Kinetics of duplex formation for individual DNA strands within a single protein nanopore

Stefan Howorka, Liviu Movileanu, Orit Braha, Hagan Bayley

Research output: Contribution to journalArticle

156 Scopus citations

Abstract

A single oligonucleotide was covalently attached to a genetically engineered subunit of the heptameric protein pore, α-hemolysin, to allow DNA duplex formation inside the pore lumen. Single-channel current recording was used to study the properties of the modified pore. On addition of an oligonucleotide 8 bases in length and with a sequence complementary to the tethered DNA strand, current blockades with durations of hundreds of milliseconds occurred, representing hybridization events of individual oligonucleotides to the tethered DNA strand. Kinetic constants for DNA duplex formation at the single molecule level were derived and found to be consistent with established literature values for macroscopic duplex formation. The resultant equilibrium constant for duplex formation in the nanopore was found to be close to the experimentally derived constant for duplex formation in solution. A good agreement between the equilibrium constants for duplex formation in the nanopore and in solution was also found for two other oligonucleotide pairs. In addition, the nanopore recordings revealed details of the kinetics difficult to obtain by conventional methods, like surface plasmon resonance, which measure ensemble properties. By investigating the temperature dependence of DNA duplex formation at the single molecule level, the standard enthalpy and entropy of the interaction could be obtained.

Original languageEnglish (US)
Pages (from-to)12996-13001
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume98
Issue number23
DOIs
StatePublished - Nov 6 2001
Externally publishedYes

ASJC Scopus subject areas

  • General

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