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Using novel ferronucleosides to study and regulate DNA replication in cells

Primary Supervisor: Dr Nikolas J Hodges, School of Biosciences

Secondary supervisor: Prof James HR Tucker, School of Chemistry

PhD project title: Using novel ferronucleosides to study and regulate DNA replication in cells.

University of Registration: University of Birmingham

Project outline:

Background: Replication of nucleic acid polymers like DNA is an essential process for all known forms of life. Furthermore, dysregulation and errors in DNA-replication are strongly linked with human diseases including cancer, as well as the process of cellular ageing. Therefore, an understanding of DNA-replication, its regulation and how this may be manipulated in cells is not only of fundamental importance to understanding the fundamental “rules of life” but also offers practical opportunities for development of new therapeutic strategies to treat disease. This is exemplified by the fact that currently, most existing anti-cancer treatments target aberrant DNA-replication in one way or another. Ferronucleosides are analogues of naturally occurring nucleosides that are based on the organometallic complex ferrocene. They have been shown to have many interesting chemical and cellular properties as well as potential applications including anti-bacterial, anti-viral and anti-cancer agents.

The Hodges and Tucker laboratories have developed and begun to study the cellular properties of a novel ferronucleoside called TUC1 a mimetic of thymidine. We have already shown that TUC1 is a potent inhibitor of DNA replication in cells and have observed this at the single molecule level directly DNA-fibre fluorography. However, currently the mechanism of action of TUC1 at the molecular level remains to be fully elucidated.

Aims and methodology: The objective of this project is to understand how TUC1 inhibits DNA replication at the molecular level both experimentally and by using molecular modelling. We will then apply this information to investigate how TUC1 can be used to study and regulate the process of DNA-replication in cells.

Identifying the target(s) of TUC1: Preliminary molecular docking studies have suggested that TUC1 can bind to DNA polymerase, an interaction that is further thermodynamically stabilised in the presence of double stranded DNA. This will be investigated further using more detailed simulations and a range of polymerases and DNA targets. To investigate this experimentally we will study if TUC1 can inhibit DNA replication in cell free DNA-replication assays. Potential specificity for different DNA-polymerases (bacterial, human, viral) will be investigated. We will also investigate if TUC1 is able to directly interact with dsDNA and other nucleic acid structures (Y-shaped junctions, ssDNA, RNA, G-quadraplexes) using gel shift assays.

Using TUC1 to study DNA interactions in cell free and cellular systems: One of the current aims of our laboratory is to use click chemistry to create fluorescent analogues of TUC1 that can then be used as “molecular probes” to visualise TUC1 directly and to study how it interacts with replicating DNA-strands. Fluorescent TUC1 analogues in combination with DNA-fibre fluorography using cell free systems (calf thymus DNA) and cellular systems will be used to study this process and also to identify interactions of TUC1 with protein components of the replication machinery like DNA-polymerase. Through collaboration with Prof Zoe Pikramenou, we are also planning to develop TUC1-fluorescent gold nanoparticle complexes as another novel mechanism for imaging interactions of TUC1 with DNA.

References:

  1. Rana et al 2021, A novel ferronucleoside that targets DNA replication in pancreatic cancer cells. DOI: https://doi.org/10.21203/rs.3.rs-741964/v1.
  2. Ismail et al 2020, Effect of Regio-chemistry and Methylation on the Anti-cancer Activity of a Ferrocene‐Containing Organometallic Nucleoside Analogue. https://doi.org/10.1002/cbic.202000124.
  3. Ismail et al, 2020, Organometallic nucleoside analogues: effect of the metallocene metal atom on cancer cell line toxicity. https://doi.org/10.1039/C9DT04174E

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology & Integrated Understanding of Health: Ageing & Pharmaceuticals

    Techniques that will be undertaken during the project:

    • Gel shift assays
    • Single molecule DNA fibre fluorography
    • In vitro DNA replication assays
    • Confocal microscopy
    • Cell culture

    Contact: Dr Nikolas J Hodges, University of Birmingham