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Protease-based synthetic sensing and signal amplification.

ABSTRACT: The bottom-up design of protein-based signaling networks is a key goal of synthetic biology; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based molecular switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially autoinhibited proteases that can be activated following site-specific proteolysis and also demonstrate the modular design of an allosterically regulated protease receptor following recombination with an affinity clamp peptide receptor. Notably, the receptor's mode of action can be varied from >5-fold switch-OFF to >30-fold switch-ON solely by changing the length of the connecting linkers, demonstrating a high functional plasticity not previously observed in naturally occurring receptor systems. We also create an integrated signaling circuit based on two orthogonal autoinhibited protease units that can propagate and amplify molecular queues generated by the protease receptor. Finally, we present a generic two-component receptor architecture based on proximity-based activation of two autoinhibited proteases. Overall, the approach allows the design of protease-based signaling networks that, in principle, can be connected to any biological process.

SUBMITTER: Stein V

PROVIDER: S-EPMC4234609 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Protease-based synthetic sensing and signal amplification.

Stein Viktor V Alexandrov Kirill K

Proceedings of the National Academy of Sciences of the United States of America 20141029 45

The bottom-up design of protein-based signaling networks is a key goal of synthetic biology; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based molecular switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially ...[more]

PMID: 25355910

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Project description:A surface plasmon resonance (SPR)-enhanced optical signal using a nanoslit array and acridine orange (AO) dye system at a flexible poly(dimethylsiloxane) (PDMS) substrate was achieved in this work and demonstrated a simple sensing scheme to directly detect SARS-CoV-2 nucleic acid via DNA hybridization. A simple nanoimprinting pattern transfer technique was introduced to form uniform reproducible nanoslit arrays where the dimensions of the slit array were controlled by the thickness of the gold film. The plasmon-exciton coupling effect on the optical enhancement of different dye molecules, i.e., AO, propidium iodide (PI), or dihydroethidium (DHE) attached to the nanoslit surfaces, was examined thoroughly by measuring the surface reflection and fluorescence imaging. The results indicate that the best overlap of the plasmon resonance wavelength to the excitation spectrum of AO presented the largest optical enhancement (∼57×) compared to the signal at flat gold surfaces. Based on this finding, a sensitive assay for detecting DNA hybridization was generated using the interaction of the selected SARS-CoV-2 ssDNA and dsDNA with AO to trigger the metachromatic behavior of the dye at the nanoarray surfaces. We found strong optical signal amplification on the formation of acridine-ssDNA complexes and a quenched signal upon hybridization to the complementary target DNA (ct-DNA) along with a blue shift in the fluorescence of AO-dsDNAs. A quantitative evaluation of the ct-DNA concentration in a range of 100-0.08 nM using both the reflection and emission imaging signals demonstrated two linear regimes with a lowest detection limit of 0.21 nM. The sensing method showed high sensitivity and distinguished signals from 1-, 2-, and 3-base mismatched DNA targets, as well as high stability and reusability. This approach toward enhancing optical signal for DNA sensing offers promise in a general, rapid, and direct vision detection method for nucleic acid analytes.

Dataset Information

Protease-based synthetic sensing and signal amplification.

Publications

Protease-based synthetic sensing and signal amplification.

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