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

preprint
revised on 04.09.2020 and posted on 07.09.2020 by Sydnee Green, Benjamin Wigman, Sepand Nistanaki, Hayden Montgomery, Christopher G. Jones, Hosea Nelson
The union of transition metal catalysis with native biochemistry presents a powerful opportunity
to perform abiotic reactions within complex biological systems.(1,2) 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/µL) 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.

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.

Funding

Packard Foundation

Pew Charitable Trusts

UCLA AIDS Institute

Bristol Myers Squibb

History

Email Address of Submitting Author

hosea@chem.ucla.edu

Institution

UCLA

Country

USA

ORCID For Submitting Author

0000-0002-4666-2793

Declaration of Conflict of Interest

None

Exports