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. 2024 Jul 11;52(19):11626–11640. doi: 10.1093/nar/gkae584

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© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 2. — Solution confocal-based FRET measurements accurately report on ssDNA end-to-end distances. (A) FRET efficiencies were calculated based on single molecule measurements of Cy3 (donor) and Cy5 (acceptor) fluorescence. Data were collected on a series of ssDNA substrates (30 pM) of increasing lengths. Energy transfer events were detected as bursts of photons as the molecules transited the confocal volume. The mean of the distribution is plotted in panel A as a function of length of poly-thymidine (dT_xx). (B) The end-to-end distances were calculated based on a R₀ value of 5.4 nm for the Cy3/Cy5 pair and Eq. (3) described in the Methods. The dotted lines represent a 95% confidence interval for the linear fit.