Precise characterization of the conformation fluctuations of freely diffusing DNA: beyond Rouse and Zimm.
We studied the dynamics of single freely diffusing fluorescence-labeled double-stranded lambda-phage DNA molecules using dual-color 3-dimensional feedback tracking microscopy and intramolecular fluorescence correlation spectroscopy. Our technique is independently sensitive to the molecule's diffusion coefficient D and radius of gyration R(g) and is concentration insensitive, providing greater precision for characterizing the molecule's intramolecular motion than other methods. We measured D = 0.80 +/- 0.05 microm(2)/s and R(g) approximately 420 nm, consistent with the Kirkwood-Riseman prediction for a flexible polymer with strong hydrodynamic interactions (HI), but we find the statistics of intramolecular motion inconsistent with the Zimm model for such a polymer. We address a dispute in the experimental literature, finding that previous measurements on double-stranded DNA likely lacked the sensitivity to distinguish between the Zimm model and the HI-free Rouse model. Finally, we observe fluorescence fluctuations with a correlation time of over 2 s that cannot be explained by either model and propose that they may be signatures of excluded volume interactions.
McHale
Kevin
K
Edward L. Ginzton Laboratory, Stanford University, Stanford, California 94305, USA. mchalekl@niddk.nih.gov
Mabuchi
Hideo
H
D010582:Bacteriophage lambda / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry*; D004058:Diffusion; D005453:Fluorescence; D021621:Imaging, Three-Dimensional; D008853:Microscopy; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011108:Polymers / Q000737:chemistry*
D004279:DNA, Viral; D011108:Polymers