RNA cleaving tris(2-aminobenzimidazoles) have been attached to DNA oligonucleotides via disulfide or amide bonds. The resulting conjugates are effective organocatalytic nucleases showing substrate and site selectivity as well as saturation kinetics. The benzimidazole conjugates also degrade enantiomeric RNA. This observation rules out contamination effects as an alternative explanation of RNA degradation. The pH dependency shows that the catalyst is most active in the deprotonated state. Typical half-lifes of RNA substrates are in the range of 12-17 h. Thus, conjugates of tris(2-aminobenzimidazoles) can compete with the majority of metal-dependent artificial nucleases.

Transglutaminases (TGases) catalyse the transamidation of glutamine residues with primary amines. Herein we report the first FRET-based activity assay for the direct detection of the ligation (transamidation) reaction mediated by tissue TGase (TG2). This novel assay was then used in a microtiter plate-based screen of a library of 18 potential amine substrates. From this screen it was discovered that propargyl amine serves as an excellent substrate for TG2. Subsequently, propargyl amine and 2-azidoethyl amine were validated independently as TG2 substrates with K(M) values of 44 ± 4 μM, and 0.99 ± 0.06 mM, respectively. In a proof-of-principle protein labelling experiment, the protein casein was selectively functionalized with propargyl amine using TG2 and subsequently fluorescently labelled through a dipolar cycloaddition reaction with an azido-fluorescein conjugate. This application demonstrates the strong potential of using TG2 for site-specific protein modification through a combination of enzymatic and bioorthogonal chemistry.

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