DOI: 10.1073/pnas.231434698. Kinetics of duplex formation for individual DNA strands within a single protein nanopore

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

10.1073/pnas.231434698

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

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.

Bibliography

Howorka, S., Movileanu, L., Braha, O., & Bayley, H. (2001). Kinetics of duplex formation for individual DNA strands within a single protein nanopore. Proceedings of the National Academy of Sciences, 98(23), 12996â13001.

Authors 4

Stefan Howorka (first)
Liviu Movileanu (additional)
Orit Braha (additional)
Hagan Bayley (additional)

References 48 Referenced 180

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Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:31 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 9:45 a.m.)
Indexed	1 month, 2 weeks ago (July 16, 2025, 9:02 a.m.)
Issued	23 years, 10 months ago (Oct. 23, 2001)
Published	23 years, 10 months ago (Oct. 23, 2001)
Published Online	23 years, 10 months ago (Oct. 23, 2001)
Published Print	23 years, 10 months ago (Nov. 6, 2001)

Funders 0

None

BibTeX

@article{Howorka_2001, title={Kinetics of duplex formation for individual DNA strands within a single protein nanopore}, volume={98}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.231434698}, DOI={10.1073/pnas.231434698}, number={23}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Howorka, Stefan and Movileanu, Liviu and Braha, Orit and Bayley, Hagan}, year={2001}, month=oct, pages={12996–13001} }

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