A single molecule study of the conformational dynamics of nucleic acids in different environments

The selection of 5′ splice sites in pre-mRNA transcripts containing multiple splice sites is poorly understood. RNA flexibility and how it affects splice site selection has not currently been investigated on a molecular level. However, secondary structures in proximity to the splice site and RNA-protein/small molecule binding events has been shown to shift splicing using ensemble fluorescence methods. Evidence suggests that using small molecules which target cancerous mutations caused by incorrectly spliced genes such as induced myeloid leukaemia cell differentiation and β-cell lymphoma proteins may have therapeutic application. Single molecule experiments are not limited by ensemble averaging and so can be used to study the conformational flexibility of individual RNA molecules in functional splicing conditions.

Single molecule fluorescence resonance energy transfer was used in this thesis to monitor the conformational flexibility of different nucleic acids including DNA (Holliday junction and molecular beacon) and RNA (adenovirus and β-cell lymphoma sequences) structures. The experiments were performed under different conditions using various methods of surface passivation and surface tethering (direct tethering and encapsulation in lipid vesicles). The experiments outlined here indicate two main things: firstly, that monitoring nucleic acids which are freely diffusing in lipid vesicles (100 nm) is more representative of physiological conditions. Single molecules in lipid vesicles demonstrated more homogeneous conformational dynamics than those samples which had been tethered directly to the imaging surface. Secondly, we have shown that the G-quadruplex putative to the Xs splice site in the β-cell lymphoma gene forms and is stabilised by ellipticine derivative, GQC-05 in HeLa cell nuclear extract. This suggests that the stabilisation of the quadruplex and alteration to mRNA flexibility upon binding likely underlies its ability to shift splicing of β-cell lymphoma, from an anti- to a pro-apoptotic isoform. The single molecule results presented here complement previous literature and combined suggest that GQC-05 is a candidate pro-drug molecule for the therapeutic relief of tumours.