Abstract
Lack of access to safe drinking water is a global problem, and methods to reliably and easily detect contaminants could be transformative. We report the development of a cell-free in vitro transcription system that uses RNA Output Sensors Activated by Ligand Induction (ROSALIND) to detect contaminants in water. A combination of highly processive RNA polymerases, allosteric protein transcription factors and synthetic DNA transcription templates regulates the synthesis of a fluorescence-activating RNA aptamer. The presence of a target contaminant induces the transcription of the aptamer, and a fluorescent signal is produced. We apply ROSALIND to detect a range of water contaminants, including antibiotics, small molecules and metals. We also show that adding RNA circuitry can invert responses, reduce crosstalk and improve sensitivity without protein engineering. The ROSALIND system can be freeze-dried for easy storage and distribution, and we apply it in the field to test municipal water supplies, demonstrating its potential use for monitoring water quality.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1451-1459 |
| Number of pages | 9 |
| Journal | Nature biotechnology |
| Volume | 38 |
| Issue number | 12 |
| DOIs | |
| State | Published - Dec 2020 |
Funding
We thank A. Thompson (Northwestern University) for managing the experimental reagents and equipment used in this study; Z. Yang (Northwestern University) for assistance with FAAS measurements; N. Donghia (Wyss Institute) for helpful discussions on lyophilization of cell-free reactions; S. Pshenychny (Recombinant Protein Production Core at Northwestern University) for assistance in protein purification; J. Webster, R. Gruenberg, J. Machado and E. Plasencia-Campos (CSU, Chico) for assisting with field work; and J. Bussan, F. Lantz and B. Golenia (Northwestern University Research Shop) for assistance in the development of the handheld illuminator. J.K.J. and M.S.V. were supported in part by Northwestern University’s Graduate School Cluster in Biotechnology, System, and Synthetic Biology, which is affiliated with the Biotechnology Training Program. P.R.C. was supported in part by the National Science Foundation (NSF) Synthetic Biology REU (grant no. DBI-1757973). This work was also supported by funding from the Pew Charitable Trusts (to D.A.C.), the National Institutes of Health (grant no. R35 GM118157 to D.P.G.), CONICYT/FONDECYT (grant no. 1161337 to P.A.P.), ANID/FONDAP (grant no. 15110020 to P.A.P.), NSF CAREER (grant no. 1452441 to J.B.L.), NSF MCB RAPID (grant no. 1929912 to J.B.L., J.-F.G. and S.J.M.), support from the Crown Family Center for Jewish and Israel Studies at Northwestern University (to J.B.L.) and Searle Funds at the Chicago Community Trust (to J.B.L.).
ASJC Scopus subject areas
- Biotechnology
- Bioengineering
- Applied Microbiology and Biotechnology
- Molecular Medicine
- Biomedical Engineering
