Probing DNA surface attachment and local environment using single molecule spectroscopy

We have employed single molecule fluorescence spectroscopy, using a total internal reflection geometry and wide-angle detection, to study the attachment of singly fluorescently labeled DNA to a silica surface by either a streptavidin-biotin of a covalent linkage. In both cases the DNA is highly monodispersed with no evidence for aggregation. The covalent coupling gave higher signal-to-noise than the streptavidin-biotin Linkage and was therefore studied in more detail. Two components in the photobleaching times, corresponding to different states of the tetramethyl rhodamine probe, were observed: a short and long component with populations in the ratio 6.7:1. Only rarely was interconversion between these two states detected during the 30-s observation time of the experiment. Hybridization experiments using a complementary strand of DNA labeled with a different fluorophore gave a low level of colocalized fluorescence, indicating a significant fraction of the surface attached DNA was not available for hybridization. These results are consistent with the surface attached DNA spending significant time collapsed on the surface.