Resolving Atomic-resolution Nucleic Acid Ensembles Using Solution State Nuclear Magnetic Resonance

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Nucleic acid molecules do not fold into static 3D structures but rather adopt various 3D conformations that interconvert over a wide range of timescales from pico-seconds to seconds under solution conditions. These conformational transitions are oftentimes involved in many fundamental biological processes, such as nucleic acid recognition and catalysis. A collection of these inter-converting 3D conformations with their Boltzmann-weights is referred to as a ‘dynamic ensemble’. Determining dynamic ensembles is important for elucidating the biological roles of nucleic acids, but this remains very difficult due to the enormous gap between the data required to describe an ensemble versus the experimental data that we can bring to bear. This dissertation develops new methods to determine nucleic acid dynamic ensembles at atomic resolution using solution state nuclear magnetic resonance (NMR) spectroscopy and applies it to three model systems.We developed a new approach to determine the ground state ensembles of RNAs with specific application to the helix-junction-helix motif the HIV-1 transactivation response element (TAR). The approach directly generates starting ensembles from RNA secondary structures using a structure prediction method, Rosetta’s Fragment Assembly of RNA with Full-Atom Refinement (FARFAR). The ensemble is then refined by using NMR residual dipolar couplings (RDCs). By testing the ensemble accuracy using quantum calculations of chemical shifts, comparison to existing crystal structures and atomic mutagenesis, we demonstrated that by starting from a FARFAR ensemble, a more accurate ground state ensemble for TAR is obtained relative to a previously determined ensemble generated using molecular dynamics (MD) simulations. We applied a similar approach to determine dynamic ensembles for lowly populated short-lived states of nucleic acids with specific application to A-T Hoogsteen base pairs (bps) in duplex DNA. We describe a strategy to resolve the dynamic ensembles of such low-abundant short-lived conformational states by combining chemical mutagenesis, NMR relaxation dispersion (RD) and RDCs, MD simulations and quantum calculations of chemical shifts. The dynamic ensembles reveal key structural features of Hoogsteen bps: the DNA helix is more constricted and kinked towards the major groove direction and this is accompanied by local sugar and backbone deformations. These unique structural fingerprints could subsequently be used to identify 13 A(syn)-T and 4 G(syn)-C+ Hoogsteen bps in protein-DNA complexes in the Protein Data Bank (PDB) which were mismodeled as Watson-Crick, revealing a greater tendency to form Hoogsteen bps near chemically or structurally stressed DNA regions. NMR methods have also been developed to study the hybridization kinetics of DNA and RNA duplexes. This non-invasive approach relies on NMR RD to measure the kinetics of nucleic acid hybridization and structurally assign the melted species of DNA and RNA duplexes at high temperature. With this approach, we show that the epitranscriptomic modification m6A slows the annealing rate of RNA duplexes, without substantially affecting the melting rate, potentially explaining how m6A slows down a variety of biologically important transitions such as the tRNA selection during mRNA translation, and the NTP incorporation during DNA replication and reverse transcription.






Shi, Honglue (2021). Resolving Atomic-resolution Nucleic Acid Ensembles Using Solution State Nuclear Magnetic Resonance. Dissertation, Duke University. Retrieved from


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