Chemocatalytic Amplification Probes Enable Transcriptionally-Regulated Au(I)-Catalysis in E. coli and Sensitive Detection of SARS-CoV-2 RNA Fragments

Creators: Green, Sydnee A.; Wigman, Benjamin; Nistanaki, Sepand K.; Montgomery, Hayden R.; Jones, Christopher G.; Nelson, Hosea M.

Abstract

The union of transition metal catalysis with native biochemistry presents a powerful opportunity to perform abiotic reactions within complex biological systems. However, several chemical compatibility challenges associated with incorporating reactive metal centers into complex biological environments have hindered efforts in this area, despite the many opportunities it may present. More challenging than chemical compatibility is biocommunicative transition metal catalysis, where the reactivity of the metal species is regulated by native biological stimuli, akin to natural biocatalytic processes. Here we report a novel Au(I)-DNAzyme that is activated by short nucleic acids in a highly sequence-specific manner and that is compatible with complex biological matrices. The active Au(I)-DNAzyme catalyzes the formation of a fluorescent molecule with >10 turnovers. This functional allostery, resulting in chemocatalytic signal amplification, is competent in complex biological settings, including within recombinant E. coli cells, where the catalytic activity of the Au(I)-DNAzyme is regulated by transcription of an inducible plasmid. We further demonstrate the potential of this transition metal oligonucleotide complex as a highly sensitive and selective hybridization probe, permitting the detection of attomolar concentrations (ca. 60 molecules/ L) of SARS-CoV-2 RNA gene fragments in simulated biological matrices with ≥85% accuracy. Notably, this sensitive detection platform avoids expensive and poorly-scalable biochemical components (e.g. post-synthetically modified oligonucleotides or enzymes) and utilizes small molecule fluorophores, inexpensive Au salts and oligonucleotides composed of canonical bases. This discovery highlights promising opportunities to perform abiotic catalysis in complex biological settings under transcriptional regulation, as well as a chemocatalytic strategy for PCR-free, direct-detection of RNA and DNA.

Additional Information

The content is available under CC BY NC ND 4.0 License. Financial support for this work was generously provided by the David and Lucile Packard Foundation (to H.M.N.), the Pew Charitable Trusts (to H.M.N), Bristol Myers Squibb (to H.M.N.), the UCLA AIDS institute (to H.M.N.) and the National Science Foundation (DGE-1650604 to B.W. and C.G.J.). S.A.G. thanks the Department of Chemistry and Biochemistry UCLA Fellowship for funding. B.W. thanks the Christopher S. Foote Fellowship for funding. S.K.N. thanks the USPHS National Research Service Award (T32GM008496). The authors thank the UCLA Molecular Instrumentation Center for NMR and mass spectrometry instrumentation. This material is based on work supported by the National Institutes of Health under instrumentation (1S10OD016387-01). We would like to thank Dr. Mark Arbing for assistance and the UCLA-DOE Protein Expression Core for E. coli cell transformation and expression. We would like to thank Amir Nasajpour for assistance with cell imaging. We would like to acknowledge Professor F. Dean Toste (UC Berkeley) for useful discussions. Author Contributions. H.M.N. conceived of project and designed experiments. S.A.G., B.W., S.K.N., and H.R.M. designed and conducted experiments. C.G.J. performed statistical analyses. H.M.N., S.A.G., B.W., S.K.N., and C.G.J. prepared the manuscript. Data Availability. Further fluorescence measurements, NMR spectra, cell images, and thermal stability measurements can be found in the SI Appendix.

Attached Files

Submitted - chemocatalytic-amplification-probes-enable-transcriptionally-regulated-au-i-catalysis-in-e-coli-and-sensitive-detection-of-sars-co-v-2-rna-fragments.pdf

Supplemental Material - cc-covid-paper-si-final-1.pdf

Files

chemocatalytic-amplification-probes-enable-transcriptionally-regulated-au-i-catalysis-in-e-coli-and-sensitive-detection-of-sars-co-v-2-rna-fragments.pdf

Files (18.4 MB)

Name	Size	Download all
chemocatalytic-amplification-probes-enable-transcriptionally-regulated-au-i-catalysis-in-e-coli-and-sensitive-detection-of-sars-co-v-2-rna-fragments.pdf md5:3733635024fd8f351a377530ff7e0792	10.3 MB	Preview Download
cc-covid-paper-si-final-1.pdf md5:3914cadbc4017b14efbfc64332753bc5	8.2 MB	Preview Download

Additional details

	All versions	This version
Views	0	0
Downloads	0	0
Data volume	0 Bytes	0 Bytes