Assembly Of Quadruplex DNAs And Construction Of Quadruplex Supramolecules

It has been known that two single-stranded DNA molecules can associate to form double helixes via the complementary GC and AT base pairs linked by Watson-Crick hydrogen bonds.With the development of the research,more DNA structures have been resolved by NMR and X-ray diffraction?XRD?.These mainly include two kinds of quadruplex structures: G-quadruplex and I-motif,which are associated by G-rich and C-rich strands containing tandem G-and C-stretches,respectively.The Gquadruplex is connected by Hoogsteen hydrogen bonds with mono-or divalent cations in the central channel to enhance its stability,which is first reported by Gellert et al.in 1962.In contrast,the I-motif connected by a series of hemi-protonated cytosine pairs?C·CH+?is composed of two parallel duplexes,which are alternatively intercalated in a head-to-tail way,and it is first proposed by Leroy et al.in 1993.Since the sequences that are able to form quadruplex DNA structures can be found in many important regions of human chromosome and the formation and dissociation of these structures may play a significant role in physiological activities,more researchers begin to pay attention to and study the quadruplex structures.Additionally,it is interesting to find that the structural switch between double helix DNA structures and quadruplexes could be realized by controlling experimental conditions.We hold that the transformation may be applied to the operation of "DNA machine" and "DNA probe".Based on the above research background,the work in this thesis is mainly focus on basic properties of duplex and quadruplex DNA structures and the construction of various types of DNA nanostructures,and it is carried out as the following aspects:a.First of all,we studied the formation and dissociation of tetra-stranded I-motifs by DNA with the sequence d?XnC4Ym??X and Y represent thymine,adenine or guanine,and n,m range from 0 to 2?by using ESI mass spectrometry,UV spectrometry and circular dichroism.According to previously reported formation pathway of four-stranded G-quadruplexes,we further provide a direct evidence of a strand-by-strand formation and dissociation pathway of the four-stranded I-motif and some multi-strand I-motifs are observed in this process.Our results also show that formation of the triple strands is the rate-limiting step in solution,while the trimeric ions are abundant when the tetramolecular ions are subjected to collision-induced dissociation?CID?in the gas phase,suggesting different dissociation behaviors of the tetrameric I-motif in the gas phase and in solution.b.In this work,several short oligonucleotides d?XGmX?and d?XCm X??X represents thymine or adenine,and m ranges from 4 to 6?,which were able to form interstrand quadruplexes,were mixed selectively and annealed under different conditions to study their associations and structural competitions by using ESI-MS and UV spectrometry.There are many factors that affect the strand association,including different terminal bases of quadruplexes and G-or C-repeats of different lengths.Our results demonstrate that the formation of quadruplex structures is a random strand by strand association process.However,with enough incubation time for these mixtures of various homologous strands,initially associated hybrid tetramers will shift into self-assembled conformations,and the length of G-repeats or C-repeats shows more significant effect on the stability of quadruplex structures than that of terminal residues.Accordingly,we can obtain the self-associated tetrameric species generated from the mixtures of various homologous G-or C-strands efficiently by altering the length of G-or C-repeats.c.On the basis of reports in literature,a five-cytosine tetra-stranded I-motif can bear ten-base flanking residues.Inspired by these results,different lengths of guaninerepeats are added at the 3' or 5' ends of the cytosine-repeats,which means that a series of oligomers d?XGiXC₅X?and d?XC₅XGiX??X =A,T or none;i < 5?are designed to study the impact of G-repeats on the formation of tetrameric I-motifs.Our data demonstrate that assemblies of these oligonucleotides are polymorphic,but may be controlled by solution pH and counter ion species,and the sequences d?TGi AC₅?selected here can form tetrameric I-motifs in large quantities.This leads to the design of two oligonucleotides d?TG4AC₇?and d?TGBrGGBrGAC₇?that self-assemble to form quadruplex supramolecules under certain conditions.d?TG4AC₇?forms supramolecules under acidic conditions in the presence of K+ that are mainly Vshaped or ring-like containing parallel G-quadruplexes and antiparallel I-motifs.d?TGBrGGBrGAC₇?forms long linear quadruplex wires under acidic conditions in the presence of Na+ that consist of both antiparallel G-quadruplexes and I-motifs.d.There are reports in the literature that “Holliday Junction” DNA structures?HJs?are essential intermediates in the processes of genetic recombination and play an important role in the gene expression.Additionally,these structures are promising materials to construct DNA nanostructures.In this part,we design four DNA sequences that can form partly mismatched HJs called as the anti-cruciform DNA structures.These anti-cruciform DNA structures have two flexible single-stranded branches,which can link themselves to be nanoarchitectures.If the arms of these anticruciform DNA structures are composed by G-rich and C-rich complementary DNA sequences,the formed nanoarchitectures may be transformed into two other DNA nanostructures containing “bimolecular G-quadruplexes and antiparallel duplexes” and “bimolecular I-motifs and antiparallel duplexes” by controlling the solution pH and counter ion species?this work need to be further explored?.