the signal-to-noise ratio of G4-based MB was 5.5.The G3-based MB response reached the maximum value in 30 min,while the G4-based MB needed 60 min to reach the maximum response value.This study shows that compared with G4-based MB,G3-based MB has higher sensitivity and faster response speed,and then discussed the mechanism of G3-based MB's high sensitivity and fast response.In addition,the effects of stem length and base sequence on the response signals of G3-based MB were systematically studied,and the structure of G3-based MB was rationally designed.This study shows that G3,which is shorter than G4,is more suitable for the construction of MB stems.2.Design of split G3-based MB and research on sensor performanceIn this study,a DNA sequence that forms G3 was split into two short G-rich fragments,is designed to the two ends of MB respectively.In solution,the G-rich sequences at both ends of the end self-assemble to form a G3 structure,thus obtaining a stem-loop nucleic acid structure,namely,split G3-based MB.This work systematically explores the effect of the splitting mode of G3 on MB responses.The results show that when the splitting mode is 6+3,the fluorescence signal response of the split G3-based MB is the largest.Fluorescence spectroscopy and circular dichroic spectroscopy(CD)were used to study the interaction between G3-based MB and ThT of different splitting types,and the mechanism of the effect of splitting mode on the response of these molecular beacons was discussed.Under optimized conditions,the linear range of split G3-based MB for detection of target DNA was 0.01 nM-350 nM,and the detection limit was 14 pM(3?).In addition,the sensitivity of target DNA detection was further improved by combining the split G3-based MB with the enzym-assisted chain substitution signal amplification reaction,and the detection limit was up to 45 aM.The stem structure of this kind of molecular beacon is G3 structure,which is different from the DNA base pair formed by Watson-Crick hydrogen bond in conventional molecular beacon,so that this kind of split G3-based MB has some new characteristics.This study provides a new idea for the construction of non-labeled MB.