Using nucleolytic toxins as restriction enzymes enables new RNA applications

Author:

Rothweiler Ulli1ORCID,Gundesø Sigurd Eidem1ORCID,Mikalsen Emma Wu12,Svenning Steingrim1ORCID,Singh Mahavir3ORCID,Combes Francis4ORCID,Pettersson Frida J4,Mangold Antonia1,Piotrowski Yvonne1,Schwab Felix1,Lanes Olav1,Striberny Bernd Ketelsen1ORCID

Affiliation:

1. ArcticZymes Technologies ASA , Sykehusveien 23, 9019  Tromsø , Norway

2. UiT – The Arctic University of Norway, Faculty of Biosciences , Fisheries & Economics, Muninbakken 21, 9019  Tromsø , Norway

3. Molecular Biophysics Unit, Indian Institute of Science , Bengaluru  560012 , India

4. Department of Biotechnology and Nanomedicine , SINTEF AS, Richard Birkelands vei 3, N-7034  Trondheim , Norway

Abstract

Abstract 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.

Funder

Research Council of Norway

Publisher

Oxford University Press (OUP)

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