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Molecular tools that target RNA at specific sites allow recoding of RNA information and processing. SNAP-tagged deaminases guided by a chemically stabilized guide RNA can edit targeted adenosine to inosine in several endogenous transcripts simultaneously, with high efficiency (up to 90%), high potency, sufficient editing duration, and high precision. We used adenosine deaminases acting on RNA (ADARs) fused to SNAP-tag for the efficient and concurrent editing of two disease-relevant signaling transcripts, KRAS and STAT1. We also demonstrate improved performance compared with that of the recently described Cas13b-ADAR.

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Quadruplex nucleic acids are widespread in genomes. They influence processes such as transcription, translation, replication, recombination, and the regulation of gene expression. Several synthetic ligands have been demonstrated to target quadruplex nucleic acids. However, only very few metabolites have been reported to interact with quadruplexes. In principle, an intracellular metabolite that selectively binds to four-stranded sequences could modulate quadruplex formation, stability, and thus functions in a riboswitch (or deoxyriboswitch) manner. Here we report quadruplex interactions with flavin derivatives such as FMN and FAD. The affinities were highest with parallel quadruplexes, with low (14-20 μm) dissociation constants. Taking into account combined intracellular flavin concentrations of 243 μm in E. coli, the observed interactions in principle open up the possibility of flavin levels affecting gene expression and other processes by modulating quadruplex formation.

A design strategy for high-affinity aptamers of complex biomolecules is presented. We developed an RNA with FAD-binding properties by combining known ATP-and FMN-aptamers.Cooperative binding of FAD was shown by SPR spectroscopy and fluorescence assays. The strategy should be transferable to several other biomolecules.Flavin adenine dinucleotide (FAD) is an important redox cofactor involved in crucial metabolic pathways. Several flavoenzymes utilize flavins for metabolic reactions for example during glycolysis, the citric acid cycle, and fatty acid synthesis and oxidation.1 Furthermore, FAD plays a role as cofactor in numerous proteins not involved in redox processes. 2,3 FAD contains a riboflavin (vitamin B 2 ) moiety, which forms from a tricyclic heteronuclear isoalloxazine and the polyalcohol ribitol. Riboflavin is a precursor of FAD and forms flavin mononucleotide (FMN) when a phosphate group is present at the ribitol moiety. Addition of an adenosine monophosphate to FMN results in FAD (Fig. 1A).Flavins have been the target of RNA aptamers since the early days of the SELEX technology. [4][5][6] Aptamers are short, single-stranded nucleic acids able to form complex threedimensional structures. They specifically recognize small molecules with low molecular weight 7 and display high affinity as well as selectivity for their targets. 8,9 The first aptamers were obtained using SELEX in the early 1990s. 4,10 Aptamers specifically binding FAD would have the potential to interfere with metabolic pathways. So far, such aptamers were originally isolated for FMN: they recognize the isoalloxazine ring and reportedly do bind FAD as well, albeit with lower affinity (0.5 vs. 0.7 µM in a buffer with 5 mM MgCl 2 ). 11 The secondary structure of FMN-aptamers isolated by Burgstaller and Famulok 11 consists of a highly conserved internal loop that is flanked by two stabilizing stems. Chain reversal is achieved by incorporation of a hairpin loop. The NMR structure of this aptamer showed that the binding is based on a specific intercalation of the isoalloxazine ring to the helix in the internal loop. The rest of the molecule, especially the adenosine part of FAD, is not involved in the binding to the aptamer and is therefore freely accessible. 12 In addition, FAD aptamers isolated by other groups only showed interaction with the isoalloxazine moiety. 13 In our study, we aimed at the creation of a more specific, high-affinity FAD aptamer. For this purpose we combined two known aptamers to enable them to bind FAD in a cooperative manner. The concept of linking two aptamers together has been explored before, for example a bi-partite aptamer has been constructed by linking two DNA aptamers that recognize two different surface receptor proteins. 14 Dual aptamer constructs recognizing a single molecule where realized targeting CD28 15 and thrombin proteins. 16 In this work we target for the first time a compound of comparable low molecular weight with two different aptamers. The two targeted substructures of FAD, the isoalloxazine ring ...