Molecular recognition with DNA nanoswitches: Effects of single base mutations on structure

C. P. Mountford, A. H. Buck, C. J. Campbell, P. Dickinson, E. E. Ferapontova, J. G. Terry, J. S. Beattie, A. J. Walton, P. Ghazal, A. R. Mount, Jason Crain

Research output: Contribution to journalArticlepeer-review

Abstract

This paper investigates the properties of a simple DNA-based nanodevice capable of detecting single base mutations in unlabeled nucleic acid target sequences. Detection is achieved by a two-stage process combining first complementary-base hybridization of a target and then a conformational change as molecular recognition criteria. A probe molecule is constructed from a single DNA strand designed to adopt a partial cruciform structure with a pair of exposed (unhybridized) strands. Upon target binding, a switchable cruciform construct (similar to a Holliday junction) is formed which can adopt open and closed junction conformations. Switching between these forms occurs by junction folding in the presence of divalent ions. It has been shown from the steady-state fluorescence of judiciously labeled constructs that there are differences between the fluorescence resonance energy transfer (FRET) efficiencies of closed forms, dependent on the target sequence near the branch point, where the arms of the cruciform cross. This difference in FRET efficiency is attributed to structural variations between these folded junctions with their different branch point sequences arising from the single base mutations. This provides a robust means for the discrimination of single nucleotide mismatches in a specific region of the target. In this paper, these structural differences are analyzed by fitting observed time-resolved donor fluorescence decay data to a Gaussian distribution of donor-acceptor separations. This shows the closest mean separation (approximately 40 angstrom) for the perfectly matched case, whereas larger separations (up to 50 angstrom) are found for the single point mutations. These differences therefore indicate a structural basis for the observed FRET differences in the closed configuration which underpins the operation of these devices as biosensors capable of resolving single base mutations.

Original languageEnglish
Pages (from-to)2439-2444
Number of pages6
JournalJournal of Physical Chemistry B (Soft Condensed Matter and Biophysical Chemistry)
Volume112
Issue number8
DOIs
Publication statusPublished - 28 Feb 2008

Keywords

  • DNA
  • recombinant DNA
  • translocation

Fingerprint Dive into the research topics of 'Molecular recognition with DNA nanoswitches: Effects of single base mutations on structure'. Together they form a unique fingerprint.

Cite this