Small Molecule Detection Via Allosteric Actuation of Strand Displacement Circuits

Bacterial allosteric transcription factors (aTFs) evolved in bacteria to sense a variety of small molecules [1]. Recently, biosensing platforms have been developed using purified native and engineered aTFs coupled with different signal transduction and amplification strategies [2-4]. We have developed an innovative biosensing platform for detection of small molecules based on the allosteric control of an endonuclease-driven toehold mediated strand displacement (TMSD) circuit [5]. We built one-step biosensors for the rapid and reliable detection of two antibiotics spiked in municipal water with nanomolar sensitivity.

Moreover, we harnessed the programmability and modularity of the platform to further enhance the TMSD circuit with the addition of a second signal amplifying cycle for detection of uric acid. Through in silico design of the nucleic acid substrates of the two-cycle system, the biosensor’s response could be fine-tuned in a wider range and detectable within 15 minutes of reaction. We successfully measured uric acid in spiked artificial saliva within the normal human salivary uric acid level and in spiked human serum comparable to a commercial uric acid meter.

References:

[1] Camilli, A., & Bassler, B. L. Science, 311(5764), 1113-1116 (2006).
[2] Jung, J. Nature Biotechnology, 38(12), 1451–1459 (2020).
[3] Iwasaki, R. S., & Batey, R. T. Nucleic Acids Research, 48(17), e101. (2020).
[4] Yao, Y., et al. Applied Microbiology and Biotechnology, 102(17), 7489–7497. (2018).
[5] Rodríguez-Serrano, A. F., & Hsing, I. M. ACS Synthetic Biology, 10(2), 371-378 (2021).

Acknowledgements: Funding support from RGC GRF#16306519, HKPFS from the HKSAR, and Conacyt Mexico is acknowledged.