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Assessment of External Guide Sequences’ (EGS) Efficiency as Inducers of RNase P-Mediated Cleavage of mRNA Target Molecules

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Bacterial Regulatory RNA

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1737))

Abstract

RNase P is a ribozyme consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. It was initially identified as the enzyme that mediates cleavage of precursor tRNAs at the 5′-end termini to generate the mature tRNAs. An important characteristic of RNase P is that its specificity depends on the structure rather than the sequence of the RNA substrate. Any RNA species that interacts with an antisense molecule (called external guide sequence, EGS) and forms the appropriate structure can be cleaved by RNase P. This property is the basis for EGS technology, an antisense methodology for inhibiting gene expression by eliciting RNase P-mediated cleavage of a target mRNA molecule. EGS technology is being developed to design therapies against a large variety of diseases. An essential milestone in developing EGSs as therapies is the assessment of the efficiency of antisense molecules to induce cleavage of the target mRNA and evaluate their effect in vivo. Here, we describe simple protocols to test the ability of EGSs to induce cleavage of a target mRNA in vitro and to induce a phenotypic change in growing cells.

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Acknowledgments

This work was supported by Public Health Service grant 2R15AI047115-04 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. A.J. was funded in part by LA Basin Minority Health and Health Disparities International Research Training Program (MHIRT) 5T37MD001368.

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Authors and Affiliations

  1. Center for Applied Biotechnology Studies, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA, USA

    Saumya Jani, Alexis Jackson, Carol Davies-Sala, Kevin Chiem, Alfonso Soler-Bistué & Marcelo E. Tolmasky

  2. Fundación Instituto Leloir, IIBBA-CONICET, and FCEyN, University of Buenos Aires, Aires, Argentina

    Alexis Jackson, Carol Davies-Sala, Alfonso Soler-Bistué & Angeles Zorreguieta

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  1. Saumya Jani
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  2. Alexis Jackson
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  3. Carol Davies-Sala
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  4. Kevin Chiem
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  5. Alfonso Soler-Bistué
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  6. Angeles Zorreguieta
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  7. Marcelo E. Tolmasky
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Correspondence to Marcelo E. Tolmasky .

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Editors and Affiliations

  1. Université Paris Diderot & LLB/CEA/CNRS, Gif-sur-Yvette, France

    Véronique Arluison

  2. Laboratorio de Bioquímica, Microbiología e Interacciones, Biológicas en el Suelo, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes-CONICET, Bernal, Argentina

    Claudio Valverde

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Jani, S. et al. (2018). Assessment of External Guide Sequences’ (EGS) Efficiency as Inducers of RNase P-Mediated Cleavage of mRNA Target Molecules. In: Arluison, V., Valverde, C. (eds) Bacterial Regulatory RNA. Methods in Molecular Biology, vol 1737. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7634-8_6

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  • DOI: https://doi.org/10.1007/978-1-4939-7634-8_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7633-1

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