title: Accurate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity.
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.
authors.affiliation: Institut für Physikalische Chemie, Lehrstuhl für Molekulare Physikalische Chemie, Heinrich-Heine-Universität, Düsseldorf, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Sindbert
authors.forename: Stanislav
authors.initials: S
authors.lastname: Kalinin
authors.forename: Hien
authors.initials: H
authors.lastname: Nguyen
authors.forename: Andrea
authors.initials: A
authors.lastname: Kienzler
authors.forename: Lilia
authors.initials: L
authors.lastname: Clima
authors.forename: Willi
authors.initials: W
authors.lastname: Bannwarth
authors.forename: Bettina
authors.initials: B
authors.lastname: Appel
authors.forename: Sabine
authors.initials: S
authors.lastname: Müller
authors.forename: Claus A M
authors.initials: CA
authors.lastname: Seidel
mesh_terms: D002232:Carbocyanines / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D031541:Fluorescence Resonance Energy Transfer; D006834:Hydrazines / Q000737:chemistry*; D015394:Molecular Structure; D012313:RNA / Q000032:analysis*
chemical_list: C417561:Alexa 488 hydrazide; D002232:Carbocyanines; D006834:Hydrazines; C085321:cyanine dye 5; D012313:RNA; D004247:DNA