The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer.

The photo-physical properties of 2-(1-ethynylpyrene)-adenosine (PyA), a fluorescent probe for RNA dynamics, were examined by solvation studies. The excited-state dynamics display the influence of the vicinity on the spectral features. Combining improved transient absorption and streak camera measurements along with a new analysis method provide a detailed molecular picture of the photophysics. After intramolecular vibrational energy redistribution (IVR), two distinct states are observed. Solvent class (protic/aprotic) and permittivity strongly affect the properties of these states and their population ratio. As a result their emission spectrum is altered, while the fluorescence quantum yield and the overall lifetime remain nearly unchanged. Consequently, the hitherto existing model of the photophysics is herein refined and extended. The findings can serve as basis for improving the information content of measurements with PyA as a label in RNA.

Photochromic switches are essential for the control and manipulation of nanoscale reactions and processes. The expansion of their application to aqueous environments depends strongly on the development of optimized water-soluble photoswitches. Here we present a femtosecond time-resolved investigation of the photochromic reactions (transition between the open and the closed form) of a water-soluble indolylfulgimide. We observe a pronounced effect of the protic nature of water as a solvent on the ultrafast ring-opening reaction. Typically, the excited state of the closed form has a larger dipole moment than the ground state, which leads to stabilization of the excited state in polar solvents and hence a lifetime (3 ps) longer than in non-polar solvents (2 ps). However, in water, despite the increased solvent polarity and the increased excited state dipole moment, the opposite trend for the excited state lifetime is observed (1.8 ps). This effect is caused by the opening of a new excited state deactivation pathway involving proton transfer reactions.

Abstract. We examine the photoinduced excited state dynamics of pyrene modified adenosine, a versatile probe for folding and hybridization of ribonucleic acids. Measurements in different solvents revealed complex ultrafast dynamics, but high robustness since the overall fluorescence quantum yield ( f ) is hardly affected. The result is a strong fluorescent RNA-probe whose spectral properties change in a defined way upon environmental changes.

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