The Three Dimensional Structure Preliminary Study On Phosphorothioation Modified DNA In Solution

DNA is the carrier of genetic information.And the ever-changing epigenetic phenomenon in nature is the masterpiece of the changing DNA,which consist of four different types of nucleotides.Not only the sequence changing of DNA can cause the change of the genetic information,but also the change of the various chemical modifications in DNA.The study on DNA and its modification is an eternal issue that has been studied for a long time but is still new,because it's the the only way which must be passed for biologists and chemists to uncover the genetic phenomenon and explore the secret of life.There are two types of chemical modification in DNA.One is DNA base modification,represented by methylation modification.The other is DNA backbone modification,represented by phosphorothioate(PT)modification.The main research object of this thesis is DNA PT modification.This modification is the first physiological modification described on the DNA backbone.And its chemical nature is a sulfur modification on DNA backbone,in which a non-bridging P-O bond is changed into a non-bridging P-S bond,and exists as Rp conformationin physiological conditions.Moreover,this DNA PT modification belongs to a kind of post-replication modification,many pathogenic bacteria associated with human disease is also reported the presence of the DNA phosphorothioation.This paper mainly solved the three dimensional structures of PT modified DNA,whoes sequence was first discovered in Streptomyces lividans,and its corresponding non-modified native DNA via 2D NMR technology.The main works in the first part of this thesis are the separation and confirmation of the Rp and Sp conformation of the PT modified DNA.We synthesized the PT modified DNA and non-modified native DNA from the company respectively.Then we pre-processed the crude products.And next we obtained a large scale Rp and Sp conformation of the PT modified DNA via High Performance Liquid Chromatography(HPLC)technology.Last,we confirmed the Rp and Sp conformation of the PT modified DNA based on the information of retention time,ultraviolet spectra(UV),molecular weight and other characteristics of standard substance.The second part of this thesis reported the three-dimensional structure of PT modified DNA in solution via biomolecular NMR magnetic resonance spectroscopy.Based on this technology,we directly assigned the NMR signals of DNA in solution without 13C and 15N isotope-labeled.The Mainly involving NMR experimental methods:2D 1H-1H NOESY,2D 1H-1H TOCSY,2D 1H-1H DQF-COSY,31P-1H HETCOR spectra,pre-saturated(Presat)suppression of water and so on.The three-dimensional structure of PT modified DNA in solution we reported is significant for us to study the influence of the PT modification.And our works may be a fundamental for scientists to further study the mechanism of DNA PT modification.