Structure guided fluorescence labeling reveals a two-step binding mechanism of neomycin to its RNA aptamer.

The ability of the cytidine analog Çmf to act as a position specific reporter of RNA-dynamics was spectroscopically evaluated. Çmf-labeled single- and double-stranded RNAs differ in their fluorescence lifetimes, quantum yields and anisotropies. These observables were also influenced by the nucleobases flanking Çmf. This conformation and position specificity allowed to investigate the binding dynamics and mechanism of neomycin to its aptamer N1 by independently incorporating Çmf at four different positions within the aptamer. Remarkably fast binding kinetics of neomycin binding was observed with stopped-flow measurements, which could be satisfactorily explained with a two-step binding. Conformational selection was identified as the dominant mechanism.

Spin the light off: rapid internal conversion into a dark doublet state quenches the fluorescence of an RNA spin label.

The spin label Çm and the fluorophore Çmf are close isosteric relatives: the secondary amine Çmf can be easily oxidized to a nitroxide group to form Çm. Thus, both compounds can serve as EPR and fluorescence labels, respectively, and their high structural similarity allows direct comparison of EPR and fluorescence data, e.g. in the context of investigations of RNA conformation and dynamics. Detailed UV/vis-spectroscopic studies demonstrate that the fluorescence lifetime and the quantum yield of Çmf are directly affected by intermolecular interactions, which makes it a sensitive probe of its microenvironment. On the other hand, Çm undergoes effective fluorescence quenching in the ps-time domain. The established quenching mechanisms that are usually operational for fluorophore-nitroxide compounds, do not explain the spectroscopic data for Çm. Quantum chemical calculations revealed that the lowest excited doublet state D1, which has no equivalent in Çmf, is a key state of the ultrafast quenching mechanism. This dark state is localized on the nitroxide group and is populated via rapid internal conversion.

The Three Possible 2-(Pyrenylethynyl) Adenosines: Rotameric Energy Barriers Govern the Photodynamics of These Structural Isomers.

This article presents a comprehensive study of the photophysics of 2-(2-pyrenylethynyl) adenosine and 2-(4-pyrenylethynyl) adenosine, which are structural isomers of the well-established fluorescent RNA label 2-(1-pyrenylethynyl) adenosine. We performed steady-state and ultrafast transient absorption spectroscopy studies along with time-resolved fluorescence emission experiments in different solvents to work out the interplay of locally excited and charge-transfer states. We found the ultrafast photodynamics to be crucial for the fluorescence decay behavior, which extends up to tens of nanoseconds and is partially multi-exponential. These features in the ultrafast dynamics are indicative of the rotational energy barriers in the first excited state.

Tetracycline determines the conformation of its aptamer at physiological magnesium concentrations.

Synthetic riboswitches are versatile tools for the study and manipulation of biological systems. Yet, the underlying mechanisms governing its structural properties and regulation under physiological conditions are poorly studied. We performed spectroscopic and calorimetric experiments to explore the folding kinetics and thermodynamics of the tetracycline-binding aptamer, which can be employed as synthetic riboswitch, in the range of physiological magnesium concentrations. The dissociation constant of the ligand-aptamer complex was found to strongly depend on the magnesium concentration. At physiological magnesium concentrations, tetracycline induces a significant conformational shift from a compact, but heterogeneous intermediate state toward the completely formed set of tertiary interactions defining the regulation-competent structure. Thus, the switching functionality of the tetracycline-binding aptamer appears to include both a conformational rearrangement toward the regulation-competent structure and its thermodynamic stabilization.

Three-State Fluorescence of a 2-Functionalized Pyrene-Based RNA Label.

The pyrene-based RNA-fluorescence label 2-(2-pyrenylethynyl) adenosine (2PyA) shows triexponential fluorescence, which depends strongly on the excitation wavelength. Most strikingly, a structured, long-lived fluorescence is observed in solution at room temperature after excitation into the S2 state, which is shifted hypsochromically by 30 nm compared to excitation into the S1 state. This very unusual behavior is investigated in detail with steady-state and time-resolved emission spectroscopy, ultrafast transient absorption spectroscopy, and quantum chemical calculations with both wave functions (CC2-level) and density-functional theory (DFT). 2PyA is found to emit simultaneously from two different intramolecular charge transfer states (mesomeric and twisted, MICT and TICT) which are populated most efficiently via the S1 state and a pyrene-like locally excited (LE) state. Rotational momentum derived from excess excitation energy is required to populate twisted LE configurations. Therefore, the LE state is most efficiently accessible via excitation to the S2. The stabilization of the different substates is related to two distinct reaction coordinates: the adenine-pyrene distance and the adenine-pyrene tilt angle, respectively.

Charge reduction and thermodynamic stabilization of substrate RNAs inhibit RNA editing.

African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds.

Discrimination between FRET and non-FRET quenching in a photochromic CdSe quantum dot/dithienylethene dye system.

A photochromic Förster resonance energy transfer (FRET) system was employed to disentangle the fluorescence quenching mechanisms in quantum dot/photochromic dye hybrids. In the off-state of the dye the main quenching mechanism is FRET whereas the moderate quenching in the on-state is due to non-FRET pathways opened up upon assembly.