50 Single-molecule studies of human telomeric G-quadruplexes and the effect of oxidative damage

Abstract

In the human genome, telomeric DNA has tandem repeats of the sequence 5′-TTAGGG terminating with a 3′ single-stranded overhang of 100–200 bases. These guanine-rich DNA sequences can fold into tetrastranded structures, known as G-quadruplexes. The precise fold of the G-quadruplex structure is dictated by the metal ions present which we studied through the use of the α-hemolysin ion channel. Being electrophoretically driven into the cis side of the α-hemolysin, the hybrid fold (K+) entered the vestibule mouth leading to current blockages for the duration of the time the DNA resided in the vestibule. Due to the polymorphic nature of the hybrid folds, the recorded current signatures could be correlated with the major structural topologies that exist for this fold in solution (e.g. hybrid-1, hybrid-2, and triplex; Mashimo et al., 2010). The hybrid folds were not capable of traversing to the trans side of the nanopore, while the triplex could achieve translocation. The basket fold (Na+) was also able to enter into the vestibule causing current blockages that were indicative of the orientation in which they entered into the vestibule. When the basket fold entered tail first, slow translocation events were observed. In contrast, the propeller fold (∼3.9 nm, 0.05 M K+/5 M Li+) exceeds the protein channel orifice (∼3.0 nm) producing only swift and small disturbances to the open channel current. Secondly, oxidative damage to the telomeric sequence is proposed to contribute to telomere shortening, dysfunction, and cell aging (Epel et al., 2004). Locations of the oxidative damages have different effects on the G-quadruplex folding that produced significant changes in their nanopore behavior. Placement of the guanine oxidation product, 8-oxoguanosine (OG), in a top or bottom tetrad results in destabilization of that layer, whereas the presence of OG in a middle tetrad leads to complete unfolding of the G-quadruplex. These behaviors were determined by their translocation times which correlated with the folding’s free energy supported by NIH GM093099.