Author(s): Alexander Rich
Mentor(s): Rodolfo Probst,
Institution U of U
In the 1980s and 90s, Natural Product Sciences Inc. (NPS) amassed one of the world’s largest venom collections to aid in the discovery of new pharmaceutical compounds. This collection, comprising venoms from spiders, snakes, scorpions, and other organisms, was left unorganized, unlabeled, and unused at the University of Utah for nearly 30 years after NPS was sold. Recently, we acquired this collection and recognized its untapped potential. First, we dedicated significant time to organizing the 1,539 samples. The collection has limited information detailing the species of origin of the venoms, hindering further study into the venoms’ bioactivity and potential for drug development. Subsequently, we developed a cost-effective, in-house RNA-to-DNA barcoding pipeline to identify species without a physical voucher, starting with spider venoms due to their abundance in the collection and their potential for future neurotoxin-derived pharmaceuticals. Our pipeline consists of multiple steps to achieve species identification. First, highly degraded messenger RNA (mRNA) is extracted from each venom sample to generate complementary DNA (cDNA) via reverse transcription. Using minibarcode primers, we then amplify a 313 base pair segment of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene with polymerase chain reaction (PCR). The resulting DNA is pooled and sequenced using nanopore technology, followed by sequence demultiplexing and comparison against the NCBI (National Center for Biotechnology Information) nucleotide database to identify the source species for each venom sample. Our pipeline successfully identified the species of 24 out of the first 25 spider venom samples processed. Identified genera include Geolycosa (burrowing wolf spiders), Heteropoda (Huntsman spiders), Alopecosa (fox spiders), and Aphonopelma (tarantulas). These results demonstrate that the venom RNA-to-DNA pipeline reliably identifies species without vouchers, with a high success rate. By providing accurate species identifications, this pipeline will enable further bioactivity assays to elucidate the mechanisms of action and potential therapeutic applications of venom-derived compounds.