New fluorescent probes for nucleic acid quadruplex structures in cells

Project completed 2015.

Supervisors:
Professor Michael Hannon, School of Chemistry
Dr Nik Hodges, School of Biosciences
Dr Hamid Dehghani, School of Computer Science

As a result of unusual base pairing; alternative secondary structures are known to form in single stranded regions of nucleic acid containing repeats of bases. These unusual DNA structures are found in the telomeres of DNA and are also believed to be present in the promoter regions of some genes, where they may act as roadblocks to proteins processing single-stranded DNA templates transiently formed during replication. Likewise, the use of single-stranded messenger RNA (as the substrate for translation) by transcriptional machineries may also be affected. Whilst these structures are recognized to interfere with the transfer of genetic information, it is not yet understood whether they participate in natural regulatory mechanisms within the cell, or are simply toxic.

The most well-studied non-B-DNA structures are called G-quadruplexes (abbreviated G4). These four-stranded assemblies form by fold-over of single-stranded sequences containing guanine repeats. Held together by H-bonding between the bases, guanine quartets n-stack on top of one another with a monovalent metal cation sandwiched between the quartets. These G4 structures are important to study because of their role in gene activation and cancer immortality. Consequently, it is of great value to develop techniques that allow them to be visualised in cells.

Pioneering research into the structural recognition of G4 by small anthraquinone derivatives was completed by S. Neidle, L. Hurley et al. Since then, there has been a large focus on targeting these structures using small molecules in order to understand their biological function. A further objective of this field of research is to discover new targeted pharmacological agents which are able to act in a sequence or structure specific manner on DNA or RNA and exhibit anticancer properties. Up to now, the vast majority of research has focused on studying the formation of G4 structures in vitro, with their formation in vivo being supported by mostly indirect evidence such as biochemical and molecular genetics data. Recently, evidence of the presence of G4 structures in DNA within the cellular environment has been provided by S. Balasubramanian et al. using a G4 specific antibody that stabilises the structure upon binding. Building upon all previous work, the first requirement of this project is to develop a highly specific quadruplex-binding fluorescent dye.