The Study Of Conformational Dynamics Of I-motif Structure Based On Single-molecule Methods

Regulation of gene expression is critical for the viability of cells.Over expression or under expression of specific genes can lead to catastrophic events in vivo,which may eventually lead to cell death;or in the case of cancer,make cells immortal.Chromosomal DNA contains not only genetic information,but also its own processing information.Based on existing researches,we have realized that the classical B-type DNA helix structure,as a transmitter of genetic information,has essential functions such as accurately inheriting genetic information to the next generation and guiding protein synthesis.Other different kinds of non-canonical DNA structures,such as left-hand Z-type DNA,hairpin,triplex,quadruplex and so forth,orderly regulate the functions of the genome in vivo.Among these higher order structures of nucleic acids,G-quadruplex(G4)formed by Hoogsteen hydrogen bonds has been extensively studied in recent years.Its complementary sequence can also form a type of quadruplex through semi-protonated cytosine pairs,called i-motif.In the year of 2018,specific i Mab antibodies confirmed the existence of i-motif in human cell nucleus under physiological conditions.The results of cell biology and bioinformatics show that the formation of i-motif in vivo is cell-cycle dependent,and they mostly exist in telomeres and the promoter of proto-oncogenes.Due to its vital roles in regulating expression of oncogenes such as Bcl2,c-MYC and KRAS,i-motif has gradually become a new target for anti-cancer therapy.Furthermore,the structural variability and sensitive p H dependence also make i-motif widely used in nanomachines and pharmaceutical delivery systems.However,compared with the in-depth and extensive researches on the G4 structures,our understanding of the dynamic folding and regulation mechanism of i-motif is still very limited.Here,we mainly use single-molecule fluorescence resonance energy transfer(sm FRET)technology,combined with traditional biochemical experimental methods,such as circular dichroism(CD),FRET-melting,DNA polymerase blocking experiments,studied the inherent folding kinetics of i-motif structures under different p H conditions and monovalent cation concentrations;and explored the effects of complementary sequences to i-motif structures.On this basis,we selected specific proteins to determine their interaction mode with i-motif structures.Our experimental results finally showed that:(1)i-motif with multiple states are more dynamic than the complementary G4 structures,forming at least four intermediate structures which are in dynamic equilibrium under certain conditions.Even under optimal conditions,the stability of i-motif structures is far lower than that of G4s.(2)Monovalent cations display opposite effects on the thermal stability of i-motif structures in different buffer systems.Specifically,K~+disrupts i-motif structures formed in MES and Bis-Tris buffers;however,K~+stabilizes i-motif structures in PB,SSC and SCB buffers.(3)The free G4 strands in an acidic environment do not affect i-motif structures,but under neutral or alkaline conditions,a part of i-motif structures spontaneously form duplex with the G4 strands.Due to the steric hindrance,G4 structures in the same position of the double strands interfere with the formation of i-motif sturctures to a certain extent regardless of the buffer conditions.(4)A low concentration(10 nM)of RPA can potently disrupt i-motif structures and maintain them in the durative linearized state.(5)Several helicases can directly unfold i-motif structures into various intermediate states under acidic condition without relying on ATP and ss DNA tail,but this mode is not suitable for duplex or G4 structures.(6)ATP regulates the interactions between helicases and i-motif structures differentially.Our results systematically illustrate the stability and status of i-motif DNA under different external environments and the existence of complementary G4 chains,which are of great significance for a more comprehensive understanding of i-motif structure in vivo and in vitro.More importantly,we discovered the unique interaction mode between i-motif and helicases at the single-molecule level for the first time,which expanded our insights on cell regulation mechanisms.As an anti-cancer target and a special bio-nanomaterial,our results also have great value and inspiration for the application of i-motif structures.