Effects Of PIWI/MID Domain Of Argonaute Protein On The Association Of MiRNAi's Seed Base With The Target And The Thermodynamics And Kinetics Of The Pseudoknot

(1)Effects of PIWI/MID domain of Argonaute protein on the association of miRNAi's seed base with the targetRNA interference(RNAi)is a relative new technology that is revolutionizing the way that researchers study mammalian gene expression.RNA interference is the double strand RNA was converted into 18?30 nt single strand RNA,and then it combined with the Argonaute protein family to form RISC silencing complex structure.Two types of small RNAs,microRNA(miRNA)and small interfering RNA(siRNA)both can participate in the process of RNA interference.These small RNAs can directly bind to the enzyme to form a complex,which can knock-down or silence the message RNA(mRNA)gene expression through the base pair.Based on this phenomenon,people can design some drugs to inhibit or silence the expression of disease-causing genes to achieve therapeutic purposes.Therefore,RNA interference has achieved extensive attention and it is the method to exploit the new drugs.In the process of the RNA interference,the small RNAs act as the guide RNA,and the 5'-end of the guide RNA can recognize and select the target mRNA.The core region of the guide RNA is the second nucleotide to the seventh nucleotide from the 5'-end,broadly speaking,including the eighth nucleotide,which is the seed region.Many experiments indicated that the base pair between the nucleotides in the seed region and the target play a key role on silencing mRNAs,so studying the thermodynamics and kinetics propensities of the base pair between the nucleotides in the seed region and the target is very meaningful.However,Molecular dynamics simulations provide a microscopic model method on the molecular scale.It can be used to simulate the motion of the molecule,and observe the details of molecular motion and then the motion trajectory can be obtained.It is a powerful tool to study the condensed matter systems.In this paper,we used molecular dynamics simulation to study the effect of the PIWI/MID domain of the Argonaute protein on the association of the miRNA's seed base with the target mRNA.We used the high temperature long term molecular dynamics simulation and chose several different simulated temperatures,and then we can obtain the trajectories of the guide RNA and target RNA at several different simulated temperatures.When the simulation time reached the association-dissociation transition equilibrium between the guide RNA and the target RNA,and then we counted the association probability and dissociation probability.Based on the relationship between the association-dissociation probabilities at each simulated temperature,we can fit the entropy change and the enthalpy change of the association of the guide RNA on the target RNA,and then the change of entropy and enthalpy can be obtained.In this work,we used the molecule dynamics simulation to study the thermodynamic and kinetic properties of the third nucleotide over the seed region associated on the target in the presence of the Argonaute protein of the PIWI/MID domain.The following results were obtained:In the presence of the PIWI/MID domain,the entropy and enthalpy changes of the association of the seed base to the target are smaller than those in the absence of protein.Structurally,due to the presence of the protein,the movement space of the base in the seed region decreased,so the conformation number decreased.On the other hand,on account of the presence of the protein,it blocked the contact between the seed region of miRNA and the solution was blocked,and the interaction between the base in the seed region and water and ions reduced.Because the association entropy between the seed region and the target decreased,it lead to the increase of binding affinity,which indicating that the nucleotides of miRNA in the seed region are prearranged.According to the simulated trajectory at each temperature,we counted the numbers and time on the association state and dissociation state,and then the average lifetime was obtained.It showed that the average lifetime of the dissociation state showed a strong dependence on temperature,while the average lifetime of the association state showed a weak dependence on temperature,which is consistent with the experimental results.That is to say,the closed state and the open state have different temperature dependence behavior.The PIWI/MID domain of the Argonaute protein decreased the association/dissociation barrier between the base in the seed region and the target.The presence of the PIWI/MID domain would lower the association barrier resulting from the unfavorable entropy loss and the dissociation barrier coming from the disruption of hydrogen bonding and base-stacking interactions.(2)Thermodynamic and kinetic properties of H-type pseudoknotRNA structure prediction is the major discussion topic on research.In non-coding RNA structures,pseudoknots'structure is the most typical structure,and they play an important role in translation,viral genome structure and ribosomes'active sites.RNA pseudoknots can fold into a specific structure mainly depends on the rules and interactions of base pair and the interaction of the loop region.The thermodynamic parameters of the base pairing have been measured in the experiment,but the thermodynamic parameters of the loop region have not been given.In the RNA pseudoknot structure prediction,different algorithms are used to calculate the thermodynamic parameters of the loop region,but the accuracy is not very high.In order to study the effect of the loop region on the pseudoknot folding,we used molecular dynamics simulation to study the thermodynamic and dynamic properties of the pseudoknot structure.The method was similar to the first work,we used long-term high-temperature molecular dynamics simulation,the base pairs of the pseudoknot reached the open-close switch equilibrium,and molecular trajectories at several simulated temperature are obtained.Based on the trajectories obtained from the simulation,we got the following conclusions:In terms of thermodynamics:according to analysis the simulated trajectories at several temperatures,the base pair of the closed probability and the open probability can be calculated,and the enthalpy change and entropy change at the open state and the closed state were obtained by fitting the curve of ln(po/pc)and 1/T.Then we used the empirical formula to quantitatively calculate the entropy change and the enthalpy change of the stem region in the presence of the loop region.Based on the calculated result,we found the entropy change and enthalpy change when the stem region of the pseudoknot is folded is different with one single base pair.In terms of dynamics,the average life of the open state of the pseudoknot is less affected by temperature,and the average life of a closed state is greatly affected by temperature.On account of the presence of the loop region,the open rate k_of the base pair at stem region increased,and the closed rate k+decreased.