Tuesday, March 1, 2016
topological polymorphism of chromatin fibers may be utilized by cells in the process of transcription, which is known to generate different levels of DNA supercoiling upstream and downstream from RNA polymerase. Genomewide analysis of the NRL distribution in the active and silent yeast genes is consistent with this assumption.
RNA Structure and Dynamics 2017-Pos Board B161 Characterizing the Structural and Energetic Properties of tRNA during Translocation through a Nanopore Prasad Bandarkar, Huan Yang, Meni Wanunu, Paul Whitford. Northeastern University, Boston, MA, USA. The fidelity of mRNA translation depends not only upon the codon-anticodon pairing between the mRNA and the tRNA, but also upon the structural aspects of the tRNA that are far from the anticodon. Therefore many experimental techniques aim to probe the conformational properties of different tRNA molecules. Electrophoretic translocation of biomolecules through a solid-state nanopore represents a label-free single molecule technique that may be used to identify molecules that have distinct structural, or dynamic properties. Recent investigations have attempted to distinguish individual tRNA species by characterizing the associated pore translocation times and blockage currents. These nanopores are large enough to accommodate the anticodon loop, though full translocation requires deformation of the molecule from its traditional L-shaped structure. Here, we apply MD simulations using a simplified structure-based energetics model of the molecule to study the structural changes in tRNA during translocation. We have implemented a simplified force-field between the nanopore walls and the molecule that mimics the steric effect of the pore. Through the use of umbrella sampling and replica exchange we calculated the mean first passage time (MFPT) over an effective free-energy barrier for different tRNA species. These calculations have also implicated transient unfolding of specific residues during translocation. Since the MFPT values are in agreement with the experimental dwell times, these result suggest that nanopore experiments specifically monitor partial unfolding of tRNA. These results provide a structural/energetic interpretation of current experiments, which can help refine methods for interpretation of single-molecule detection using nanopores. 2018-Pos Board B162 Synonymous Mutations Reduce Genome Compactness in Icosahedral ssRNA Viruses Luca Tubiana1, Anze L. Bozic2, Cristian Micheletti3, Rudolph Podgornik4. 1 Computational Physics, University of Vienna, Wien, Austria, 2Max Planck Institute for Biology of Ageing, Cologne, Germany, 3SISSA-ISAS, Trieste, Italy, 4University of Ljubljana, Ljubljana, Slovenia. Recent studies have shown that single-stranded (ss) viral RNAs fold into more compact structures than random RNA sequences with similar chemical composition and identical length. Based on this comparison, it has been suggested that wild-type viral RNA may have evolved to be atypically compact so as to aid its encapsidation and assist the viral assembly process. To further explore the compactness selection hypothesis, we systematically compare the predicted sizes of >100 wild-type viral sequences with those of their mutants, which are evolved in silico and subject to a number of known evolutionary constraints. In particular, we enforce mutation synonynimity, preserve the codon-bias, and leave untranslated regions intact. It is found that progressive accumulation of these restricted mutations still suffices to completely erase the characteristic compactness imprint of the viral RNA genomes, making them in this respect physically indistinguishable from randomly shuffled RNAs. This shows that maintaining the physical compactness of the genome is indeed a primary factor among ssRNA viruses’ evolutionary constraints, contributing also to the evidence that synonymous mutations in viral ssRNA genomes are not strictly neutral.  Biophysical journal 108 (1), 194-202. 2019-Pos Board B163 Mimicking Ribosomal Vectorial Unfolding of RNA Pseudoknot in a Protein Channel Xinyue Zhang1, Xiaojun Xu2, Zhiyu Yang3, Andrew J. Burcke1, Kent S. Gates4, Shi-Jie Chen2, Li-Qun Gu1. 1 Department of Bioengineering, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, USA, 2Department of Physics, Department of Biochemistry, and Informatics Institute, University of Missouri-Columbia, Columbia, MO, USA, 3Department of Chemistry, University of Missouri-Columbia, Columbia, MO, USA, 4Department of Chemistry, Department of Biochemistry, University of Missouri-Columbia, Columbia, MO, USA.
Pseudoknots are a fundamental RNA tertiary structure found in almost all classes of RNAs. They not only play a key role in diverse biological mechanisms, but also provide an excellent model for understanding RNA folding/unfolding processes involved in their functions. Molecular force spectroscopic approaches such as optical tweezers can track the pseudoknot’s unfolding intermediate states by pulling the molecule from both 5’ and 3’-ends, but the pseudoknot unfolding kinetic pathway may be different from that in vivo, which proceeds in the translation direction from the 5’ to 3’ end. Here we developed a ribosome-mimicking, nanopore pulling assay for dissecting the vectorial unfolding mechanism of pseudoknots. The unfolding pathway of pseudoknot in the nanopore, either from the 5’ to 3’ end or in the reverse direction, can be controlled by a DNA lead that is attached to the pseudoknot at the 5’ or 3’ ends. The different nanopore conductance between DNA and RNA translocation serves as a marker for the position and structure of the unfolding RNA in the pore. With this design, we identify that the pseudoknot unfolding is a two-step, multi-state, metal ion-regulated process, and along different pathways dependent on the pulling direction. Notably, the unfolding in both directions are rate-limited by the unzipping of the first helix domain, which is Helix-1 in the 5’/3’ direction and Helix-2 in the 3’/5’ direction, suggesting that the initial unfolding step in either pulling direction needs to overcome an energy barrier contributed by the non-canonical triplex base-pairs and coaxial stacking interactions for the tertiary structure stabilization. These findings provide new insights into RNA vectorial unfolding mechanism. Potentially, the nanopore assay could be adapted to study different pseudoknots and riboswitches, shedding light on their biological functions including frameshifting and anti-virus drug design. 2020-Pos Board B164 Junction Topological Constraints in Hairpin Ribozyme Folding Alex Morriss-Andrews1, Anthony M. Mustoe2, Charles L. Brooks III1. 1 Chemistry, University of Michigan, Ann Arbor, MI, USA, 2Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. The hairpin ribozyme is an RNA enzyme found in plant viruses that catalyzes cleavage and ligation reactions in RNA replication. The active site is formed when two loop domains located on separate helical arms are in contact. We conduct coarse-grained molecular dynamics simulations of the hairpin ribozyme using the TOPRNA model. By modifying the topology of the junction between the two active arms we determine how topology, junction stress, and conformational entropy bias folding propensity. Modifications include using 2- through 5-way junctions, adding a small loop region at the junction, unpairing one base pair at one or both arms, and shortening or lengthening one of the arms (changing the phase of helix-helix alignment). The TOPRNA model is ideal for answering these topological questions and, using only three beads per residue, it is efficient for throughput. This work is done in collaboration with the Walter group at the University of Michigan, who are conducting single molecule experiments on these hairpin ribozyme constructs, using FRET microscopy to determine their folding propensities. These measurements combined with our simulation data will allow us to isolate the contribution of topology in hairpin ribozyme folding. 2021-Pos Board B165 A Combined Molecular Docking/Dynamics Approach to Study Selectivity and Binding Affinity of L-Stereoisomer RNA Aptamer Towards CCL2 and Related Chemokines Senthilkumar Kailasam. Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, AB, Canada. CC chemokine ligand 2 (CCL2)- also known as Monocyte Chemoattractant Protein 1- is a primary chemoattractant in monocytes. It has a high-affinity interaction with chemokine receptor 2 (CCR2) and causes calcium mobilization and chemotaxis. In inflammatory processes and cancer, CCL2 is highly expressed. Preventing CCL2 function by interfering with CCL2-CCR2 binding can alleviate complications seen in many inflammatory diseases. Many oligonucleotide-based aptamers that target proteins are currently being developed to block the interaction of the target protein with other molecules. An L-stereoisomer oligonucleotide aptamer (emapticap pegol), targeting CCL2 was identified by Noxxon Pharma AG using an in vitro process known as ‘‘systematic evolution of ligands by exponential enrichment’’ (SELEX). This L-RNA aptamer was used to treat patients with diabetic nephropathy and completed Phase IIa of clinical trials. The mode of action of the drug was deciphered using a combination of binding and structural data; however, this data could not fully explain the observed selectivity profile of the L-aptamer. In this work, using molecular dynamics simulation approach, we were able to understand the dynamic behaviour of L-aptamer in free form and in complex with