Using nucleolytic toxins as restriction enzymes enables new RNA applications.

Over the past five decades, DNA restriction enzymes have revolutionized biotechnology. While these enzymes are widely used in DNA research and DNA engineering, the emerging field of RNA and mRNA therapeutics requires sequence-specific RNA endoribonucleases. Here, we describe EcoToxN1, a member of the type III toxin-antitoxin family of sequence-specific RNA endoribonucleases, and its use in RNA and mRNA analysis. This enzyme recognizes a specific pentamer in a single-stranded RNA and cleaves the RNA within this sequence. The enzyme is neither dependent on annealing of guide RNA or DNA oligos to the template nor does it require magnesium. Furthermore, it performs over a wide range of temperatures. With its unique functions and characteristics, EcoToxN1 can be classified as an RNA restriction enzyme. EcoToxN1 enables new workflows in RNA analysis and biomanufacturing, meeting the demand for faster, cheaper, and more robust analysis methods.