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Immobilized RNA switches for the analysis of complex chemical and biological mixtures

Nature Biotechnology volume 19, pages 336–341 (2001)Cite this article

Abstract

A prototype biosensor array has been assembled from engineered RNA molecular switches that undergo ribozyme-mediated self-cleavage when triggered by specific effectors. Each type of switch is prepared with a 5′-thiotriphosphate moiety that permits immobilization on gold to form individually addressable pixels. The ribozymes comprising each pixel become active only when presented with their corresponding effector, such that each type of switch serves as a specific analyte sensor. An addressed array created with seven different RNA switches was used to report the status of targets in complex mixtures containing metal ion, enzyme cofactor, metabolite, and drug analytes. The RNA switch array also was used to determine the phenotypes of Escherichia coli strains for adenylate cyclase function by detecting naturally produced 3′,5′- cyclic adenosine monophosphate (cAMP) in bacterial culture media.

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Figure 1: A representative RNA molecular switch that is activated by cobalt.
Figure 2: Selective immobilization and function of the AR1 molecular switch on gold surfaces.
Figure 3: A prototype RNA array constructed using engineered molecular switches.
Figure 4: Assessment of analytes in biological samples using the prototype RNA array.
Figure 5: Quantitative assessment of cAMP in E. coli 7043 culture medium.

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Acknowledgements

We are grateful to Mark Reed and James Klemic for the production of gold-coated surfaces, and Michael Tarlov for helpful suggestions regarding gold–thiol immobilization. Funding for this work was provided by grants from the National Institutes of Health, the Defense Advanced Research Projects Agency (DARPA), and the Yale Diabetes and Endocrinology Research Center. R.R.B. is the recipient of a Hellman Family Fellowship and a fellowship from the David and Lucile Packard Foundation.

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  1. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, 06520-8103, CT

    Sukeerthi Seetharaman, Maris Zivarts, Narasimhan Sudarsan & Ronald R. Breaker

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Correspondence to Ronald R. Breaker.

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Seetharaman, S., Zivarts, M., Sudarsan, N. et al. Immobilized RNA switches for the analysis of complex chemical and biological mixtures. Nat Biotechnol 19, 336–341 (2001). https://doi.org/10.1038/86723

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