Fluorescence Properties Of G-quadruplex Dimer/thioflavin T Complexes

As an important biological macromolecule,nucleic acid can be assembled into many structures to play a significant effect on its nature and function.G-quadruplex(G4)DNA is a special secondary structure of nucleic acids,which is composed of guanine-rich sequences.At the core of the G-quadruplex structure are the planar G-tetrads composed of four guanines that are held together by Hoogsteen bonding and further stabilized by G-tetrad stacking.G-quadruplex could combine with Thioflavin T(ThT)to produce strong fluorescence.But the G4/ThT has lower quantum yield and poor system stability.G4 also have polymer structures,and there are few reports of interactions between G4 polymers and ThT.In this paper,we systematically studied the G4 dimer on the enhancement effect of ThT fluorescence and selected a G4 dimer sequences as G4 dimer/ThT fluorescence probe for further application to construct label-free fluorescent biosensors.The main researches are described as follows:1.G-quadruplex dimer/Thioflavin T complex fluorescence properties studyAs a recently identified higher-order G4 structure,G4 dimer possess unique structure and biological function.In this work,we incidentally found that the two tandem repeats of PW17 sequence(a G4-forming DNA sequence)can directly fold into a stable G4 dimer,and the G4 dimer can dramatically enhance the fluorescent intensity of thioflavin T(ThT).The fluorescence intensity of the G4 dimer/ThT is about 9-fold that of the corresponding G4 monomer/ThT.Compared with the common G4/ThT system,the G4 dimer/ThT exhibited more stable and intense fluorescence emission.On the basis of these findings,the G4 dimer/ThT was used as fluorescence indictor to construct one arched DNA probe for label-free detection of DNA.The arched DNA probe showed lower detection limit than the traditional G4-based molecular beacon probe.This study demonstrates that dimeric G4 is an effective nucleic acid scaffold for lighting up ThT.2.G-quadruplex dimer/Thioflavin T combined with rolling circle amplification for highly sensitive detection of DNARolling circle amplification is one of the nucleic acid signal amplification method at constant temperature.A traditional RCA reaction follows a linear amplifcation manner with a relatively limited efciency and lower base utilization.In this study,the complementary sequence of G4 dimer(D-PW17)with the recognition site of endonuclease at both ends was introduced into the circular DNA template.With the help of Phi29 polymerases and dNTPs,the target DNA hybridized with the circular DNA template and triggered the rolling circle amplification reaction,producing a large number of D-PW17 sequences.At the same time,due to the cleavage of endonuclease,a large number of target analogues have been produced,which participate in the next rolling circle amplification reaction and generate more D-PW17 sequences.Finally,the generated D-PW17 sequence combined with ThT,which generate strong fluorescence emission.Because of the exponential amplification,the detection limit of this method is 10 aM,which is much lower than that of the rolling circle amplification system using G4 monomer/ThT as the signal probe.In this study,a high sensitivity,good selectivity and free-labeled method for the highly sensitive detection of DNA was proposed.