A light-responsive RNA aptamer for an azobenzene derivative.

Regulation of complex biological networks has proven to be a key bottleneck in synthetic biology. Interactions between the structurally flexible RNA and various other molecules in the form of riboswitches have shown a high-regulation specificity and efficiency and synthetic riboswitches have filled the toolbox of devices in many synthetic biology applications. Here we report the development of a novel, small molecule binding RNA aptamer, whose binding is dependent on light-induced change of conformation of its small molecule ligand. As ligand we chose an azobenzene because of its reliable photoswitchability and modified it with chloramphenicol for a better interaction with RNA. The synthesis of the ligand 'azoCm' was followed by extensive biophysical analysis regarding its stability and photoswitchability. RNA aptamers were identified after several cycles of in vitro selection and then studied regarding their binding specificity and affinity toward the ligand. We show the successful development of an RNA aptamer that selectively binds to only the trans photoisomer of azoCm with a KD of 545 nM. As the aptamer cannot bind to the irradiated ligand (λ = 365 nm), a light-selective RNA binding system is provided. Further studies may now result in the engineering of a reliable, light-responsible riboswitch.

A combined optical and EPR spectroscopy study: azobenzene-based biradicals as reversible molecular photoswitches.

Azobenzene compounds are known as versatile examples for photoswitchable systems because of their isomeric cis- and trans-configurations. The switching between these isomers can be reversibly controlled by light excitation. In this study we characterize two members of this class by joining the azobenzene moiety with each two paramagnetic nitroxide spin labels. Two different linkers were chosen to tune the molecular properties. The combined approach using optical and EPR spectroscopy proved the reversibility of photoexcitation and high fatigue resistance. Furthermore, depending on the nature of the linker, PELDOR distance measurements monitored clearly the photo-induced structural changes of the azobenzene unit. Thus, a powerful concept is presented resulting from the combination of these two complementary spectroscopic techniques.

Ultrafast Dynamics of Photoisomerization and Subsequent Unfolding of an Oligoazobenzene Foldamer.

Investigating and deciphering the dynamics of photoswitchable foldamers provides a detailed understanding of their photoinduced conformational transitions, resembling similar processes in photoresponsive biomacromolecules. We studied the ultrafast dynamics of the photoisomerization of azobenzene moieties embedded in a foldamer backbone and the resulting conformational helix-coil transition by time-resolved femtosecond/picosecond pump-probe spectroscopy in the visible and infrared region. During E → Z photoisomerization of the azobenzenes, the complexity of the photoinduced conformational transition of the pentameric foldamer 105 is reflected in distinct spectral characteristics and a 2-fold slower decay of the excited-state absorption bands compared to the monomer M (τ4,foldamer = 20 ps, τ4,monomer = 9 ps). Time-resolved IR experiments reveal the vibrational features of the monomer and the foldamer after photoexcitation, with an additional time constant for the foldamer (τ = 150 ps), indicating the initial steps of unfolding of the helical conformation, which are supported by density functional theory calculations. Our results record the overall sequence of photoinduced structural changes in the foldamer, starting from the initial ultrafast isomerization of the azobenzene unit(s) and ending with the complete unfolding on a later time scale. From our experiments, we could gain insight into the coupling of primary photoisomerization events ("cause") and secondary unfolding processes ("effect") in these oligoazobenzene foldamers.

Ultrafast Spectroscopy of Hydroxy-Substituted Azobenzenes in Water.

Ultrafast UV/Vis pump/probe experiments on ortho-, meta- and para-hydroxy-substituted azobenzenes (HO-ABs), as well as for sulfasalazine, an AB-based drug, were performed in aqueous solution. For meta-HO-AB, AB-like isomerisation behaviour can be observed, whereas, for ortho-HO-AB, fast proton transfer occurs, resulting in an excited keto species. For para-HO-AB, considerable keto/enol tautomerism proceeds in the ground state, so after excitation the trans-keto species isomerises into the cis form. Aided by TD-DFT calculations, insight is provided into different deactivation pathways for HO-AB, and reveals the role of hydroxy groups in the photochemistry of ABs, as well as their acetylation regarding sulfasalazine. Hydroxy groups are position-specific substituents for AB, which allow tuning of the timescale of thermal relaxation, as well as the amount and contribution of the keto species to photochemical processes.

Water-soluble Py-BIPS spiropyrans as photoswitches for biological applications.

The ultrafast photochemistry of a new spiropyran photoswitch (Py-BIPS) has been investigated, revealing many advantages in the application in water over the previously studied spiropyrans. Functionalized Py-BIPS derivatives are presented for the study of pH dependence, stability, toxicity, and the thermal and photochemical behavior on longer time scales in aqueous media using several spectroscopic methods. These investigations pave the way for the practical use of Py-BIPS derivatives as photoswitchable ligands of biomolecules.

Reversible photoswitching of RNA hybridization at room temperature with an azobenzene C-nucleoside.

Photoregulation of RNA remains a challenging task as the introduction of a photoswitch entails changes in the shape and the stability of the duplex that strongly depend on the chosen linker strategy. Herein, the influence of a novel nucleosidic linker moiety on the photoregulation efficiency of azobenzene is investigated. To this purpose, two azobenzene C-nucleosides were stereoselectively synthesized, characterized, and incorporated into RNA oligonucleotides. Spectroscopic characterization revealed a reversible and fast switching process, even at 20 °C, and a high thermal stability of the respective cis isomers. The photoregulation efficiency of RNA duplexes upon trans-to-cis isomerization was investigated by using melting point studies and compared with the known D-threoninol-based azobenzene system, revealing a photoswitching amplitude of the new residues exceeding 90 % even at room temperature. Structural changes in the duplexes upon photoisomerization were investigated by using MM/MD calculations. The excellent photoswitching performance at room temperature and the high thermal stability make these new azobenzene residues promising candidates for in-vivo and nanoarchitecture photoregulation applications of RNA.

Ultrafast two-dimensional infrared spectroscopy resolves the conformational change of an Evans auxiliary induced by Mg(ClO4)2.

Structure determination of reactive species is a key step in understanding reaction mechanisms. We demonstrate the application of polarization-dependent two-dimensional infrared spectroscopy (P2D-IR) as a powerful tool combining structure resolution with ultrafast time resolution. We apply this technique to investigate the substrate-catalyst complexes in a Lewis acid catalyzed Diels-Alder reaction. Using Mg(ClO(4))(2) as a Lewis acid, we found that an additional complex besides the chelate typically postulated as reactive species forms. Experimental access to this new species leads to a deeper understanding of the observed selectivities for the Diels-Alder reaction catalyzed by Lewis acids. Our findings are supported by density functional computations at the M06/6-31+G(d,p) level, including solvent corrections.

Two-dimensional infrared spectroscopy reveals the structure of an Evans auxiliary derivative and its SnCl4 Lewis acid complex.

Determining the structure of reactive intermediates is the key to understanding reaction mechanisms. To access these structures, a method combining structural sensitivity and high time resolution is required. Here ultrafast polarization-dependent two-dimensional infrared (P2D-IR) spectroscopy is shown to be an excellent complement to commonly used methods such as one-dimensional IR and multidimensional NMR spectroscopy for investigating intermediates. P2D-IR spectroscopy allows structure determination by measuring the angles between vibrational transition dipole moments. The high time resolution makes P2D-IR spectroscopy an attractive method for structure determination in the presence of fast exchange and for short-lived intermediates. The ubiquity of vibrations in molecules ensures broad applicability of the method, particularly in cases in which NMR spectroscopy is challenging due to a low density of active nuclei. Here we illustrate the strengths of P2D-IR by determining the conformation of a Diels-Alder dienophile that carries the Evans auxiliary and its conformational change induced by the complexation with the Lewis acid SnCl(4), which is a catalyst for stereoselective Diels-Alder reactions. We show that P2D-IR in combination with DFT computations can discriminate between the various conformers of the free dienophile N-crotonyloxazolidinone that have been debated before, proving antiperiplanar orientation of the carbonyl groups and s-cis conformation of the crotonyl moiety. P2D-IR unequivocally identifies the coordination and conformation in the catalyst-substrate complex with SnCl(4), even in the presence of exchange that is fast on the NMR time scale. It resolves a chelate with the carbonyl orientation flipped to synperiplanar and s-cis crotonyl configuration as the main species. This work sets the stage for future studies of other catalyst-substrate complexes and intermediates using a combination of P2D-IR spectroscopy and DFT computations.