Primary Supervisor: Professor Mike Hannon, School of Chemistry
Secondary supervisor: Pawel Grzechnik, Biosciences, UoB; Zania Stamataki, IBR, UoB
PhD project title: Supramolecular recognition of specific nucleic acid structures in the untranslated region of viral genomes
University of Registration: University of Birmingham
RNA structure targeting is a new frontier for molecular design. In particular functional RNA structures in structurally-conserved untranslated regions (UTRs) of many viruses are intriguing targets. These UTRs are not only highly structured but often share common structural elements that are functionally essential and so conserved as the virus evolves (drifts) genetically. Structure-affecting mutations in the UTR have been used to create live attenuated or inactivated vaccine strains showing their importance. In RNA viruses, such as HIVs, coronaviruses, dengue and zika, functional involvement of the UTR has been shown in either initiation of replication (for example by recruiting proteins or by direct interaction with the ribosome) or regulation of the replication cycle. This project will explore how to design new agents that bind these functional structures. By binding and interfering with them, and then studying the effects, we will learn more about the role of these structures in the viral infection cycle.
We have previously shown that nanoscale metallo-supramolecular cylinders are unique nano-drug that can bind within an RNA cavities (a perfect three-way junction (3WJ)) (see Figure) (1). These cylinders also bind bulge structures in RNA, prevent TAT protein from recognizing the binding site in the TAR sequence of HIV and arresting HIV replication in mammalian cells (2).
In this project we will develop this approach to explore new designs to recognise RNA structures in UTRs. We will focus initially on the UTRs of coronaviruses (e.g. SARS-CoV-2; and as permitted, SARS-CoV; MERS-CoV; hCoV-229E and hCoV-NL63) which as well as humans also affect a range of farmed and pet animals (such as IBV in birds, TGEV in pigs and FIPV in cats), and later explore other RNA viruses. RNA shape prediction (computation) will be used to identify the UTR structures and their positions and will be followed by molecular dynamics simulations to identifies the molecular detail of structures and suitable agents to bind them. Chemical synthesis and design will be used to prepare the new binding agents, which will be fully characterised using a range of analytical techniques. Computational predictions will be confirmed by “wet-lab” approaches. The successful candidate will establish experimental and bioinformatics pipeline for high-throughput RNA sequencing analysis SHAPE-seq, (Selective 2’-Hydroxyl Acylation Analyzed by Primer Extension Sequencing) to test the viral RNA secondary structures, and how they are affected by the interaction with the cylinders.
RNA binding studies in vitro will also employ a range of spectroscopies and complement in cellulo virology studies to assess how the RNA-binding affects different viruses inside cells.
The approach and understanding developed will represent a new roadmap for design of drugs to target RNA structural motifs across biology and nucleic acid nanoscience, that can allow us to probe the role of such structures in viral infection.
- Binding of a Designed Anti-Cancer Drug to the Central Cavity of an RNA Three-Way Junction. S. Phongtongpasuk, S. Paulus, J. Schnabl, R. K. O. Sigel, B. Spingler, M. J. Hannon, E. Freisinger, Angew. Chem. Intl Ed, 2013, 52, 11513--11516. DOI: 10.1002/anie.201305079
- Metallo supramolecular cylinders inhibit HIV-1 TAR-TAT complex formation and viral replication in cellulo. L. Cardo, I. Nawroth, P.J. Cail, J.A. McKeating, M. J. Hannon, Scientific Reports, 2018, 8, Article number 13342. DOI: 10.1038/s41598-018-31513-3.
BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology
Techniques that will be undertaken during the project:
- Molecular Dynamics simulations on RNA and drugs that recognise it
- High-throughput Illumina RNA sequencing (SHAPE-seq)
- RNA secondary structure computational analysis and prediction
- Molecular probe design and chemical synthesis
- Compound characterisation
- RNA Binding studies using spectroscopies
- Growing and handling mammalian cells and infecting them with viruses and the binding agents
- Fluorescent microscopies (high content imaging for automated enumeration of viral infection, super resolution imaging of infected cells to study viral replication)
Contact: Professor Mike Hannon, University of Birmingham