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.

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

“…For a thorough discussion of minimizing the uncertainty in k 2 using anisotropy experiments, see ref. [43]. k 2 can theoretically assume values between 0 and 4.…”

Simulation of FRET dyes allows quantitative comparison against experimental data

“…For the folded state, we compare the mean and standard deviation from the distance distribution of the simulation (black crosses and error bars) to the respective values of an accessible volume calculation 11,12 (blue). The accessible volume approach calculates all sterically possible dye positions within the linkage length and considers them as equally probable.…”

“…A straightforward approach is predicting the inter-dye distance by estimating the accessible volume of FRET dyes. [8][9][10][11][12] This does, however, not account for dynamics and the mutual dye orientation, reflected by the orientation factor κ 2 . An approach that has been used extensively for unfolded and intrinsically disordered proteins is the interpretation of FRET data in terms of analytical polymer models, 13 but such models are not applicable to folded proteins.…”

Photoacoustic molecular rulers based on DNA nanostructures

“…15,16 Due to the accurate specificity of base-pair interactions, it is possible to obtain self-assembled DNA nanoplatforms that allow accurate positioning of various moieties with sub-nanometer precision. 17,18 The decoration of these DNA constructs with fluorophores [19][20][21][22][23] has already resulted in several studies of molecular interactions based on FRET pairs using dye-dye or dyequencher combinations. These pairs have been used extensively in DNA nanostructures as .…”