Structural Features Of G-qudruplexes Studied By Mass Spectrometry And Fluorescence Spectroscopy

G-quadruplexes are non- canonical nucleic acid secondary structures. They can be fomed by G-rich DNA,RNA or other oligonucleotide analogues. G-quadruplexes have attracted much research interest because of their significant roles in biological processes, such as cancer gene disturbance, their potential function as drug design targets, and their applications in nanomachines and various biosensors. G-quadruplexes are different from the well- nown Watson-Cric base pairing pattern. They are formed when four guanines(G) of different G-tracts associate through Hoogsteen hydrogen bonds to form a G-quartet plane, followed by stacking of several G-quartets. The stacked structures have loops bridging the G-tracts and are stabilized by monovalent cations. The overall conformation of the G-quadruplex depends on several factors; these include the number and composition of the guanine and non-guanine bases in the parent oligonucleotide sequence, the nature and concentration of cations in solution, ligands, and the sample preparation procedure. It is hard to predict the unknown conformation for the structural polymorphism of G-quadruplex. Therefore, it is possible for us to understand the regular pattern of G-quadruplex folding so that we can trace them inside human cells or synthesize ligands to stabilize G-quadruplex.The focus of G-quadruplex research is on the biological expression and its nanoscale biomaterials. To examine the detailed structures of G-quadruplex, X-Ray crystallography and NMR are employed and still more methods are needed in structural analysis. It is of great significance to make progress on the combination between mass spectrometry and spectroscopy in investigating G-quadruplex structures. Our works are major in the following aspects:1. We have systematically measured G-quadruplexes structures with various conformations to determine their characterized fluorescent features. The aim is to build correlations between G-quadruplexes topologies and fluorescence emissions. The aromatic 5/6 ring overlaps at the interface of 5'-5' stacked dimers formed by two parallel G-quadruplexes have been demonstrated to produce a characteristic 385 nm florescence emission band excited at 260 nm. To further investigate the effect of base stacks within nucleic acid G-quadruplexes on generation of the particular fluorescence emission bands, a series of G-rich DNA sequences are selected. They have been previously studied by NMR forming G-quadruplexes in which bases have defined glycosidic bond angles(GBA) and stack patterns, thus the G-quadruplexes give characteristic circular dichroism spectra termed as parallel or antiparallel structure. Herein, we measure their florescence emissions excited at 260 nm. Our results show that parallel G-quadruplexes can produce characteristic fluorescence emissions especially when they stack at the interface in 5'-5' manner regardless of aromatic 5/6 ring overlaps. Stacked guanines inside G-quadruplex core that present in anti GBA can also give subtle influence on fluorescence emissions, which result in tailing peaks over 390 nm. Furthermore, simultaneous presence of two remarkable emission bands around 330 nm and 385 nm are found to be related to the overall anti guanines stacking. Some correlations between base stacks of G-quadruplexes and fluorescence emissions are obtained in present work, which is significant and useful for prediction of topological G-quadruplexes from sequences.2. Sequences in the mode of Gx Ny Gx Ny Gx Ny Gx are investigated with lack of guanines in different G-tracts. The(4n-1) and(4n-2) pattern have the tendency to form polymolecular aggregations. Their fluorescence emissions are employed to distinguish their different conformations in solution. Furthermore, more derivative sequences fluorescent emission features are measured containing short G-tracts, which inserted as GG inside sequences. However,the folding dynamics are still unclear so far by our investigation and more details of these kind of high-ordered G-quadruplexes are needed to be studied.3. Electrospray ionization mass spectrometry(ESI-MS) combined with fluorescence, circular dichroism, UV spectrophotometer, and native polyacrylamide gel electrophoresis techniques are used to study structural features of interlocked dimers formed by DNA sequence 93del(GGGGTGGGAGGAGGGT) and its derivatives. Herein, we demonstrate that the interlocked dimers can be distinguished from stacked dimers formed by sequences T30923(GGGTGGGTGGGTGGGT) andT30177(GTGGTGGGTGGGTGGGT). In addition, loop length, the base at 5?-end, and the isolation of T and TT to the first 4G tract do significantly influence the formation and topologies of interlocked dimers. Furthermore, our results suggest that the 4G tract and the 2G tract in various locations in the 93 del derivative sequence can form interlocked structure. This work not only provides new insight into the assembly of 3 + 1 interlocked DNA conformations but also demonstrates that ESI-MS combined with other analytical methods is rapid and useful for DNA structural studies.4. The mass spectrometry features of G-qudruplex are deeply researched. We first optimized the instrument conditions by tuning the ion energy and collision energy to control the ionization as well as the abundance of all peaks. We have concluded that the dimeric stacked parallel G-quadruplexes are so stable in gas phase that their structures can tolerate relatively high electric field. Via the combining ammonium ions inside G-quadruplexes, we can determine the gas-phased stability. Furthermore, structural transformation can be monitored when G-quadruplexes are incubated at high strand and ion concentrations. However, high resolution and high energy are needed to obtain this transformation. Ultimately, we have compared the stability in the gas phase to that in solution and confirmed the consistency of stability between two phases.