Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors

Abstract

Allosteric RNA devices are increasingly being viewed as important tools capable of monitoring enzyme evolution, optimizing engineered metabolic pathways, facilitating gene discovery and regulators of nucleic acid–based therapeutics. A key bottleneck in the development of these platforms is the availability of small-molecule-binding RNA aptamers that robustly function in the cellular environment. Although aptamers can be raised against nearly any desired target through in vitro selection, many cannot easily be integrated into devices or do not reliably function in a cellular context. Here, we describe a new approach using secondary- and tertiary-structural scaffolds derived from biologically active riboswitches and small ribozymes. When applied to the neurotransmitter precursors 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, this approach yielded easily identifiable and characterizable aptamers predisposed for coupling to readout domains to allow engineering of nucleic acid–sensory devices that function in vitro and in the cellular context.

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Figure 1: RNA scaffolds used for selection.
Figure 2: Selection of scaffolded aptamers that selectively bind 5-hydroxytryptophan (5HTP).
Figure 3: Analysis of potential 5HTP aptamers.
Figure 4: In vitro and intracellular performance of 5HTP and L-DOPA sensors.
Figure 5: A 5HTP-aptamer-based biosensor functions in E. coli.

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Acknowledgements

The authors thank B. Suess (Technical University Darmstadt) and A. Heckel (Goethe University Frankfurt) for the kind gift of α-methyl-5-hydroxy-L-tryptophan, R. Knight and J. Shorenstein for useful discussions about bioinformatics analysis of selections, A. Lambowitz (University of Texas at Austin) for the generous gift of the GsI reverse transcriptase, M. Yarus for the amino acid column labeling protocol, D. McKay and J. Trausch for crystallographic support, and A. Young and A. Palmer for assistance with microscopy. J. Kieft, J. Pfingsten and M. Matyjasik provided critical feedback on the manuscript. This work was supported by grants to R.T.B. from the National Science Foundation (NSF 1150834) and the National Institutes of Health (R01 GM073850) and a predoctoral training grant in Signaling and Cellular Regulation (T32 GM008759).

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E.B.P. conceived the project and either performed or participated in all experiments and in writing of the manuscript. J.T.P. performed the 5HTP and L-DOPA sensor construction, assay and interpretation. M.M.M. participated in the GR/SSIII selection, screened for crystals of the 5GR-II–5HTP complex and collected crystallographic data. R.T.B. oversaw the project, including planning of experiments, interpretation of data and writing of the manuscript.

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Correspondence to Robert T Batey.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–7 and Supplementary Figures 1–14 (PDF 11175 kb)

Supplementary Data

Representative sequences from clusters defining eight 5-hydroxytryptophan binding aptamers (5HTP) and four 3,4-dihydroxyphenylalanine binding aptamers (3,4-DHF). (ZIP 331 kb)

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Porter, E., Polaski, J., Morck, M. et al. Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors. Nat Chem Biol 13, 295–301 (2017). https://doi.org/10.1038/nchembio.2278

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