Molecular motion of DNA as measured by triplet anisotropy decay.

Abstract

We have used triplet anisotropy decay techniques to measure the internal flexibility and overall rotational motion of DNA, covering a time range from 15 ns to 200 Is.Nearly monodisperse DNA fragments 65-600 base pairs long were studied by using the intercalating dye methylene blue as a triplet probe. We found that the slow end-over-end tumbling of short DNA fragments (s165 base pairs) is as predicted for a rigid rod.As expected, a longer DNA fragment (600 base pairs) experiences slow segmental motion of its helix axis. We found that, at the earliest times, anisotropy decays more rapidly than expected for a rigid rod, suggesting that, when bound, methylene blue monitors fast internal motion of the helix. Since the rod-like end-over-end tumbling of short fragments rules out fast bending motions, we conclude that the fast components of DNA anisotropy decay are due to twisting motion of the helix, occurring with a time constant near 50 ns.Under physiological conditions, B-form DNA is an exceedingly stable structure. Recently, however, evidence has accumulated suggesting that the structure ofthe helix fluctuates substantially (1)(2)(3)(4)(5)(6)(7)(8). It has been shown by NMR techniques (1-4) that the helix experiences large fast motions in the nanosecond time range. Fast motions in the 1-to 100-ns time range have also been seen by fluorescence anisotropy methods (5-7), using as a probe ethidium bromide intercalated in DNA, and by measuring the ESR line shapes of spin-labeled intercalated dyes (8).Here we describe triplet anisotropy decay measurements of DNA internal motions. As in fluorescence anisotropy measurements, we have used an intercalating dye as a probe; however, we have monitored the anisotropy decay of the triplet rather than the singlet state by using methylene blue, a dye with a high triplet yield. Since the triplet lifetime of methylene blue is =100 pus, we can use triplet anisotropy decay techniques to measure motions over a time scale 1,000 times larger than fluorescence methods allow.MATERIALS AND METHODS In time-resolved triplet anisotropy measurements, chromophores bound rigidly to macromolecules are excited by a short pulse of linearly polarized light. For dyes with high triplet yields, a population of oriented triplet-state molecules results, oriented here meaning that the angular distribution of excited dipole moments is not spherically isotropic. Via rotational Brownian motion, the angular distribution will randomize in time, and the time dependence of the return to a spherically isotropic distribution of dipoles can be monitored either by observing the polarization of the triplet-singlet emission (phosphorescence) or by measuring the absorbance anisotropy associated with the triplet state.In the triplet-state anisotropy decay experiments described here, we have monitored the absorbance anisotropy of the depleted singlet state rather than the triplet state directly. The two techniques are interchangeable; however, singlet-state measurements are often more sensitive because of the high...

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

“…Data about the collective motion of DNA/RNA helical domains arising from structural fluctuations of the duplex (bending and twisting) are almost non-existent in the literature, especially for short duplexes. However, it can be inferred that these motions mainly cover the range from tens of nanosecond to tens of microseconds (Hogan et al 1982;Naimushin et al 2000;Orden and Jung 2008;Porschke 2007;Schwieters and Clore 2007;Shajani and Varani 2007), although motions in the range 20-400 ls have been reported (Kojima et al 2001). Together, these considerations indicate that in most cases, energy transfer captures a transient ''static'' configuration of the duplex, whereas the slow acquisition rate allows the DNA/RNA duplex and associated fluorophores to adopt all possible configurations within the experimental period used for data acquisition.…”

Corrigendum

“…In these measurements, the laser pulse has a finite width and therefore must be convoluted into the initial 20 ns of anisotropy decay simulation. When fitting our data to equations (1) and (2), we have included such a convolution (the pulse has been included as a. 20-ns gaussian excitation profile, using 100 points at 0.2-ns intervals).…”

“…4.0x 10-11 9.0X 10- 13 1.0 X 10-!2 2.0 X 10- 12 4.0x [10][11] l.Ox 10- 12 1.0 X 10-!2 2.0 X 10-12 4 Parameters are derived from a best fit of equations (1) and (2) to anisotropy decay data. 400±60, 260±40 and 800±200bp long DNA fragments were prepared as described in the legend to Fig.…”

“…1. C, Torsional spring constant calculated from equations (1) and (2); -r, exponential time constant for motion of the long helix axis estimated from a fit of the slowest part of the anisotropy decay data to a single exponential ; P, the apparent persistence length of the fragments ; E, Young's modulus, calculated from fits of data to equations (1) and (2). In these measurements, the laser pulse has a finite width and therefore must be convoluted into the initial 20 ns of anisotropy decay simulation.…”

Errata

“…Parameters are derived from a best fit of equations (1) and (2) to anisotropy decay data. 400±60, 260±40 and 800±200bp long DNA fragments were prepared as described in the legend to Fig.…”