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Abstract: A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3‐d]pyrimidine analogue of adenosine (tzA), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and tzATP to yield NtzAD+, which can be enzymatically phosphorylated by NAD+ kinase and ATP or tzATP to the corresponding NtzADP+. The latter can be engaged in NADP‐specific coupled enzymatic transformations involving conversion to NtzADPH by glucose‐6‐phosphate dehydrogenase and reoxidation to NtzADP+ by glutathione reductase. The NtzADP+/NtzADPH cycle can be monitored in real time by fluorescence spectroscopy. [ABSTRACT FROM AUTHOR]

An enzyme-mediated approach for the assembly of singly modified RNA constructs in which specific G residues are replaced with thG, an emissive isomorphic G surrogate, is reported. Transcription in the presence of thG and native nucleoside triphosphates enforces initiation with the unnatural analogue, yielding 5′-end modified transcripts that can be mono-phosphorylated and ligated to provide longer site-specifically modified RNA constructs. The scope of this unprecedented enzymatic approach to non-canonical purine-containing RNAs is explored via the assembly of several altered hammerhead (HH) ribozymes and a singly modified HH substrate. By strategically modifying key positions, a mechanistic insight into the ribozyme-mediated cleavage is gained. Additionally, the emissive features of the modified nucleoside and its responsiveness to environmental changes can be used to monitor cleavage in real time by steady state fluorescence spectroscopy. [ABSTRACT FROM AUTHOR]