A cell-free biosensor signal amplification circuit with polymerase strand recycling

Yueyi Li; Tyler Lucci; Matias Villarruel Dujovne; Jaeyoung Kirsten Jung; Daiana A. Capdevila; Julius B. Lucks

doi:10.1038/s41589-024-01816-w

A cell-free biosensor signal amplification circuit with polymerase strand recycling

Yueyi Li, Tyler Lucci, Matias Villarruel Dujovne, Jaeyoung Kirsten Jung, Daiana A. Capdevila, Julius B. Lucks^*

^*Corresponding author for this work

Chemical and Biological Engineering

Research output: Contribution to journal › Article › peer-review

Abstract

Cell-free systems are powerful synthetic biology technologies that can recapitulate gene expression and sensing without the complications of living cells. Cell-free systems can perform more advanced functions when genetic circuits are incorporated. Here we expand cell-free biosensing by engineering a highly specific isothermal amplification circuit called polymerase strand recycling (PSR), which leverages T7 RNA polymerase off-target transcription to recycle nucleic acid inputs within DNA strand displacement circuits. We first construct simple PSR circuits to detect different RNA targets with high specificity. We then interface PSR circuits to amplify signals from allosteric transcription factor-based biosensors for small molecule detection. A double equilibrium model of transcription factor–DNA/ligand binding predicts that PSR can improve biosensor sensitivity, which we confirm experimentally by improving the limits of detection by 10-fold to submicromolar levels for two biosensors. We believe this work expands the capabilities of cell-free circuits and demonstrates PSR’s potential for diverse applications in biotechnology. (Figure presented.)

Original language	English (US)
Article number	5
Journal	Nature Chemical Biology
DOIs	https://doi.org/10.1038/s41589-024-01816-w
State	Accepted/In press - 2025

Funding

We thank C. Knopp (Northwestern University) and A. Moreno (Northwestern University) for managing the experimental reagents and equipment used in this study; S. Schaffter (National Institute of Standards and Technology) for helpful discussion on TMSD circuits and T7 RNAP activity; H. Demissie (Northwestern University) for helpful discussion on lyophilization; and S.O. Kelley (Northwestern University), E.H. Sargent (Northwestern University), A.D. Ellington (University of Texas at Austin), M.C. Jewett (Stanford University), and J.L. Chavez (AFRL) for helpful discussions on miRNA target selection. Y.L. was supported by the National Institutes of Health Training Grant (T32GM008449) through Northwestern University\u2019s Biotechnology Training Program and the Ryan Fellowship and the International Institute for Nanotechnology at Northwestern University. T.L. was supported by the National Science Foundation Synthesizing Biology Across Scales NRT training program (2021900). J.K.J. was supported by Northwestern University\u2019s Graduate School Cluster in Biotechnology, System, and Synthetic Biology, which is affiliated with the Biotechnology Training Program, the Ryan Fellowship and the McCormick School of Engineering Terminal Year Fellowship. This work was also supported by Army Contracting Command (W52P1J-21-9-3023) and Defense Advanced Research Projects Agency (DARPA) (N660012324041) and the National Science Foundation (2310382) to J.B.L. The views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense, the National Science Foundation or the US Government. D.A.C. and M.V.D. were supported by Bunge & Born, Argentina, Williams Foundations and MINCYT Argentina (PICT 2022-ET-09-00049 and B81 CYTCH); D.A.C is a staff member from CONICET, Argentina; and M.V.D. is supported by a fellowship provided by CONICET, Argentina.

ASJC Scopus subject areas

Molecular Biology
Cell Biology

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10.1038/s41589-024-01816-w

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title = "A cell-free biosensor signal amplification circuit with polymerase strand recycling",

abstract = "Cell-free systems are powerful synthetic biology technologies that can recapitulate gene expression and sensing without the complications of living cells. Cell-free systems can perform more advanced functions when genetic circuits are incorporated. Here we expand cell-free biosensing by engineering a highly specific isothermal amplification circuit called polymerase strand recycling (PSR), which leverages T7 RNA polymerase off-target transcription to recycle nucleic acid inputs within DNA strand displacement circuits. We first construct simple PSR circuits to detect different RNA targets with high specificity. We then interface PSR circuits to amplify signals from allosteric transcription factor-based biosensors for small molecule detection. A double equilibrium model of transcription factor–DNA/ligand binding predicts that PSR can improve biosensor sensitivity, which we confirm experimentally by improving the limits of detection by 10-fold to submicromolar levels for two biosensors. We believe this work expands the capabilities of cell-free circuits and demonstrates PSR{\textquoteright}s potential for diverse applications in biotechnology. (Figure presented.)",

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A cell-free biosensor signal amplification circuit with polymerase strand recycling

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