Research On The Formation Of Tumor Disease-related Gene G-quadruplex And Its Recognition With Small Molecules Of Natural Products

G-quadruplexes are special secondary structures of nucleic acids that widely exist in human genome,and it has been suggested that these structures could be involved in the celluar processes,such gene transcription,cell growth and apoptosis.DNA or RNA that fold G-quadrulexes are uaually guanine-richsequences,such as human telomere sequence and the promoter regions of certain oncogenes.Since G-quadruplexesrepresent potentially important targets in cancer relif and drugdevelopment,information about the formation,properties,recoginition and biological behaviours of G-quadruplexes may provide useful guidance on tumorprevention and treatment.The DNA chose in paper were three different G-richsequences,including promoter of Brcal gene,human telomere and the promoter of Kras gene.Electrospray ionization mass spectrometry(ESI-MS),cirular dichroism(CD)and dimethyl sulfate(DMS)footprinting were performed to investigate the formationand recognition of these G-quadruplexes.The main contents and results are as followed:1.Thestudy focused on the G-rich sequence(SO)of the promoter of Brcal gene,and two sequences(S1 and S2)were chose as target according to the sequencecharacteristic of SO.The formation and properties of Brcal G-quadruplexes were studied by ESI-MS,CD and DMS experiments.The crescent natural products chelerythrine(P1)and jatrorrhizine(P2)were found to have high affinity to the G-quadruplexes and induce the transformation of the conformation.Further studies showed that the parallel structure of Brcal G-quadruplexes was not as stable as the anti-parallel;maybe the small molecules facilitate the G-quadruplexes shift to the conformation that was more stable.2.The telomere sequences were chose as target,and E SI-MS was used to investigate the effect of NH4+ to the formation of the G-quadruplexes.When 150 mM NH4OAc was added to the DNA solution,the signal-to-noise of the ESI-MS spectra improved significantly,and the peaks of DNA shifted to the low charge states and possessed few adducts.Iindicating the formation of G-quadrupex.In the CD experiment,the characteristic absorption at 290 nm and 260 nm of S1 in NH4OAc solution(which was not appeared in pure water)suggested the formation of G-quadruplexes.Among the Six small molecules of natural products selected,the molecule homoharringtonine(P1)was found to have the highst binding affinity to the G-quadruplexes.More importantly,P1 poessessed good selectivity between the G-quadruplexes and the duplex DNA.The aotodock analysis showed that P1 binds to the exteral groove of G-qudruplex thougth hydrogen bonding and Van der Waals force.It is likely that the suitable size of P1 and the hydrogen bonding between the moleules enhance the high affinity and selectivity of P1 to the G-quadrulexes.3.ESI-MS and CDspectrometry were used to investigate the formation and transformation of G-quadruplexes formed by Kras gene.Results showed that the native type of Kras sequence(KS1)could fold into stable G-quadruplexes structures with three tetrads.Further,the mutant experiment was performed to investigate the contribution of different guanine(G)of KS1 to the formation of G-quadruplexes.The results indicated that the sixth G(form 5' to 3')of KS1 didn't involve in the formation of G-tetrad,and when it was replaced by thymine(T),the conformation and stability of G-quadruplexesremained basically unchanged.The eighteenth and nineteenth G(form 5'to 3'),which showed a big difference to the formation of G-quadruplexes,and the mutant induced the conformation of G-quadruplexes transformed into anti-parallel and the stability decreased obviously.