Biophysical study of DNA at single molecule level using solid-state nanopores

Abstract

Since the discovery of deoxyribonucleic acid (DNA) over 140 years ago, this biomolecule still remains one the most studied macromolecules in nature. The modifications and interactions associated with this duplex biopolymer have been shown to play fundamental roles in cellular machinery. This research project exploited sets of single-molecule detection techniques in parallel with conventional molecular biology methodologies to study i) chemical modification (methylation) of the DNA and its functional role and ii) electrostatic interaction between two homologous DNA duplexes. In this project solid-state nanopores were utilised as a novel approach to probe structural and conformational changes of linear and circular DNA. To begin with, the effect of DNA methylation level in breast cancer cell-lines was investigated. Using solid-state nanopore sensors and a methyl specific antibody (5’-mc), the methylated and unmethylated regions of FOXA1 (a gene associated with breast cancer development) promoter were differentiated. Simultaneously, the methylation level of this gene was evaluated in various breast cancer cell-lines and confirmed the impact of DNA methylation in gene silencing. In addition, using atomic force microscopy analysis, the binding affinity of the antibody to the methylated DNA was determined Furthermore, by employing the same methodologies, the presence of an electrostatic recognition step in homologous segments of a bacteria plasmid within the framework of Kornyshev-Leikin theory was investigated. However to this end, the verification of this model was inconclusive. Nevertheless it was serendipitously found that the plasmid with homologous regions was dimerised and then formed a single loop. This finding would be the motivation behind further experiments to gain a better understanding of the possible sequence dependence of the DNA topology and configuration during cloning and amplification procedures. Furthermore, using a combination of various techniques, the biophysical properties of the monomeric and dimeric plasmids were characterised. Overall, the combined findings of the mentioned projects provided remarkable insights on the molecular biophysics of DNA-DNA and DNA-protein interactions within the framework of the central dogma of molecular biology.