Base Flipping within the ?-Hemolysin Latch Allows Single-Molecule Identification of Mismatches in DNA.

ABSTRACT: A method for identifying and differentiating DNA duplexes containing the mismatched base pairs CC and CA at single molecule resolution with the protein pore ?-hemolysin (?HL) is presented. Unique modulating current signatures are observed for duplexes containing the CC and CA mismatches when the mismatch site in the duplex is situated in proximity to the latch constriction of ?HL during DNA residence inside the pore. The frequency and current amplitude of the modulation states are dependent on the mismatch type (CC or CA) permitting easy discrimination of these mismatches from one another, and from a fully complementary duplex that exhibits no modulation. We attribute the modulating current signatures to base flipping and subsequent interaction with positively charged lysine residues at the latch constriction of ?HL. Our hypothesis is supported by the extended residence times of DNA duplexes within the pore when a mismatch is in proximity to the latch constriction, and by the loss of the two-state current signature in low pH buffers (<6.3), where the protonation of one of the cytosine bases increases the stability of the intrahelical state.

SUBMITTER: Johnson RP 

PROVIDER: S-EPMC4828915 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature