Optical Spectroscopy and Single-Molecule Microscopy Combined to Study the Conformation of Confined Polymers
B. Akerman, Kenneth D. Cole
Optical microscopy is a powerful tool to study the global behavior of individual polymers, which we here combine with optical spectroscopy to study the conformation of confined polymers also at sub-optical length scales. Circular DNA molecules (52 and 220 kbp) are impaled on single gel fibers in agarose gels and stretched by an electric field ( 75 V/cm). Combined fluorescence microscopy and polarised UV-spectroscopy data on the coil-deformation show that the DNA-helix is less field-aligned than theoretically predicted for an unconfined polymer at the same apparent coil extension. This observation suggests that the gel obstacles deflect the DNA-path through the gel at sub-optical length scales. An analysis based on the reptation model indicate that the DNA chains are fully stretched along the path at all fields, while the average degree of field-alignment of the path itself increases with increasing field in agreement with biased reptation. The effective DNA-charge is evaluated to be (0.25 0.05)e per base-pair, in agreement with previous measurements in agarose gels. These conclusions are supported by studies of linear DNA-molecules which instead are anchored by covalent end-tethering to the gel fibers through a streptavidin-biotin bridge. The results demonstrate a microscopy-spectroscopy approach to characterize confined polymers at all length scales.