Assembly And Structural Diversity Of Guanine-Rich DNA Molecules

It is well-known that DNA sequences can form double-helix structures by Watson-Crick hydrogen bonds.However,in fact,DNA molecules can also form other structures,including double-stranded DNA,A-DNA,Z-DNA,?-DNA,triple-stranded DNA,C⁺·GC and CG·G,four-stranded DNA,G-quadruplex,and I-motif.G-quadruplexes are tetra-stranded nucleic acid structures formed by G-rich oligonucleotide sequences.Four guanine bases can associate into a G-quartet through Hoogsteen hydrogen bonds,and then two or more G-quartets further assemble into G-quadruplex via hydrophobic stacking.The stacking of G-quartets can be stabilized by monovalent or divalent cations like K⁺,NH₄⁺,Sr₂⁺.G-quadruplex has important biological significance in eukaryotic cell telomeres,immunoglobulin switch region,and genes promoter region.As building materials,DNA molecules also have important applications in the design of DNA molecular probes,molecular machines and other special nanostructures.This paper focuses on the studies of DNA structures formed by G-rich DNA sequences and main contents are shown bellow:In the first chapter of the thesis,we first introduced the secondary structures of DNA,including double-stranded DNA,triple-stranded DNA,and four-stranded DNA.Then we introduced the common techniques and methods for the study of DNA structure.In this section we also introduced the assembly of DNA molecules and techniques for DNA macromolecular structures.Finally,we put forward our research ideas.In the second chapter of the thesis,we confirmed that the sequence d?CGACGA??S1?under acidic conditions?pH 4.5?can form a stable homologous parallel double-stranded ?-DNA,and under alkaline conditions?pH 9.0?,it associated the sequence d?TCGTCG??CS1?to form stable antiparallel double-stranded B-DNA,the sequence d?AGCAGC?could not form ?-DNA under acidic conditions.Based on this,two DNA sequences were designed:d?CGACGAT₂G₆T₂AGCAGC??SG2?and d?GCTGCTT₂G₆T₂TCGTCG??CSG2?.The structures formed by the sequences in distinct acidic and alkaline solution with the presence of different cations were studied.ESI mass spectrometry?ESI-MS?,UV spectrometry,circular dichroism?CD?,native gel electrophoresis,and atomic force microscopy?AFM?were used to explore the structure formed by the short sequence and the macromolecular structure.Generally,SG2,CSG2 can form four-stranded parallel G-quadruplexes with[G₆]₄ as the core.In acidic potassium acetate solution,SG2 form dimeric quadruplexes through the association of 5'-?CGA?_n sequence.Under alkaline conditions?pH 9.0?,terminal sequences of SG2 form Watson-Crick base pairs.The further association of SG2 and CSG2 gives supramolecular structures,the size of the structure can been controlled by changing pH value.This is the first time putting forward the idea that combines parallel double-stranded ?-DNA and G-quadruplex to form a DNA nanostructure.This work provides meaningful information on the specific interactions between bases and the construction of biological macromolecules.The third chapter explores the structural diversity of a specific DNA sequences d?TGGGGTCCCCT?in various solution conditions.The structures were investigated by using ESI mass spectrometry,UV spectrometry,circular dichroism and Raman spectrometry,etc.Our results show that,under alkaline conditions with Li⁺,NH₄⁺,Na⁺and K⁺,d?TG₄TC₄T?formed dominated anti-parallel bimolecular B-DNA and hairpin structure A-DNA.Under acidic conditions,the experimental results show that whether in the buffer solutions containing lithium ion?Li⁺?,ammonium ion?NH₄⁺?,or sodium?Na⁺?,majority of the structures are also B-DNA and hairpin structure A-DNA.But in the presence of potassium ions,a large number of four-stranded parallel G-quadruplex structures are formed particularly.It is known that G-quadruplexes can be stabilized by potassium ions best compared to other monovalent cations.In addition,in acidic solution,the terminal C·C⁺base pair promotes the formation of parallel double-stranded structure,which further stabilized theG-quadruplexpolymerization.Therefore,largeamountsofparallel G-quadruplexes can be detected under acidic potassium conditions.