Photochromic nucleosides were designed that combine the structural features and molecular recognition properties of nucleic acids with the light-sensitivity of diarylethenes. Target compounds 1a-c consist of a 7-deazaadenosine unit that is linked to a thiophene as the second aryl functionality via a 1,2-cyclopentenyl linker. These nucleoside analogues undergo a reversible electrocyclic rearrangement, generating strongly colored closed-ring isomers upon irradiation with UV-light, while exposure to light in the visible range triggers the cycloreversion to the colorless opened-ring form. UV-vis spectroscopy, HPLC, and (1)H NMR measurements revealed recognition of complementary thymidine and up to 97% conversion to the thermally stable closed-ring isomers after illumination with UV-light. The required wavelength for ring closure was found to vary depending on the substituents attached to the thiophene moiety. In a first design step, we used this important feature of diarylethenes to shift the switching wavelength from initially 300 nm (1a) to 405 nm (1cH(+)). In a second step, we generated a pair of orthogonal switches, differing enough in their respective switching wavelengths to be controlled independently in the same sample. Finally, a molecular switch was developed that showed both photochromism and acidichromism, thereby illustrating the possibility to gate the spectral properties to multiple stimuli. These new photochromic nucleosides represent useful building blocks for the generation of light-sensitive nucleic acids either by inducing conformational changes upon isomerization or by exploring the different spectral properties of the closed and opened isomers, for example, for use as reversible fluorescence quenchers.

In a prior report we introduced a novel class of photochromic nucleosides (PCNs) that combine the structural features of adenine with the photochromic properties of diarylethenes. Herein, we translated this concept to the nucleoside guanosine, generating reversibly switching guanosine‐like PCNs. These switches consist of a 7‐deazaguanosine unit and a second aryl functionality, which are linked through a cyclopentene unit. Irradiation of the open‐form isomer with light at 300 nm induces a pericyclic reaction that can be reversed with visible light. In addition to optical stimulation, these switches respond to light‐independent stimuli, such as the presence of acid or complexation with metal ions.

Here we report the first example of a photoactivatable ribozyme which catalyzes a bimolecular reaction of two small organic molecules with multiple turnover, under control of a photo-cleavable protecting group by exploiting the structural significance of a single hydrogen bond.

Chemical probing is a common method for the structural characterization of RNA. Typically, RNA is radioactively end-labelled, subjected to probing conditions, and the cleavage fragment pattern is analysed by gel electrophoresis. In recent years, many chemical modifications, like fluorophores, were introduced into RNA, but methods are lacking that detect the influence of the modification on the RNA structure with single-nucleotide resolution. Here, we first demonstrate that a 5′-terminal 32P label can be replaced by a dye label for in-line probing of riboswitch RNAs. Next, we show that small, highly structured FRET-labelled Diels–Alderase ribozymes can be directly probed, using the internal or terminal FRET dyes as reporters. The probing patterns indeed reveal whether or not the attachment of the dyes influences the structure. The existence of two dye labels in typical FRET constructs is found to be beneficial, as ‘duplexing’ allows observation of the complete RNA on a single gel. Structural information can be derived from the probing gels by deconvolution of the superimposed band patterns. Finally, we use fluorescent in-line probing to experimentally validate the structural consequences of photocaging, unambiguously demonstrating the intentional destruction of selected elements of secondary or tertiary structure.

Enzymology at the single-molecule level by using fluorescence resonance energy transfer (smFRET) offers unprecedented insight into mechanistic aspects of catalytic reactions. Implementing spatiotemporal control of the reaction by using an external trigger is highly valuable in these challenging experiments. Here, we have incorporated a light-cleavable caging moiety into specific nucleotides of the Diels-Alderase (DAse) ribozyme. In this way, the folding energy landscape was significantly perturbed, and the catalytic activity was essentially suppressed. A careful smFRET efficiency histogram analysis at various Mg(2+) ion concentrations revealed an additional intermediate state that is not observed for the unmodified DAse ribozyme. We also observed that only a fraction of DAse molecules returns to the native state upon cleavage of the caged group by UV light. These constructs are attractive model RNA systems for further real-time single-molecule observation of the coupling between conformational changes and catalytic activity.