Single-particle tracking for DNA tether length monitoring.
We describe a simple single-particle tracking approach for monitoring the length of DNA molecules in tethered particle motion experiments. In this method, the trajectory of a submicroscopic bead tethered by a DNA molecule to a glass surface is determined by videomicroscopy coupled to image analysis. The amplitude of motion of the bead is measured by the standard deviation of the distribution of successive positions of the bead in a given time interval. We were able to describe theoretically the variation of the equilibrium value of the amplitude of the bead motion with the DNA tether length for the entire applicable DNA length range (up to approximately 3500 bp). The sensitivity of the approach was illustrated by the evidence obtained for conformational changes introduced into a Holliday junction by the binding of the Escherichia coli RuvA protein. An advantage of this method is that the trajectory of the tethered bead, rather than its averaged motion, is measured, allowing analysis of the conformational dynamics of DNA chains at the single-molecule level.
Pouget
Noëlle
N
Institut de Pharmacologie et Biologie Structurale (UMR CNRS 5089), 205 route de Narbonne, 31077 Toulouse Cedex, France.
Dennis
Cynthia
C
Turlan
Catherine
C
Grigoriev
Mikhail
M
Chandler
Michaël
M
Salomé
Laurence
L
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases; D045566:DNA, Cruciform / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000378:metabolism; D018715:Microscopy, Video; D008863:Microspheres; D009038:Motion; D009068:Movement; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D004247:DNA; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases