Regulation of mRNA translation by a photoriboswitch

Abstract

Optogenetic tools have revolutionized the study of receptor-mediated processes, but such tools are lacking for RNA-controlled systems. In particular, light-activated regulatory RNAs are needed for spatiotemporal control of gene expression. To fill this gap, we used in vitro selection to isolate a novel riboswitch that selectively binds the trans isoform of a stiff-stilbene (amino-tSS)–a rapidly and reversibly photoisomerizing small molecule. Structural probing revealed that the RNA binds amino-tSS about 100-times stronger than the cis photoisoform (amino-cSS). In vitro and in vivo functional analysis showed that the riboswitch, termed Werewolf-1 (Were-1), inhibits translation of a downstream open reading frame when bound to amino-tSS. Photoisomerization of the ligand with a sub-millisecond pulse of light induced the protein expression. In contrast, amino-cSS supported protein expression, which was inhibited upon photoisomerization to amino-tSS. Reversible photoregulation of gene expression using a genetically encoded RNA will likely facilitate high-resolution spatiotemporal analysis of complex RNA processes.

Molecular photoswitches in aqueous environments

“…125 However, recently, a few stiff stilbene derivatives were equipped with solubilizing groups and characterized in DMSO-water mixtures, buffer solutions and even in vivo. 116,[141][142][143] The compounds were reversibly switchable over several cycles, 141 however, photo-induced oxidation was observed in some cases (cf. Fig.…”

Photoswitches for controllable RNA binding: a future approach in the RNA-targeting therapy

“…The peculiarities of the aptamer-ligand interactions allowed to obtain systems with highly efficient, light-controllable switching of the ligand binding state (''ON/OFF binding''). Thus, four examples of photoswitchable binders for RNA aptamers were reported that are based on the reversible photoinduced transformations of spiropyran 4, 42 benzodihydropyrene 5, 43 azobenzene 6, 44 and stiff-stilbene 7 45 (Schemes 2c, 3 and Table 1). The RNA aptamers that bind specifically only to one of the two forms of the ligands 4-6 were selected in vitro from RNA aptamer libraries by the Systematic Evolution of Ligands by EXperimental enrichment (SELEX) procedure 46 i.e.…”

“…In the case of stiffstilbene 7, the selection was performed using an RNA pool derived from a bacterial SAM-1 riboswitch by replacing its ligand-binding domain with a random region of 45 nucleotides and partially randomizing the terminator and anti-terminator hairpins. 45 As a result, the selected RNA aptamer was isolated as a fused construct together with a riboswitch expression platform. The reversible photoswitching between the isomers of ligands 4-7 gave the systems an advantage of accomplishing repeated binding/dissociation cycles with RNA.…”

“…43 In the second example, a synthetic riboswitch comprising the stiff-stilbene 7-binding aptamer has been successfully implemented into a bacterial plasmid that allowed to perform reversible photocontrol of the Fluc gene expression in the modified E. coli in vivo. 45 Nevertheless, all the studied aptamer models were selected from the randomized RNA libraries and had, therefore, no therapeutic relevance, thus reducing the applicability of these systems for real human disease models.…”

Stiff‐Stilbene Photoswitches: From Fundamental Studies to Emergent Applications

“…[71] In another recent study,Míšek and Luptak described that the photocontrol of binding of as tiff-stilbene amino alcohol to m-RNAc an be used to regulate protein expression. [72]…”