Single-molecule detection of a guanine(C8) - thymine(N3) cross-link using ion channel recording

摘要

The capability to identify and sequence DNA damage within the context of the genome is an important goal for medical diagnostics. However, currently available methods are not suitable for this purpose. Ion channel nanopore analysis shows promise as a potential single-molecule method to sequence genomic DNA in such a way that also allows detection of base or backbone modifications. Recent studies in human cell lines demonstrated the occurrence of a new DNA cross-link between guanine(C8) and thymine(N3) (5′-G*CT*-3′). The current work presents immobilization and translocation studies of the 5′-G*CT*-3′ cross-link in a single-stranded oligodeoxynucleotide using the α-hemolysin (α-HL) ion channel. A 3′-biotinylated DNA strand containing the 5′-G*CT*-3′ cross-link was incubated with streptavidin that allowed immobilization of the DNA in the β-barrel of α-HL. In this experiment, the 5′-G*CT*-3′ cross-link was placed near the sensitive constriction zone of α-HL, yielding a 2.5% deeper blockage to the ion current level when compared to the unmodified strand. Next, free translocation of a cross-link-containing strand was studied, and an inverse relationship of the time constant with respect to an increase in the applied voltage was found, indicating that the cross-link can easily fit into the β-barrel and traverse through the ion channel. However, a modulation in the ion current level was not observed. These studies suggest that higher resolution ion channels or mechanisms to slow the translocation process, or both, might ultimately provide a mechanism for single-molecule sequencing for G-T cross-links.