| Background:Guanine-rich sequences can fold into stable secondary structures called G-quadruplexes（G4s）,which bear unique biological functions.G4s have been demonstrated to exist in the human genome through BG4 and 1H6 antibodies.Subsequently,more than700,000 potential G4-forming sequences in the human genome have been found by high-throughput sequencing methods,and these potential G4-forming sequences are mostly enriched in replication origins,oncogene promoters,and telomere,thus suggesting G4s are highly associated with the biological processes,such as DNA replication,transcription of oncogenes（BCL2,MYC,RET,VEGF,and C-KIT）,and telomere maintenance.Hence,from a pharmacological perspective,designing small molecule compounds for G4s targeting aims to induce G4-mediated cancer cell apoptosis.Moreover,targeting G4s for the treatment of cancers has also been a prominent goal for scientists for many years.The folding/unfolding kinetics of G4s may affect the biological functions of cells,such as DNA replication and transcription.Therefore,it is necessary to explore the folding/unfolding kinetics of G4s and the molecular mechanisms of compounds on folding/unfolding kinetics of G4s.However,due to the complexity of G4 topologies,it is difficult to distinguish and quantify various G4s in a solution environment by traditional biochemical methods.The aforementioned problems can be resolved using single molecule magnetic tweezers.Hence,studying the kinetics of G4s at the single molecule level is necessary for understanding their functions in physiologically relevant conditions.Objective:The goal of this thesis is to explore the folding/unfolding kinetics of various G4s and the regulatory roles of compounds on the kinetics of G4s in double-stranded DNA.These results will provide insights for understanding the functions of G4s in vivo and designing compounds for G4s targeting.Moreover,these findings are also important for developing novel G4-based DNAzymes,biosensors,aptamers,and nanodevices.Methods:In this thesis,the folding/unfolding kinetics of various G4s and the molecular mechanisms of compounds on the kinetics of G4s have been studied by combining single molecule magnetic tweezers and traditional biochemical methods.Firstly,the formation of G4s was characterized by traditional biochemical methods,such as circular dichroism and nuclear magnetic resonance.We next analyzed the folding/unfolding kinetics of various G4s using force-ramp experiments by single molecule magnetic tweezers.In addition,the regulatory roles of a series of classical G4s targeting compounds on the folding/unfolding kinetics of G4s in double-stranded DNA have also been studied.Conclusions:1.We showed that the hybrid-stranded Bcl2-2345 G4 is a kinetically favored structure with faster folding and unfolding rates.In contrast,the parallel-stranded Bcl2-1245 G4 has much slower folding and unfolding rates.In addition to the previously reported Bcl2-2345and Bcl2-1245 G4s,our results showed a hybrid-stranded Bcl2-1234 G4 can also form in the Pu39 sequence for the first time.2.The parallel-stranded intramolecular G4s exhibit extreme slow unfolding rates(on the order of 10-5-10-7 s-1),indicating that unfolding such stable structures will need in days.We also measured the loop-length-dependent folding/unfolding rates of G4s,suggesting that the inverse correlation between thermodynamic stability and loop lengths of G4s is due to the decrease of folding probability and the apparent folding rates.The parallel-stranded intramolecular G4s exhibiting extreme slow unfolding rates can also extend to the non-canonical G4s,thereby revealing a general applicability.3.The formation of telomere G4 could be strongly induced by bisquinoline compound360A.The main reason is 360A can significantly accelerate the folding rate and slow the unfolding rate of telomere G4 but has no significant effect on the folding and unfolding rates of double-stranded DNA,thus resulting in balance tipped towards the formation of G4. |
PDF Full Text Request