Real-time monitoring of interactions between naturally occurring proteins and DNA (or RNA) quadruplexes using whispering gallery mode resonators.

Protein-DNA interactions (PDIs) are critical for regulating many cellular processes including transcription, replication and DNA repair. PDIs are traditionally characterised based on methods including electrophoretic mobility-shift assays and nuclease footprinting. More recently, microarray-based approaches have appeared allowing for rapid, high-throughput characterisation of in vitro DNA-binding specificities. However, fewer methods are available to accurately measure interaction affinities. Among them, Surface Plasmon Resonance (SPR) technology remains the gold standard in direct biomolecular interaction sensing. It suffers, however, from a large number of drawbacks including limitations due to mass transport affecting kinetic analysis, non-specific interactions of the ligands with the matrix or elevated running cost. Herein, we propose to develop an easy-to-use, cheap and highly sensitive sensor for monitoring PDIs in real time. We will characterise the interactions between selected proteins and DNA (or RNA) targets immobilised on a whispering gallery mode (WGM) resonator. WGM sensing offers attractive prospects over more conventional technologies. Advantages of WGM sensors include real-time sub-second acquisitions, label-free, can be made using CMOS processing methods and accessibility to a non-expert (which implies that it can be used for point-of-care diagnostic applications). The operating mechanism of WGM sensing is simple: essentially resonance modes in dielectric optical micro-cavities can be used to detect minute changes in the surrounding local environment. Binding of an analyte to the cavity, or even a change in conformation of analyte already bound, will result in a shift in the resonance wavelength. Importantly real-time information such as association, dissociation, folding, and unfolding trajectories can easily be obtained making this an exceptionally valuable technique.
Within the scope of this PhD, we will be focusing on biologically relevant PDIs between naturally occurring proteins and four-stranded G-quadruplex structures (or G4) found at the end of telomeres and also widespread in human gene promoters. Formation of intramolecular G4s was first proposed to occur at the 3'-end of telomeres, thus preventing telomere maintenance by the enzyme telomerase in tumour cells. Convergent bioinformatics studies subsequently revealed the high prevalence of putative G-quadruplex forming sequences across the human genome, with a strong enrichment in untranslated regions, within 1kb upstream of the transcription start site (TSS). Severe conditions like cancer, fragile X syndrome, Bloom syndrome and Werner syndrome are related to genomic defects that involve G-quadruplex forming sequences. For this reason, G-quadruplex recognition and processing (e.g. stabilisation or unwinding) by naturally occurring proteins represent a key target to modulate physiological or pathological pathways. A large number of small molecules have appeared in the literature that bind G4s in a structure-specific manner and show biological activity both in vitro and in vivo. However, very little is known about the way these proteins recognise G4s. Whilst most G4-targeting therapeutic small molecules are assessed based on their ability to bind and/or stabilise these structures, little is known about their ability to interfere with these structure-specific PDIs. This multidisciplinary project will therefore deliver valuable tools for monitoring, in real-time and with medium- to high-throughput, the interaction between proteins and naturally occurring DNA and RNA G4s at the telomeres or in gene promoters. It will also deliver small molecules drugs that can interfere with these PDIs and as a consequence have great therapeutic (e.g. anti-cancer) potential via either inhibition of telomerase activity in cancer cells or specific transcriptional regulation of proto-oncogenes.