Expanding fluorescent base analogue labelling of long RNA by in vitro transcription

To track RNA inside living cells and to study its natural interactions and mechanisms is vital in RNA research and the development of RNA-based therapeutics. In this work, we show that the fluorescent adenine analogue 2CNqA-triphosphate (2CNqATP) is accepted by the polymerase of a standard in vitro transcription, which results in fluorescently labelled long RNA. We demonstrate that the labelling degree can be tuned by the fractional exchange of natural ATP for 2CNqATP in the in vitro transcription reaction. Furthermore, in a thorough spectroscopic characterization, we present how the fluorescence lifetime and quantum yield change upon incorporation at different labelling degrees and how this in turn affects overall brightness per RNA strand. After transfecting human cells with the corresponding mRNA, we demonstrate its applicability in live-cell confocal microscopy by simultaneously imaging 2CNqA-labelled RNA and the mCherry reporter protein. We quantified the reporter gene expression using flow cytometry and observed a decrease in mRNA translatability with increasing 2CNqA incorporation. In a benchmark study with Cy5-labelled RNA we reveal that the effect on translatability is similar for both fluorophores. Moreover, we present a general statistical analysis highlighting the importance of considering the effects of unlabelled RNA being translated in cell studies at too low labelling degrees and discuss how this might affect experimental outcome. Despite a decreased protein synthesis, we show that 2CNqATP can be conveniently used to efficiently label and image long RNA, making it a promising fluorescent label in, for example, drug-delivery studies.