Crown ether-electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules.
DNA abasic (AP) sites are one of the most frequent lesions in the genome and have a high mutagenic potential if unrepaired. After selective attachment of 2-aminomethyl-18-crown-6 (18c6), individual AP lesions are detected during electrophoretic translocation through the bacterial protein ion channel α-hemolysin (α-HL) embedded in a lipid bilayer. Interactions between 18c6 and Na(+) produce characteristic pulse-like current amplitude signatures that allow the identification of individual AP sites in single molecules of homopolymeric or heteropolymeric DNA sequences. The bulky 18c6-cation complexes also dramatically slow the DNA motion to more easily recordable levels. Further, the behaviors of the AP-18c6 adduct are different with respect to the directionalities of DNA entering the protein channel, and they can be precisely manipulated by altering the cation (Li(+), Na(+) or K(+)) of the electrolyte. This method permits detection of multiple AP lesions per strand, which is unprecedented in other work. Additionally, insights into the thermodynamics and kinetics of 18c6-cation interactions at a single-molecule level are provided by the nanopore measurement.
Department of Chemistry, University of Utah, Salt Lake City, UT 84112-0850, USA.
Na
N
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Aaron M
AM
Fleming
Henry S
HS
White
Cynthia J
CJ
Burrows
D043844:Crown Ethers / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004573:Electrolytes / Q000737:chemistry*; D004586:Electrophoresis / Q000379:methods; D005821:Genetic Techniques; D006460:Hemolysin Proteins / Q000737:chemistry; D007700:Kinetics; D058608:Nanopores; D012964:Sodium / Q000737:chemistry; D013816:Thermodynamics
D043844:Crown Ethers; D004573:Electrolytes; D006460:Hemolysin Proteins; D004247:DNA; D012964:Sodium