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Probing DNA surface attachment and local environment using single molecule spectroscopy

journal contribution
posted on 2023-06-08, 07:37 authored by Mark Osborne, C L Barnes, S Balasubramanian, D Klenerman
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.

History

Publication status

  • Published

Journal

Journal of Physical Chemistry B

ISSN

1089-5647

Issue

15

Volume

105

Page range

3120-3126

Pages

7.0

Department affiliated with

  • Chemistry Publications

Notes

First measurement of mono-dispersity in random arrays of immobilized single-molecules as a step towards massively high density single-molecule DNA arrays. Experimental design, construction and imaging performed by MAO and principle author.

Full text available

  • No

Peer reviewed?

  • Yes

Legacy Posted Date

2012-02-06

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