Sequence-specific activity of Shiga toxin and other ribosome-inactivating proteins towards different nucleic acid substrates
Final Report Abstract
Ribosome inactivating proteins (RIPs) depurinate nucleic acids leaving abasic sites, however the sequence-specificity of RIPs was not studied well. The aim of the project was to establish a high-throughput method to the study sequence-specificity of RIPs. The method established uses synthetic, randomized DNA libraries, generated by solid-phase synthesis, to screen the sequence space in an unbiased fashion. The nucleic acid libraries used contained the loop region of the Sarcin-Ricin-Loop (SRL), the presumed target of RIPs in cells, or simple hairpin with a short, randomized loop region. These libraries were transcribed into RNA as well. After incubation of the libraries with RIP, the resulting abasic sites were cleaved by incubation with aniline. Cleaved and uncleaved sequences were then separated on denaturing polyacrylamide gels. The uncleaved sequences were recovered from the gel, reverse-transcribed if necessary, and converted into sequencing libraries by addition of sequencing adaptors via PCR. The libraries were sequenced using Illumina technology and the abundance of each sequence before and after RIP treatment was compared to determine the most sensitive sequences. The results obtained using saporin as a model RIP showed that it can depurinate adenine in many single-stranded sequence contexts, however it exhibited a clear preference for DNA tetraloops with the sequence ACGN. We confirmed the sequencing data by ordering single sensitive and resistant variants from each library and confirming their sensitivity towards depurination and cleavage using PAGE. We also compared the reaction kinetics of the most sensitive ACGN tetraloops with the GAGA tetraloop, which was presumed to be the target site of RIPs, based on their activity at the SRL. The kinetics demonstrated that the ACGN tetraloop is orders of magnitude more sensitive to depurination than the GAGA tetraloop. In summary, a method to screen the sensitivity of nucleic acid sequences towards depurination by RIPs was developed. Using this method, it was found that, in the absence of other factors, the DNA tetraloop ACGN was much more efficiently depurated by saporin than a GAGA tetraloop or the SRL.
Publications
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Analysis of the Sequence Preference of Saporin by Deep Sequencing. ACS Chemical Biology, 17(9), 2619-2630.
Hauf, Samuel; Rotrattanadumrong, Rachapun & Yokobayashi, Yohei
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High-throughput analysis of a sequence-specific depurination enzyme. Presented at: The 49th International Symposium on Nucleic Acids Chemistry, November 2022, Tokyo, Japan
Samuel Hauf; Rachapun Rotrattanadumrong & Yohei Yokobayashi
