Increased brightness of fluorescent uridine, qU, inside single- and double-stranded RNA

Fluorescent base analogues enable studies of nucleic acids and by expanding their range of properties the scope of potential applications broadens. The recent photophysical characterisation of quadracyclic uridine (qU) in its monomeric ribonucleotide form revealed a high fluorescence quantum yield of 27%, with an interesting dual-band character emission. In this study, we show that incorporation of qU into RNA results in loss of the dual-band character but with further increased quantum yields and longer fluorescence lifetimes, in both single- and double-stranded RNA. Additionally, we find that neighbouring bases of qU affect its photophysical properties, with quantum yields varying between 33 and 68% in single-strands and 30-51% in duplexes. Furthermore, circular dichroism and duplex melting analyses of the qU-labelled duplexes were compared to unlabelled counterparts, with results suggesting that A-form RNA conformation is maintained, albeit with destabilisation caused by qU. Spectral features indicate that incorporated qU predominantly exists in its iminol tautomer, incapable of Watson-Crick base-pairing, which likely strongly contributes to the observed destabilisation. Overall, we report one of the highest brightness values (reaching 6 000 M^{- 1} cm^{- 1}) to date for an RNA-incorporated fluorescent nucleobase, highlighting qU as a valuable label for applications such as fluorescence microscopy that require strong emission.