The folding of large RNA molecules can be approached as a hierarchical process in which pre-formed substructures assemble into the final, complex 3-dimensional structure. Important and little understood RNA building units are the multi-helix junction loops, the simplest of which are the three-way junctions (3WJ). Such structures occur frequently in large RNAs; a better knowledge of their folding rules would significantly improve our ability to predict and model the three-dimensional structures of a large variety of RNAs. In vitro selection and evolution methodologies (hereafter referred to as SELEX) will be employed to explore the molecular interactions which stabilize and define the structures of RNA 3WJ. Dr. Leontis will apply SELEX to generate, select, and amplify a library of RNA molecules capable of forming 3WJ structures, in which nucleotide positions surrounding the junction site are randomized. The most compact and thermally stable species in the RNA pool will be selected by thermal gradient gel electrophoresis and amplified reiteratively. After sufficient cycles have been completed, such that a large percentage of the pool exhibits the desired physical properties, a sampling of RNA sequences from the pool will be cloned, sequenced, and chemically and enzymatically probed to characterize the atomic interactions which stabilize the structures. Molecular modeling techniques will be employed to construct 3-dimensional atomic models of the structures of representative molecules using the probing data as a guide. Modeling studies will be carried out in collaboration with Dr. Eric Westhof, a leader in the field of RNA structure modeling. The thermal stabilities of these molecules will be measured using UV-monitored thermal analysis (""""""""UV melting"""""""") and isothermal titration calorimetry to provide thermodynamic parameters to correlate with the structural models. Further experimental verification for the 3-dimensional structures will be sought by attempting to crystallize interesting sequences. If crystals are obtained, crystallographic analysis will be pursued. Solution structures will be characterized using multi-dimensional/multi-nuclear NMR spectroscopy.
| Zirbel, Craig L; Roll, James; Sweeney, Blake A et al. (2015) Identifying novel sequence variants of RNA 3D motifs. Nucleic Acids Res 43:7504-20 |
| Cannone, Jamie J; Sweeney, Blake A; Petrov, Anton I et al. (2015) R3D-2-MSA: the RNA 3D structure-to-multiple sequence alignment server. Nucleic Acids Res 43:W15-23 |
| Abu Almakarem, Amal S; Petrov, Anton I; Stombaugh, Jesse et al. (2012) Comprehensive survey and geometric classification of base triples in RNA structures. Nucleic Acids Res 40:1407-23 |
| Novikova, Irina V; Hassan, Bachar H; Mirzoyan, Marina G et al. (2011) Engineering cooperative tecto-RNA complexes having programmable stoichiometries. Nucleic Acids Res 39:2903-17 |
| Petrov, Anton I; Zirbel, Craig L; Leontis, Neocles B (2011) WebFR3D--a server for finding, aligning and analyzing recurrent RNA 3D motifs. Nucleic Acids Res 39:W50-5 |
| Takeda, Ryuta; Petrov, Anton I; Leontis, Neocles B et al. (2011) A three-dimensional RNA motif in Potato spindle tuber viroid mediates trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana. Plant Cell 23:258-72 |
| Šponer, Ji?í; Šponer, Judit E; Petrov, Anton I et al. (2010) Quantum chemical studies of nucleic acids: can we construct a bridge to the RNA structural biology and bioinformatics communities? J Phys Chem B 114:15723-41 |
| Besseova, Ivana; Reblova, Kamila; Leontis, Neocles B et al. (2010) Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome. Nucleic Acids Res 38:6247-64 |
| Zirbel, Craig L; Sponer, Judit E; Sponer, Jiri et al. (2009) Classification and energetics of the base-phosphate interactions in RNA. Nucleic Acids Res 37:4898-918 |
| Stombaugh, Jesse; Zirbel, Craig L; Westhof, Eric et al. (2009) Frequency and isostericity of RNA base pairs. Nucleic Acids Res 37:2294-312 |
Showing the most recent 10 out of 40 publications
