Using Oxford Nanopore Technology direct RNA sequencing to identify depurination events induced by ricin and other ribosome inactivating proteins

Abstract

Depurination is a frequent modification to both DNA and RNA, in DNA causing point mutations through misincorporation, in RNA, disabling ribosomes and halting protein synthesis. Some modifications of nucleic acids can be determined by direct sequencing using Oxford Nanopore Technologies (ONT). However, the identification of modifications is often limited by noise and their variety and number. Ricin is a toxin which enters cells and depurinates an adenine base in the sarcin-ricin loop of the large ribosomal subunit. This leaves only a ribose backbone, thus inhibiting protein translation. In humans, biological threat agents and ribosome inactivating proteins, such as ricin and saporin, depurinate base 4605 on the 28S rRNA providing a single defined target to try and identify. We postulated that the depurination event could be detected using ONT direct RNA sequencing through a change in charge in the ricin loop. A software tool was developed, RIPpore, that quantified the adenine modification from direct RNA sequencing data of ribosomal RNA purified from respiratory epithelial cells exposed to ricin. This provided a novel method of directly identifying ricin exposure and a basis for the utility of ONT in detecting lesions in nucleic acids caused by depurination events.