Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing.

Deng, F; Li, Y; Yang, B; Sang, R; Deng, W; Kansara, M; Lin, F; Thavaneswaran, S; Thomas, DM; Goldys, EM

Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing.

Deng, F Li, Y Yang, B Sang, R Deng, W

Kansara, M Lin, F Thavaneswaran, S Thomas, DM Goldys, EM

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Publisher:: Springer Nature
Publication Type:: Journal Article
Citation:: Nat Commun, 2024, 15, (1), pp. 1818
Issue Date:: 2024-03-05

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Field	Value	Language
dc.contributor.author	Deng, F
dc.contributor.author	Li, Y
dc.contributor.author	Yang, B
dc.contributor.author	Sang, R
dc.contributor.author	Deng, W https://orcid.org/0000-0002-9413-0978
dc.contributor.author	Kansara, M
dc.contributor.author	Lin, F
dc.contributor.author	Thavaneswaran, S
dc.contributor.author	Thomas, DM
dc.contributor.author	Goldys, EM
dc.date.accessioned	2024-03-15T04:43:01Z
dc.date.available	2024-02-09
dc.date.available	2024-03-15T04:43:01Z
dc.date.issued	2024-03-05
dc.identifier.citation	Nat Commun, 2024, 15, (1), pp. 1818
dc.identifier.issn	2041-1723
dc.identifier.issn	2041-1723
dc.identifier.uri	http://hdl.handle.net/10453/176778
dc.description.abstract	Control of CRISPR/Cas12a trans-cleavage is crucial for biosensor development. Here, we show that small circular DNA nanostructures which partially match guide RNA sequences only minimally activate Cas12a ribonucleoproteins. However, linearizing these structures restores activation. Building on this finding, an Autocatalytic Cas12a Circular DNA Amplification Reaction (AutoCAR) system is established which allows a single nucleic acid target to activate multiple ribonucleoproteins, and greatly increases the achievable reporter cleavage rates per target. A rate-equation-based model explains the observed near-exponential rate trends. Autocatalysis is also sustained with DNA nanostructures modified with fluorophore-quencher pairs achieving 1 aM level (<1 copy/μL) DNA detection (106 times improvement), without additional amplification, within 15 min, at room temperature. The detection range is tuneable, spanning 3 to 11 orders of magnitude. We demonstrate 1 aM level detection of SNP mutations in circulating tumor DNA from blood plasma, genomic DNA (H. Pylori) and RNA (SARS-CoV-2) without reverse transcription as well as colorimetric lateral flow tests of cancer mutations with ~100 aM sensitivity.
dc.format	Electronic
dc.language	eng
dc.publisher	Springer Nature
dc.relation	http://purl.org/au-research/grants/nhmrc/1181889
dc.relation.ispartof	Nat Commun
dc.relation.isbasedon	10.1038/s41467-024-46001-8
dc.rights	info:eu-repo/semantics/openAccess
dc.subject.mesh	DNA, Circular
dc.subject.mesh	RNA
dc.subject.mesh	CRISPR-Cas Systems
dc.subject.mesh	RNA, Guide, CRISPR-Cas Systems
dc.subject.mesh	DNA
dc.subject.mesh	Helicobacter pylori
dc.subject.mesh	Nanostructures
dc.subject.mesh	Ribonucleoproteins
dc.subject.mesh	Helicobacter pylori
dc.subject.mesh	Ribonucleoproteins
dc.subject.mesh	DNA
dc.subject.mesh	DNA, Circular
dc.subject.mesh	RNA
dc.subject.mesh	Nanostructures
dc.subject.mesh	CRISPR-Cas Systems
dc.subject.mesh	RNA, Guide, CRISPR-Cas Systems
dc.subject.mesh	DNA, Circular
dc.subject.mesh	RNA
dc.subject.mesh	CRISPR-Cas Systems
dc.subject.mesh	RNA, Guide, CRISPR-Cas Systems
dc.subject.mesh	DNA
dc.subject.mesh	Helicobacter pylori
dc.subject.mesh	Nanostructures
dc.subject.mesh	Ribonucleoproteins
dc.title	Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing.
dc.type	Journal Article
utslib.citation.volume	15
utslib.location.activity	England
pubs.organisational-group	University of Technology Sydney
pubs.organisational-group	University of Technology Sydney/Faculty of Engineering and Information Technology
pubs.organisational-group	University of Technology Sydney/Faculty of Engineering and Information Technology/School of Biomedical Engineering
utslib.copyright.status	open_access	*
dc.date.updated	2024-03-15T04:42:56Z
pubs.issue	1
pubs.publication-status	Published online
pubs.volume	15
utslib.citation.issue	1

Abstract:

Control of CRISPR/Cas12a trans-cleavage is crucial for biosensor development. Here, we show that small circular DNA nanostructures which partially match guide RNA sequences only minimally activate Cas12a ribonucleoproteins. However, linearizing these structures restores activation. Building on this finding, an Autocatalytic Cas12a Circular DNA Amplification Reaction (AutoCAR) system is established which allows a single nucleic acid target to activate multiple ribonucleoproteins, and greatly increases the achievable reporter cleavage rates per target. A rate-equation-based model explains the observed near-exponential rate trends. Autocatalysis is also sustained with DNA nanostructures modified with fluorophore-quencher pairs achieving 1 aM level (<1 copy/μL) DNA detection (106 times improvement), without additional amplification, within 15 min, at room temperature. The detection range is tuneable, spanning 3 to 11 orders of magnitude. We demonstrate 1 aM level detection of SNP mutations in circulating tumor DNA from blood plasma, genomic DNA (H. Pylori) and RNA (SARS-CoV-2) without reverse transcription as well as colorimetric lateral flow tests of cancer mutations with ~100 aM sensitivity.

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http://hdl.handle.net/10453/176778