Detection and quantification of ricin-mediated 28S ribosomal depurination by digital droplet PCR

Justine L Lewis; Katherine A Shields; Damien C Chong

doi:10.1016/j.ab.2018.09.017

Detection and quantification of ricin-mediated 28S ribosomal depurination by digital droplet PCR

Anal Biochem. 2018 Dec 15:563:15-19. doi: 10.1016/j.ab.2018.09.017. Epub 2018 Sep 27.

Authors

Justine L Lewis¹, Katherine A Shields², Damien C Chong³

Affiliations

¹ Defence Science and Technology Group, 506 Lorimer Street, Fishermans Bend, Victoria, 3207, Australia. Electronic address: Justine.Lewis@dst.defence.gov.au.
² Defence Science and Technology Group, 506 Lorimer Street, Fishermans Bend, Victoria, 3207, Australia. Electronic address: Katherine.Shields@dst.defence.gov.au.
³ Defence Science and Technology Group, 506 Lorimer Street, Fishermans Bend, Victoria, 3207, Australia. Electronic address: Damien.Chong@dst.defence.gov.au.

PMID: 30267707
DOI: 10.1016/j.ab.2018.09.017

Abstract

Ricin acts to damage cells by producing a site of depurination in 28S ribosomal RNA. This depurination results in ribosome inactivation which inhibits protein synthesis and ultimately leads to cell death. We have developed a multiplexed digital droplet polymerase chain reaction assay that enables the objective measurement of toxin activity through quantitation of depurinated 28S rRNA molecules. This assay demonstrates the first use of digital PCR technology to measure ribotoxin-mediated damage. Depurination events were detected in ricin-treated lung cell cultures as early as 1 h, and within 9 h of exposure the maximum ribosomal damage of 70% was reached and was sustained for at least 24 h post-exposure.

Keywords: Depurination; Digital droplet PCR; RIP; RNA; Ricin; Toxin.

MeSH terms

Polymerase Chain Reaction / methods*
Protein Biosynthesis
RNA, Ribosomal / genetics
RNA, Ribosomal, 28S / genetics*
Ribosomes / metabolism*
Ricin / metabolism*

Substances

RNA, Ribosomal
RNA, Ribosomal, 28S
Ricin