| In addition to duplex type,cytosine(C)-rich nucleic acid sequences can fold into non-canonical imotif structures in cells.Based on their structural dynamics,they play key roles in many essential progress,such as telomerase inhibition,DNA replication,and transcriptional regulation.Considering that i-motifs can fold spontaneously,studying their unfolding mechanisms is particularly important for understanding the biological processes they regulate.In cells,the unfolding is mainly modulated by i-motif specialized proteins,such as the heterogeneous nuclear ribonucleoprotein K(hnRNP K).hnRNP K is a kind of poly(C)-binding proteins(PCBPs)and it can activate c-MYC transcription by unfolding the i-motif structure formed in Py25 which in the core sequence of the proto-oncogene c-MYC promoter.However,the molecular dynamic process and mechanismare still unclear.There are at least three questions that need to be answered.Firstly,Py25 contains five runs of cytosines: C-tracts 1,2,3,4,and 5.Whether type-1245 or type-1234 is the major structure is the subject of controversy;Secondly,the molecular mechanismof how hnRNP K unfolds c-MYC i-motif structure remains elusive due to the limitations of research methods;Finally,hnRNP K contains three KH(hnRNP K homology)domains that mediate the resolution of i-motif structures.However,it remains unclear where all three of these motifs can disrupt i-motif structures,and which motif exhibits the most activity.In recent decades,the development of single-molecule technology has greatly enhanced the ability of researchers to monitor the interaction processes between biological macromolecules in real time,which has an irreplaceable role in understanding the mechanisms of biological macromolecular interactions.Hence in this study,single-molecule fluorescence resonance energy transfer(smFRET)technology was used in combination with other bulk methods,such as circular dichroism(CD)and fluorescence spectroscopy,to comprehensively study the micro-dynamic process and molecular mechanismof hnRNP K unfolding c-MYC i-motif.The main research contents are as follows:(1)The CD spectrum technique showed that Py25 and its possible folding sequences Py25(1245)and Py25(1234)could fold to i-motif structures at pH ≤ 5.8,and the 1245-type was more thermodynamically stable than the 1234-type.Then the 1245-type i-motif was confirmed to be the major structure of Py25 by the smFRET technique.(2)Using different protein purification strategies,full-length hnRNP K and three fragments of KH were expressed and purified.The bulk fluorescence spectra showed that Py25 i-motif structure was unfolded by hnRNP K,and the higher the concentration of hnRNP K,the faster the unwinding rate of Py25 i-motif.(3)The smFRET method was used to monitor the details of hnRNP K unfolding the c-MYC i-motif in real time at the single-molecule level.The results showed that hnRNP K unfolded the i-motif structure through two intermediate states,which were assigned to the opposite hairpin and neighboring hairpin,as further confirmed by site mutations.(4)The three KH domains reacting with the c-MYC i-motif structure,it was found that all three of the KH domains could resolve the i-motif structure,and KH2 had the strongest activity.Finally,gel chromatography revealed that hnRNP K was mainly active in the dimer state.This study revealed the details of hnRNP K unfolding c-MYC i-motifs at the single-molecule level,which provided a scientific basis for people to understand the interaction between hnRNP K and i-motif,as well as a reference for the subsequent design of i-motif-related drugs. |
