Temperature and electrolyte optimization of the α-hemolysin latch sensing zone for detection of base modification in double-stranded DNA

The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12–35°C) and KCl concentration (0.15–1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼8 kJ mol−1 decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes

Base-excision repair activity of uracil-DNA glycosylase monitored using the latch zone of α-hemolysin

Nanopores have been investigated as a simple and label-free tool to characterize DNA nucleotides when a ssDNA strand translocates through the constriction of the pore. Here, a wild-type α-hemolysin protein nanopore was used to monitor DNA repair enzyme activity based on base-specific interactions of dsDNA with the vestibule constriction “latch”, a previously unrecognized sensing zone in α-hemolysin specific for dsDNA structure. The presence of a single abasic site within dsDNA that is in proximity to the latch zone (±2 nucleotides) results in a large increase in ion channel current, allowing accurate quantitation of the kinetics of base repair reactions involving an abasic site product. Taking advantage of the high resolution for abasic site recognition, the rate of uracil-DNA glycosylase hydrolysis of the N-glycosidic bond, converting 2′-deoxyuridine in DNA to an abasic site, was continuously monitored by electrophoretically capturing reaction substrate or product dsDNA in the ion channel vestibule. Our work suggests use of the nanopore as an enzymology tool and provides a means to identify single base structural changes in dsDNA