| Description |
Nanopore technology has been at the center of attention during the past decade as one of the most promising methods for next-generation DNA sequencing. It proceeds by electrically drawing an individual single-stranded DNA (ssDNA) strand through a nanoscale pore, leading to detectable changes in the electrical current. Chief advantages of this technology are the minimal requirements of expensive reagents and data storage and handling equipment. The bacterial protein a-hemolysin is the most commonly used biological nanopore for this platform, due to its excellent stability, reproducibility and precise tuning properties by site-directed mutagenesis. One of the most difficult challenges of this technology arises from the low current amplitude resolution between the DNA nucleotides. The first work presented here approaches these problems by DNA site-specific chemistry to attach detectable labels to one of the most commonly occurring lesions in cells, DNA abasic (AP) sites. Several amines were used to attach to AP site, which showed detectable current changes when the ssDNA was immobilized inside of the nanopore. However, only the 18-crown-6 (18c6) moiety produced distinct current signatures during translocation, when bound to Na+. The bulky adduct also slowed down the DNA motion to more easily recordable levels, achieving the detection of individual AP sites at a single-molecule level. 18c6 can form different shapes of complexes, dictated by the surrounding ions, which was used to precisely manipulate its electrical behaviors. Secondly, the nanocavity of this protein was used to provide insights into secondary structures of the human telomeric G-quadruplexes at a single-molecule level. The folding of the repeat sequence at the end of the chromosome was shown to have significance to genome protection, and depending on the surrounding ions, it could form various quadruplexes. The interactions of these structures and the a-HL were correlated to different current patterns when the DNA was encapsulated inside of the channel, providing better understanding into the polymorphism of the human telomere sequence. Lastly, combining the above two findings, the 18c6 label was used to detect the oxidative damage of the G-quadruplexes, and the effect on the stability of these structures were also evaluated. |
