Probing DNA surface attachment and local environment using single moleculespectroscopy

We have employed single molecule fluorescence spectroscopy, using a total i nternal reflection geometry and wide-angle detection, to study the attachme nt of singly fluorescently labeled DNA to a silica surface by either a stre ptavidin-biotin of a covalent linkage. In both cases the DNA is highly mono dispersed with no evidence for aggregation. The covalent coupling gave high er signal-to-noise than the streptavidin-biotin Linkage and was therefore s tudied in more detail. Two components in the photobleaching times, correspo nding to different states of the tetramethyl rhodamine probe, were observed : a short and long component with populations in the ratio 6.7:1. Only rare ly was interconversion between these two states detected during the 30-s ob servation time of the experiment. Hybridization experiments using a complem entary strand of DNA labeled with a different fluorophore gave a low level of colocalized fluorescence, indicating a significant fraction of the surfa ce attached DNA was not available for hybridization. These results are cons istent with the surface attached DNA spending significant time collapsed on the surface.