Optimized measurements of separations and angles between intra-molecular fluorescent markers

Kim I. Mortensen, Jongmin Sung, Henrik Flyvbjerg () and James A. Spudich ()
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Kim I. Mortensen: Stanford University School of Medicine
Jongmin Sung: Stanford University School of Medicine
Henrik Flyvbjerg: Technical University of Denmark
James A. Spudich: Stanford University School of Medicine

Nature Communications, 2015, vol. 6, issue 1, 1-9

Abstract: Abstract We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each colour-separated microscope image in a time-lapse movie and using only simple means, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space with accuracy and precision. The positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA molecules internally labelled with two fixed fluorophores, we demonstrate the accuracy and precision of our method using the known structure of double-stranded DNA as a benchmark, resolve 10-base-pair differences in fluorophore separations, and determine the unique 3D orientation of each DNA molecule, thereby establishing short, double-labelled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly.

Date: 2015
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DOI: 10.1038/ncomms9621

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