Optical Spectroscopy and Single-Molecule Microscopy Combined to Study the Conformation of Confined Polymers

Published

October 12, 2021

Author(s)

B. Akerman, Kenneth D. Cole

Abstract

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.

Citation

Macromolecules

Pub Type

Journals

Keywords

agarose, alignment, DNA, electrophoresis, fluorescence, gel, microscopy, spectroscopy, trapping

Citation

Akerman, B. and Cole, K. (2021), Optical Spectroscopy and Single-Molecule Microscopy Combined to Study the Conformation of Confined Polymers, Macromolecules (Accessed December 13, 2024)