Probing the structure and interactions of DNA using Single-Molecule Multiparameter Fluorescence Detection

Abstract

A powerful approach, combining the strengths of high-resolution singlemolecule FRET, time-resolved spectroscopy, and molecular modeling (MD) is developed and used in two studies to explore the global structure of selected DNA molecules.Branched nucleic acids are key intermediates in DNA replication, recombination, and repair and serve as building blocks in nanoscience applications. The local and global structure of a fully complementary DNA three-way junction (3WJ) in solution is presented. A Y-shaped, pyramidal structure is adopted and clearly visualised and defined. Further exploration finds no evidence for coaxial stacking of the arms in the presence of Mg2+ and the global structure is found to be maintained upon changing DNA sequence. Local structure reveals an unpairing of the bases adjacent to the branchpoint, despite the full complementarity of the molecule. This unpairing allows a nanoscale cavity to form, which has clear applications in the development of new DNA-based supramolecular receptors and nanosystems.Cisplatin is a widely used anticancer drug that works by forming cross-links with DNA. The structure of these cisplatin-DNA adducts is vital in understanding the mechanism of action of cisplatin and to aid in the development of new drugs.Cisplatin-DNA interactions are studied using FRET at the ensemble and singlemolecule level. Whilst previous research suggests a bending of DNA upon cisplatin binding, this is not observed using the MFD technique. Quenching and reaction conditions are explored as reasons for this observation. Furtherdevelopments and investigations into dyes used and single-molecule photophysics are discussed and evaluated. The technique employed in this research has proved to be robust, accurate and powerful. The MFD approach measures the color, lifetime, polarisation and intensity of fluorescence simultaneously. The accurate FRET distances determined using this experiment have been combined with MD simulations to produce global structures at the single-molecule level. Within these investigations the technique has been tested and validated using controlexperiments. As well as contributing to the research surrounding branched-DNA molecules and cisplatin-DNA interactions, these studies have exposed the limitations of ensemble measurements. The combinatory technique presented has clear applications, with many potential structures that could benefit from this powerful analysis.

Date of Award	1 Aug 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Steven Magennis (Supervisor)