Differentiation of G:C vs A:T and G:C vs G:mC Base Pairs in the Latch Zone of α-Hemolysin

Abstract

The alpha-hemolysin (α-HL) nanopore can detect DNA strands under an electrophoretic force via many regions of the channel. Our laboratories previously demonstrated that trapping duplex DNA in the vestibule of wild-type α-HL under force could distinguish the presence of an abasic site compared to a G:C base pair positioned in the latch zone at the top of the vestibule. Herein, a series of duplexes were probed in the latch zone to establish if this region can detect more subtle features of base pairs beyond the complete absence of a base. The results of these studies demonstrate the most sensitive region of the latch can readily discriminate duplexes in which one G:C base pair is replaced by an A:T. Additional experiments determined that while neither 8-oxo-7,8-dihydroguanine nor 7-deazaguanine opposite C could be differentiated from a G:C base pair, in contrast, the epigenetic marker 5-methylcytosine, when present in both strands of the duplex, yielded new blocking currents when compared to strands with unmodified cytosine. The results are discussed with respect to experimental design for utilization of the latch zone of α-HL to probe specific regions of genomic samples.

Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the α-Hemolysin Nanopore

“…The observed ~0.5 pA current difference between the nicked and repaired duplexes is likely due to one additional negative charge, the PO 4 2− on the 5’ end of the 8-mer probe, which we speculate may increase the local counter-ion concentration at the latch zone in addition to modulating the ion flux blockade via duplex structure modification, as previously reported for single base recognition. 13, 21, 51 …”

“…20 Further investigation of the latch constriction of α-HL has demonstrated that one G:C base pair can be distinguished from an A:T pair, as well as single-base mismatches (e.g., C:C or C:A), based on current blockade levels. 21–22 Recently, we also observed that mismatched base pairs induce characteristic ion current modulations in the α-HL channel. 22–23 …”

Nanopore Sensing

“…136, 137 Detection of both single base-pair replacement (e.g., GC pair replaced by AT pair) and epigenetic markers (e.g., 5-methylcytosine) has also been achieved with α-HL latch sensors. 139 …”

Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin

“…These ion channel recordings can be used to identify changes to DNA structure such as damage, 37–39 methylation 40 and mismatch 36 sites. In a typical experiment, a DNA helix, modified with a threading poly-T tail at the end of one strand, is driven into the αHL nanopore (embedded in a lipid bilayer) under an applied potential (Figure 1A).…”

“…37, 38, 40 A DNA duplex containing a single mismatched base-pair positioned at the latch constriction results in a current signature that modulates between two distinct states ( I 1 and I 2 ). In contrast, for a fully-complementary reference duplex, no modulation is observed (Figure 1B).…”