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bioRxiv · 10.1101/2020.06.18.159046

Beyond Plug and Pray: Context Sensitivity and in silico Design of Artificial Neomycin Riboswitches

Günzel, C.·Kuehnl, F.·Arnold, K.·Findeiss, S.·Weinberg, C. E.·Stadler, P. F.·Mörl, M.
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Abstract

Gene regulation in prokaryotes often depends on RNA elements such as riboswitches or RNA thermometers located in the 5’ untranslated region of mRNA. Rearrangements of the RNA structure in response, e. g., to the binding of small molecules or ions control translational initiation or premature termination of transcription and thus mRNA expression. Such structural responses are amenable to computational modeling, making it possible to rationally design synthetic riboswitches for a given aptamer. Starting from an artificial aptamer, we construct the first synthetic transcriptional riboswitches that respond to the antibiotic neomycin. We show that the switching behavior in vivo critically depends not only on the sequence of the riboswitch itself, but also on its sequence context. We therefore developed in silico methods to predict the impact of the context, making it possible to adapt the design and to rescue non-functional riboswitches. We furthermore analyze the influence of 5’ hairpins with varying stability on neomycin riboswitch activity. Our data highlight the limitations of a simple plug-and-play approach in the design of complex genetic circuits and demonstrate that detailed computational models significantly simplify, improve, and automate the design of transcriptional circuits. Our design software is available under a free license on Github.1Competing Interest StatementThe authors have declared no competing interest.List of Abbreviationsauarbitrary time unitsAURaptamer upstream regionbgaBβ-galactosidasebgaBthe β-galactosidase genedLdecoupling leaderE. coliEscherichia colieGFPthe enhanced green fluorescent proteinegfpthe enhanced green fluorescent protein geneGFPgreen fluorescent proteinLHleader hairpinLMmutated leaderMFEminimum free energymRNAmessenger RNAoLoriginal leaderPospositive controlRBSribosomal binding siteRFUrelative fluorescence unitRNAribonucleic acidRNAPRNA polymeraseRNase ERibonuclease ERppHRNA 5’ pyrophosphohydrolaserRNAribosomal RNASELEXsystematic evolution of ligands by exponential enrichmentSSCsaline sodium citrate bufferTBETris-borate-EDTATDRterminator downstream regionTSStranscription start siteUunstructured regionUTRuntranslated regionView Full Text

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Günzel, C., Kuehnl, F., Arnold, K., Findeiss, S., Weinberg, C. E., Stadler, P. F., Mörl, M.. 2020-06-18. Beyond Plug and Pray: Context Sensitivity and in silico Design of Artificial Neomycin Riboswitches. https://doi.org/10.1101/2020.06.18.159046

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