Engineering sequence-directed protein-RNA covalent linkages for compact RNA-labeling tools

The HUH-endonuclease superfamily is a diverse group of nucleases that cleave single-stranded DNA (ssDNA) and covalently link to the substrate's newly exposed 5’ end in a sequence specific manner. These nucleases have been co-opted for use in biotechnology applications as sequence-directed protein-ssDNA bioconjugation fusion partners (HUH-tags) due to their modest size and tremendous reaction efficiency. Surprisingly, a minority of these nucleases can promiscuously interact with RNA, but with dramatically low efficiency in comparison to the cognate reaction with ssDNA. Here, we present the engineering, characterization, and example applications of a set of laboratory-evolved RNA-interacting HUH-endonucleases (rHUH-tags). Yeast surface display-based protein engineering campaigns have produced engineered rHUH-tag variants with dramatically enhanced reactivity towards non-cognate RNA substrates. We demonstrate that rHUH-tags are a robust means of sequence-directed protein-RNA covalent bioconjugation in vitro and highlight their potential applications through a variety of proof-of-concept RNA labeling experiments. Finally, we demonstrate their versatility through the development of a hyper-compact RNA-editing system dubbed ‘HUH-ADAR’, which is capable of converting adenosine bases to inosine in a sequence-specific manner on transcripts of interest.