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Cited by 268

The DOI matches its Crossref record; 54 of the 54 references it cites were found in a registry or an index.

Accurate Distance Determination of Nucleic Acids via Förster Resonance Energy Transfer: Implications of Dye Linker Length and Rigidity

Sindbert, Simon, Kalinin, Stanislav, Nguyen, Hien, Kienzler, Andrea, Clima, Lilia, Bannwarth, Willi, Appel, Bettina, Müller, Sabine, Seidel, Claus A. M.·2011·Journal of the American Chemical Society
DOI 10.1021/ja105725e
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Abstract

In Förster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A detailed analysis of possible orientation effects (κ(2) problem) indicates that, for short linkers and unknown local environments, additional κ(2)-related uncertainties are clearly outweighed by better defined dye positions.

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Metadata

Authors
Sindbert, S., Kalinin, S., Nguyen, H., Kienzler, A., Clima, L., Bannwarth, W., Appel, B., Müller, S., & Seidel, C. A. M.
Venue
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Published
2011-02-03
Volume
133
Issue
8
Pages
2463-2480
ISSN
  • 0002-7863 (ISSN-L)
  • 1520-5126
DOI
10.1021/ja105725e
OpenAlex
W2009436485
Cited by
268
References
54
FWCI
8.35
Type
journal article
Language
en
Open access
closed

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