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Mapping the three dimensional structure of RNA

Primary Supervisor: Professor Martin Wills, Department of Chemistry

Secondary supervisor: Professor Yue Wan, A*STAR Singapore and Dr Christophe Corre, Department of Chemistry and School of Life sciences

PhD project title: Mapping the three dimensional structure of RNA

University of Registration: University of Warwick

Project outline:

We aim to combine organic synthetic chemistry, molecular biology, virology and computational biology tools to elucidate the 3D structure determinants behind RNA viruses such as dengue virus and coronaviruses. Most RNA viruses including Dengue, Zika and coronaviruses utilize their RNA genome, which folds into complex structures to interact with host and viral factors for genome replication and packaging. While it is possible to determine whether a base is paired or unpaired along the genomes using high throughput structure probing techniques, this information is insufficient to decipher the higher-order conformation of the virus genomes. Here, we aim to design a range of small molecular probes which are capable of binding two proximal sites in an RNA strand, within a fixed distance of each other as defined by the structure of the small molecule reagent. Following attachment, the RNA is extracted from a cell and isolated using its biotin tag (also a part of the small molecule) by streptavidin beads, converted into a cDNA library and then subsequently sequenced using high throughput sequencing. Through this approach, the three-dimensional folded structure of the RNA can be mapped in detail. Variation of the spacing using a library of reagents can map separations between regions of RNA in even greater detail. Hence, the detailed structure of the folded RNA in its native state in cells can be established.

We will first apply the method on rRNA in vivo, as this acts as a “gold-standard” to determine the sensitivity and accuracy of our strategy. We will then aim to apply the method to more complex Dengue and coronaviruses viruses, which are important human pathogens with high social and economic burden across the world.

Synergy and complimentary of expertise between the two groups, one in Warwick can one in Singapore in 1) synthetic organic chemistry and 2) cell biology and virology, is critical for this project to succeed. The (1) development of a range of new molecular probes by MW, (2) will be used by YW to map RNA 3D structures using high throughput sequencing. YW will also integrate the structural information into 3D modelling to better elucidate 3D structures in Dengue viruses and coronaviruses. During the course of the research project, the MIBTP PhD student at Warwick will be responsible for the synthesis of a range of chemical probes which will be sent to Singapore, i.e. the Warwick project will be significantly focussed on synthetic organic chemistry. Researchers in the Singapore group will evaluate these for reactions within cells through the in vivo and in vitro processes. Neither group could complete the full set of studies independently, so this collaboration is essential for the project to be a success.

Relevant references:

  1. A. Mistry, R. C. Knighton, S. Forshaw, Z. Dualeh, J. S. Parker and M. Wills, Synthesis and cycloaddition reactions of strained alkynes derived from 2,2'-dihydroxy-1,1'-biaryls, Org. Biomol. Chem. 2018, 16, 8965 - 8975.
  2. E. M. Sletten and C. R. Bertozzi, Bioorthogonal chemistry: fishing for selectivity in a sea of functionality, Angew. Chem., Int. Ed. 2009, 48, 6974–6998.
  3. R.G. Huber, X.N. Lim, W.C. Ng, A.Y.L. Sim, H.X. Poh, Y. Shen, S.Y. Lim, K. B. Sundstrom, X. Sun, J.G. Aw, H.K. Too, P.H. Boey, A. Wilm, T. Chawla, M. M. Choy, L. Jiang, P.F. de Sessions, X.J. Loh, S. Alonso, M. Hibberd, N. Nagarajan, E.E. Ooi, P.J. Bond, O.M. Sessions, Y. Wan. Structure mapping of dengue and Zika viruses reveals functional long-range interactions. 2019. Nature Communications, Mar 29;10(1):1408.
  4. J.G. Aw, A. Wilm, M. Sun, Y. Shen, X.N. Lim, KL. Boon, YS. Chan, T. Zhang, S. Tapsin, T.T. Susanto, N. Nagarajan, Y. Wan (2016) In vivo mapping of eukaryotic RNA interactomes reveals principles of higher-order organization and regulation. Molecular Cell 2016 May 19;62(4):603-17.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology

Techniques that will be undertaken during the project:

In Warwick; chemistry, synthetic organic chemistry, small molecule characterization, use of NMR, MS and normal synthetic chemistry characterisation methods.

NOTE that the MIBTP student on this project will be located in Warwick and will carry out the work below:

In detail: The Warwick-based applicant benefits from expertise in the development of synthetic chemistry methodology, much of which has been collaborative and relevant to medicinal chemistry. The Warwick group will complete the design and synthesis of an extensive range of new custom-designed molecular probes which are designed to attach to sites in the RNA by a chemical process. This will facilitate the RNA studies at Singapore, which has used prototype molecules from Warwick to secure the preliminary results which underpin this application. In the PhD project, the Warwick group will generate more complex molecular probes of varying length, which will be provided to the Singapore group for evaluation.

In Singapore; molecular biology, biochemistry, virology, computational biology.

This will be done by collaborator in Singapore and not at Warwick however the Warwick-based MIBTP student will be part of the overall project.

In detail: We aim to utilize the different molecular probes generated by MW to capture RNA helices that are in close spatial proximity. We will treat human cells with a molecular label, which intercalates into double stranded regions of RNAs. We will then fix the cells and add the molecular probes generated by MW to the cells to capture nearby labels using selective chemistry. We will then extract the RNA from the cells and identify RNA helical interactions. Chimeric mapping after deep sequencing will enable us to determine which regions close in space. We will then integrate the through-space data with structure modeling to determine whether it generates more accurate structure models using rRNA and apply that to RNA viruses.

Contact: Professor Martin Wills, University of Warwick