| Description |
The alpha-hemolysin (a-HL) protein ion channel is a potential next-generation sequencing platform that has been extensively used to study nucleic acids at a singlemolecule level. After applying a potential across a lipid bilayer, the imbedded a-HL allows monitoring of the duration and current levels of DNA translocation and immobilization. Because this method does not require DNA amplification prior to sequencing, all the DNA damage present in the cell at any given time will be present during the sequencing experiment. The goal of this research is to determine if these damage sites give distinguishable current levels beyond those observed for the canonical nucleobases. Because DNA cross-links are one of the most prevalent types of DNA damage occurring in vivo, the blockage current levels were determined for thyminedimers, guanine(C8)-thymine(N3) crosslinks and platinum adducts. All of these crosslinks give a different blockage current level compared to the undamaged strands when immobilized in the ion channel, and they all can easily translocate across the a-HL channel. Additionally, the a-HL nanopore technique presents a unique opportunity to study the effects of DNA cross-links, such as thymine-dimers, on the secondary structure of DNA G-quadruplexes folded from the human telomere sequence. Using this singlemolecule nanopore technique we can detect subtle structural differences that cannot be easily addressed using conventional methods. The human telomere plays crucial roles in maintaining genome stability. In the presence of suitable cations, the repetitive 5'-TTAGGG human telomere sequence can fold into G-quadruplexes that adopt the hybrid fold in vivo. The telomere sequence is hypersensitive to UV-induced thymine-dimer (T=T) formation, and yet the presence of thymine dimers does not cause telomere shortening. The potential structural disruption and thermodynamic stability of the T=T-containing natural telomere sequences were studied to understand how this damage is tolerated in telomeric DNA. The a-HL experiments determined that T=Ts disrupt double-chain reversal loop formation but are well tolerated in edgewise and diagonal loops of the hybrid G-quadruplexes. These studies demonstrated the power of the a-HL ion channel to analyze DNA modifications and secondary structures at a single-molecule level. |
