Thermodynamic And Kinetic Properties Of A Single Base Pair Under Mechanical Force

RNA plays diverse biological functions by adaptively acquiring different threedimensional structural conformations in response to specific conditions of the cell environment.Many RNAs regulate gene expression through conformation arrangements and the formation of biologically relevant structures is often kinetically controlled.The unfolding of RNA in biological cells by helicases or ribosomes is similar to unfolding by force,but the mechanism is still not clear.Furthermore,single-molecule technology experiments applying a force along a chosen direction to a single molecule have been widely used to control the folding reactions and study mechanical response.But it is still hard to be used to study a single base pair.CRISPR-Cas9 is an RNA-guided enzyme that leverages the sequence specificity of the guide RNA to bind and cleave complementary DNA sequences.Upon site-specific recognition of a protospacer adjacent motif(PAM)in the target DNA strand,the DNA binds to the sg RNA-Cas9 complex with the 20-nucleotide(nt)guide segment complementary base pairing,sequentially replaces the DNA duplex with the invasive RNA strand to form an RNA-DNA hybrid.Experiments have revealed that the sequence specificity of the PAM and the 11-nt seed sequence at the 3′ end of the guide RNA is critical to target recognition and cleavage,but the mechanism is still unclear.It was found that the CRISPR/Cas9-mediated cleavage efficiency and off-target events were correlated with binding free energy changes and binding kinetics.Therefore,understanding the thermodynamic and kinetic properties of the closing and opening of a single base pair is vital for a quantitative understanding of the biological functions of many RNA and RNA/DNA molecules.The thermodynamics and kinetics of single base pair under mechanical force were investigated through the constant force stretching molecular dynamics simulations.Furthermore,the effect of the Cas9 protein on the seed-target base pair of the sg RNA-DNA hybrid duplex was also obtained.The main research results are as follows:(1)Thermodynamics and kinetics of RNA base pairThe thermodynamic properties and kinetic mechanism of the opening and closing of RNA terminal base pair AU under mechanical force were studied by constant force stretching molecular dynamics simulation.It was found that high mechanical force results in overstretching,and the open state is a high-energy state.The enthalpy and entropy change of the base-pair opening-closing transition were obtained and the results were in good agreement with the nearest-neighbor model at low forces.The average lifetimes of the closed state and open state have a strong correlation with temperature and force,while the transition path time shows weak temperature and force dependences.The position of the transition state for the base pair opening-closing transition under mechanical force was determined,and the force does not change the position of the transition state.The free energy barrier of opening a base pair without force is the enthalpy increase,due to the disruption of hydrogen bonding and base-stacking interactions.The work done by the force from the closed state to the transition state would decrease the barrier and increase the opening rate.The free energy barrier of closing the base pair without force results from the entropy loss,due to the accompanying restriction from the torsional angles,the viscosity of the solvent,and so on.The work done by the force from the open state to the transition state would increase the barrier and decrease the closing rate.(2)Thermodynamics and kinetics of RNA/DNA hybrid base pairThe thermodynamic properties and kinetic mechanism of the binding and dissociation of RNA/DNA hybrid base pair under mechanical force were studied by constant force stretching molecular dynamics simulation.The entropy and enthalpy changes of the base pair of the RNA/DNA hybrid during the dissociation process were obtained,which were in good agreement with the experiments.The free energy barrier of dissociating a base pair without force is the enthalpy increase,and the work done by the force from the binding state to the transition state would decrease the barrier and increase the dissociation rate.The free energy barrier of binding the base pair without force results from the entropy loss,and the work done by the force from the dissociated state to the transition state would increase the barrier and decrease the binding rate.The transition rates are strongly dependent on the temperature and force,while the transition path times are weakly dependent on temperature.(3)The effect of the Cas9 protein on the seed-target base pair of the sg RNA/DNA hybridThrough stretching molecular dynamics simulation,we studied the thermodynamics and kinetics of the binding-dissociation process of the seed base and the target DNA base with Cas9 protein,and elucidated the mechanism that how the seed region affects the cleavage efficiency.The results showed that in the presence of the Cas9 protein,the enthalpy change and entropy change in binding-dissociation of the seed base with the target are smaller than those without the Cas9 protein.The reduction of entropy penalty upon association with the protein resulted from the pre-organization of the seed base in an Aform helix,and the reduction of the enthalpy change was due to the electrostatic attraction of the positively charged channel with the negative target DNA.The binding barrier coming from the entropy loss and the dissociation barrier resulting from the destruction of the base pair in the presence of the Cas9 protein were lower than those without protein,which indicates that the seed region is crucial for efficiently searching the correct target by accelerating the binding rate and dissociating fast from the wrong target.