Structural Features Of DNA G-quadruplexes Studied By Surface-enhanced Raman Spectroscopy

DNA is a very important genetic material in an organism,which is closely related to gene transmission.There are various types of DNA secondary structures.In addition to the classic B-type double helix structure,the G-quadruplexes structures formed by DNA sequences rich in guanine(G)have attracted more and more attention in recent years.Studies have shown that telomere DNA sequences can form G-quadruplexes and inhibit telomerase activity,which is a very attractive drug target for cancer therapeutic intervention.Traditional methods of nucleic acid structure analysis mainly include X-ray crystal diffraction(XRD)and nuclear magnetic resonance(NMR),which can provide structural information at the molecular level,but require high purity and concentration of samples,as well as time consuming and expensive.However,simple detection methods such as circular dichromatography(CD)can only provide structural profile information.Thus,the existing analytical techniques cannot satisfy the needs of nucleic acid structure analysis.Since 1970s,Surface Enhanced Raman Spectroscopy(SERS)detection technology has developed for many years.Besides the advantages of low cost,simple and quick analysis,the sensitivity of the analysis is greatly improved while retaining detailed information on the stretching vibrations of the chemical bonds in molecules.Many breakthroughs have been made in unlabeled detection of nucleic acids by SERS method.For example,Bell et al.added magnesium sulfate to the silver sol system to make nucleic acid molecules close to the surface of substrate and directly obtained nucleic acid molecular signals.After that,Ren Bin et al.added iodide ion to the silver sol system to reduce the interference signal.Our group used silver sol(Ag IANPs)containing aluminum and iodide ion as the substrate to further improve the sensitivity of SERS analysis of nucleic acid molecules,and obtained abundant SERS spectra reflecting secondary structural characteristics such as G-quadruplexes.Due to the rich structural characteristics of G-quadruplexes,the correlation between the structure and SERS spectrum has not been completely established.In this paper,we selected the G-quadruplexes structures formed by human telomere DNA sequences and the special G-quadruplexes structures containing the adenine coplanar G-quartet(G4)as the research objects,and studied the relationship between the structural characteristics of G-quadruplexes structures and Raman spectroscopy.This paper is divided into three chapters,the main content of each chapter is as follows:The first chapter is the introduction.In this chapter,the author describe the structural characteristics,diversity and biological functions of G-quadruplexes and G-quadruplexes in human telomeres in detail,and summarize the mechanism and methods of detecting G-quadruplexes by SERS.Finally,aiming at the scientific problems that need to be solved in the SERS analysis of G-quadruplexes structures,the idea and significance of the topic selection of this paper are proposed.The second chapter is the study of G-quadruplexes containing G4 with coplanar adenine by SERS.The SERS with Ag IANPs as substrate was employed by the author for studying eight DNA G-quadruplexes,five of which consist of adenine coplanar G4.The results of the study showed that,the intensities of the peaks from adenine ring breath(ν_A)were particularly enhanced when adenine were coplanar with G4,which was higher than the intensity of guanine ring breath(ν_G).Furthermore,the peak intensity ratio ofν_Aandν_Gvs.the ratio of the numbers of adenine on G4 to the sum of numbers of adenine and guanine show very good linear relationship in the quantitative detection spectrum.Thus,based on the peak intensities ofν_Atoν_Gand their ratio in the SERS spectrum,a rapid,low cost,simple and sensitive platform for the recognition of whether DNA G-quadruplexes have adenine coplanar G4or not are proposed.In addition,this work also provides important information on the oritantions of G-quadruplexes molecules in the“Hot Spots”.The third chapter is the study of structural features of human telomere G-quadruplexes by SERS.The author used SERS method to study four G-quadruplexes formed by human telomere DNA sequences.The number of G4s were same but the configuration of(TTA)loops were different in these G-quadruplexes.When the(TTA)segment forms a double-chain loop rather than a diagonal loop,the intensity of the ring breathing vibration peak of thymine(d T)in SERS spectrum is enhanced greatly.This indicates that the double-chain loop formed by the(TTA)segment is closer to the G4 than the diagonal loop.Therefore,we proposed a low-cost,highly sensitive,simple and quick method to identify different configuration of(TTA)loops in human telomere G-quadruplexes.We hope that the detection method of G-quadruplexes by SERS in this paper can play an important role in the study of the G-quadruplexes structures.