Using Single Molecule Force Spectroscopy To Study The Effects Of Ligands On The Stability And Dynamics Of SAM-I Riboswitch

Riboswitches are important gene regulatory elements in organisms.The riboswitch stabilizes its structure in a certain"on"or"off"conformation by binding ligands,and affects the expression of downstream genes.The key questions to understand the function of riboswitches include:1)how the ligand affects the structural stability of the riboswitch,and 2)whether the riboswitch can reach equilibrium between the two conformations before downstream genes are expressed.By measuring the changes in the structure and thermodynamic stability of the riboswitch under the conditions of magnesium ions and ligands,as well as the kinetics of the formation of the riboswitch structure,the above problems can be well explained.Traditional methods usually measure the static structure of a molecule in a specific conformation,and lack information on molecular structure transition and dynamics.The single-molecule method can measure the intermediate state of the structural transition and correspond to the specific structure of the molecule.It is also very beneficial for the measurement of kinetics and is an important supplement to the traditional method.Using the advantages of single-molecule force spectroscopy based on atomic force microscopy(AFM)in the measurement of molecular intermediate states,we can understand the differences in the intermediate state and structure of the riboswitch under the conditions of magnesium ions and ligands,as well as state transitions and energy variation information,which helps to explain the relationship between structural changes and functions.Although the excellent time resolution of AFM is very conducive to measuring the intermediate state,the traditional single-molecule force spectroscopy based on AFM is easy to ignore the intermediate state information of RNA molecules due to insufficient accuracy and stability at low force(5-30 pN)levels.At the same time,AFM probes usually use non-specific attachment to measure force spectra,which reduces the efficiency of force spectroscopy data acquisition.Here,we use the focused ion beam(FIB,focused ion beam)to process the probe to improve the stability and accuracy of the measurement,so that more intermediate states of the riboswitch can be observed,and the specific attachment of the AFM probe to the molecule can efficiently obtaine interpretable force spectrum data,which facilitates our energy and dynamic analysis.Based on the improvement of the AFM measurement method,we measured the intermediate state,energy and kinetic parameters in the unfolding process of the SAM-I riboswitch.The unfolding path shows that the SAM-I riboswitch has six intermediate states:F,P4,P2P3,P2,P2?part,and U,corresponding to the P1 structure,binding pocket and pseudoknot structure,P4 structure,P3structure,and P2 structure.Although the SAM-I riboswitch did not show a significant difference in the intermediate state due to insufficient measurement accuracy under the conditions of magnesium ion and ligand,it showed a significant change in the unfolding force of the intermediate state F,indicating that the ligand has a positive effect on F?P4,which include The P1 structure,binding pocket and tertiary structure.By measuring the free energy change?G during the unfolding process,it is found that the ligand increases?G by about 9±7 kal/mol,indicating that the ligand may increase the overall free energy of the riboswitch by stabilizing the structure of the F?P4 transition.Based on the kinetic analysis results of the transition of the F state during the unfolding process,the addition of the ligand reduces the dissociation rate constant₀ of the F state from 0.05±0.09^-1to 0.01±0.01^-1,indicating The ligand raises the energy barrier for F-state unfolding.The experimental results support that the ligand helps the SAM-I riboswitch to perform the function of gene regulation by stabilizing these three structures.However,the analysis of the folding speed of the riboswitch requires the experimental results of folding dynamics.The higher resolution force spectroscopy we have developed makes it possible for AFM to measure the force spectra of riboswitches,which enhances the applicability of AFM in the study of riboswitches.In the future,if the resolution of the AFM measurement can be further improved,we can reconstruct the free energy spectrum of the folding and unfolding of more intermediate states of the SAM-I riboswitch,and give a complete picture of the energy and dynamics of the state transition,so that it can be more Clearly explain the regulatory function of the SAM-I riboswitch.