Study Of The Folding Kinetics Of G-triplex DNA

Telomere is a special structure at the end of eukaryote linear chromosome.It protects the chromosome and keeps the completeness of genetic information.It has an important connection with cell lifespan and the developing of cancer.G-quadruplex?G4?DNA is a special DNA structure formed by guanine-rich DNA sequences.This structure has wide distribution in telomeres,promoters of gene and so on.G4 is believed to have vital function in these locations,but its structure and folding pathway are still not very clear.G-triplex DNA is an important folding intermediate of G4,and its structure and folding kinetics are still not very clear.This paper is just focused on the structure and folding kinetics of G-triplex DNA by using single-molecule fluorescence resonance energy transfer?smFRET?method.smFRET is widely used single-molecule technique in biology.The energy transfer efficiency and the distance between donor and acceptor have a negative correlation,and this characteristic is used as a high-precision ruler to study the structure changes in biomacromolecules.In our paper,we first optimized the algorithm of smFRET data processing.The optimized algorithm is more automatic and objective in dots finding,mapping,data filtering and fitting process,and the results have higher SNR.The donor and acceptor fluorescence intensities have a negative correlation.By calculating the coefficient of the association,the optimized algorithm is able to pick out the high-quality data.The optimized algorithm also uses global fitting of Baum-Welch algorithm to fit all E_FRET-t curves under the same experiment conditions.Once the initial parameters are inputted,the algorithm will get the final probability transfer matrix and states.The optimized algorithm will get the accurate positions and percentage of each state faster than traditional algorithm.By dealing with the same experiment video of G4 folding in K⁺solution,the optimized algorithm has a higher signal to noise ratio,yields more data and the result shows the variation of G4 DNA structure in K⁺more clearly.The optimized algorithm increases the accuracy and efficiency of data processing in smFRET experiments.Based on the previous work,we start to study the structure and folding kinetics of G-triplex DNA by using the smFRET method.By switching locations of labeling sites of fluorescent dye molecules,we discovered that G-triplex has both parallel and anti-parallel structures in K⁺solution.Using cicular dichroism?CD?spectrum measurements,we find that the folding speeds of both parallel and anti-parallel G-triplex will be slowed down by proximal DNA,and when the proximal DNA is on the5'end of G-triplex sequence,the folding process of anti-parallel G-triplex will be restrained.Further smFRET experiment verified this conclusion.Based on these results we proposed the folding pathway of G-triplex sequence in K⁺solution.This pathway is a completion to former researches.The new pathway can explain the folding pathway of parallel G4 and the influence of proximal DNA on the G4 folding process.