Studies On DNA Structural Properties,Assembly And Application Of G-quadruplex

With the gradual increase understanding on DNA secondary structure,in addition to the classic double helix structure,more and more DNA structural topologies have been discovered,including G-quadruplexes,which can be folded by the guanine-rich(G-rich)sequences widely distributed in the region of promoter and telomeres.And the rapid development of modern instrument technology also allows us to observe the dynamic change of the G-quadruplex in the living cell,which has been found to be related with the cell cycle.In addition to playing an important role in the field of life science,this structure has received attention from more and more researchers by virtue of its excellent electrical conductivity and structural rigidity.Accompanied by the maturity of in vitro DNA synthesis technology,people can design any sequences they want.Some carefully designed sequences can form nanostructures containing G-quadruplexes with specific patterns or functions under certain conditions,such as DNA nanowires with conductive prospects and DNA nanogels that can recognize and kill the tumor cells with the help of the G-quadruplex forming aptamer(AS1411).Besides,G-quadruplex can also combine with hemin to form catalase-like enzymes,catalyze reactions that leads to the enhancement of chemiluminescence,or combine with other porphyrin molecules to produce strong fluorescence,so they are often used for bioanalysis.Therefore,it is very necessary to accurately predict the structures formed by G-rich DNA sequences.Except for the well-known classic G-quadruplex containing only G-tetrad,the A and C bases in the sequence can not only serve as a loop connecting adjacent G-tetrad but sometimes also participate in the structure by forming hydrogen bonds with other bases,which increases the difficulty of prediction of DNA structures.Based on the above research background,this paper starts from the study of the structural properties of the G-quadruplex,focusing on the following aspects to study the influence of the A and C bases on the formation of the G-quadruplex under different conditions,and explore the application of G-quadruplex in bioanalysis and nanotechnology.a.In this work,the effect of terminal adenine on the formation and stability of tetramolecular G-quadruplexes(G4s)have been studied by electrospray ionization mass spectrometry(ESI-MS),UV(ultraviolet)and NMR(nuclear magnetic resonance)spectroscopy.Several evidences suggested that the sequences d(AG_nA)(n=4 or 5)form stable uncompleted tetramolecular G4 at acidic condition which is different from the canonical one in the neutral condition.After mixing d(AG₄A)and d(AG₅A)in acidic NH₄OAc solution,the random association of DNA strands with unequal G-repeats was observed which is abnormal.In addition,hydrolysis of guanine has also been observed in acidic condition that may occur for unpaired bases rather than complete G4.Thus,a new G4 topology containing incomplete G-quartet is proposed that is very stable and particularly presents in acidic condition.The information presented in this study is expected to be helpful for understanding of possible G4s in acidic environment.b.According to our existing research and literature reports,A and C base can significantly influence the configuration of G-quadruplex by forming hydrogen bond with G base.Based on this,we replaced the TTA tracts in d(G₃TTAG₃)sequence truncated from human telomere sequences with CAC,CTC,TTT and TAT,and studied the effect of C and A bases on the formation of DNA structure respectively.Compared with other sequences that mostly form tetramolecular antiparallel G-quadruplexes,we found that d(G₃CACG₃)can form a parallel DNA nanowire which is even Na⁺dependent.Through the analysis of its thermal difference spectroscopy(TDS)and one-dimensional nuclear magnetic spectroscopy(1D-NMR),it is found that the structure may contain two hydrogen bonding modes,GC and AG.If the specific structures of d(G₃CACG₃)and related sequences can be resolved,it will help us understand the reasons for the formation of nanowires and provide experience for constructing DNA nanostructures.Therefore,we are trying to solve this problem with the help of X-ray crystallography,but the conditions for growing crystal need to be further optimized.c.DNA gel has been widely used in bioprinting,construction of ion nanopores,cell culture and drug delivery,etc.due to its excellent biocompatibility,stimulus responsiveness,injectability and adjustable mechanical properties.Referring to the previously reported strategy of assembling DNA gels with Y-DNA units,we designed three different types of DNA gels as the research targets and developed a highly sensitive surface-enhanced Raman spectroscopy method to analyze the microstructure of DNA gels.By using aluminum ions as the aggregating agent and dichloromethane as the interface agent,the negatively charged silver nanoparticles are adsorbed on the surface of the DNA networks,which greatly improves the intensity of the Raman signal.In the Raman spectra,we can observe not only the ring breathing vibration bands of the four bases,but also the characteristic peaks of the newly formed structures,such as glycosidic angle and hydrogen bonds,etc.,which provide a new way for the characterization of DNA nanostructures.d.The intrinsic fluorescence of the interlocked bimolecular DNA G-quadruplex(93del4T)was utilized to achieve label-free and non-toxic detection of target DNA.Similar to double-stranded probes that have been reported,the designed probe consists of a single stranded DNA template that hybridizes with the sequence of 93del4T at 5'end forming partial complementary duplex and leaving single strand overhang at 3'terminus to recognize target DNA molecule.Once the target DNA hybridizes with the probe to form another part of duplex at 3'terminus,exonuclease?will catalyze the stepwise removal of nucleotides from the blunt 3'terminus,consecutively liberating target DNA and sequence of 93del4T.The released target DNAs can recycle to bind more probes and release more 93del4T that can fold into G-quadruplexes to amplify fluorescence signals.As a result,quantitative detection of target DNA has been achieved according to fluorescent signal intensities.In comparison to other methods,this strategy is simple,rapid,inexpensive and toxic-free.At the same time,it also extends new application value and direction for G-quadruplex with self-fluorescence.