Förster resonance energy transfer within the neomycin aptamer.

Förster resonance energy transfer (FRET) measurements between two dyes is a powerful method to interrogate both structure and dynamics of biopolymers. The intensity of a fluorescence signal in a FRET measurement is dependent on both the distance and the relative orientation of the dyes. The latter can at the same time both complicate the analysis and give more detailed information. Here we present a detailed spectroscopic study of the energy transfer between the rigid FRET labels Çmf (donor) and tCnitro (quencher/acceptor) within the neomycin aptamer N1. The energy transfer originates from multiple emitting states of the donor and occurs on a low picosecond to nanosecond time-scale. To fully characterize the energy transfer, ultrafast transient absorption measurements were performed in conjunction with static fluorescence and time-correlated single photon counting (TCSPC) measurements, showing a clear distance dependence of both signal intensity and lifetime. Using a known NMR structure of the ligand-bound neomycin aptamer, the distance between the two labels was used to estimate κ2 and, therefore, make qualitative statements about the change in orientation after ligand binding with unprecedented temporal and spatial resolution. The advantages and potential applications of absorption-based methods using rigid labels for the characterization of FRET processes are discussed.

Protein-Specific, Multicolor and 3D STED Imaging in Cells with DNA-Labeled Antibodies.

Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two-color STED imaging of whole cells.