Strategies to improve multi-enzyme compatibility and coordination in one-pot SHERLOCK

Simple item page

Simple item page

Full item details

creativework.keywords - en
Zoonotic and emerging pathogens
Zoonosis
creativework.keywords - fr
Pathogènes zoonotiques et émergents
Zoonoses
dc.contributor.author
Li, Hongzhao
Kielich, Dominic M. S.
Liu, Guodong
Smith, Greg
Bello, Alexander
Strong, James E.
Pickering, Bradley S.
dc.date.accepted
2023-05-23
dc.date.accessioned
2025-03-05T20:46:48Z
dc.date.available
2025-03-05T20:46:48Z
dc.date.issued
2023-06-30
dc.date.submitted
2023-11-12
dc.description.abstract - en
While molecular diagnostics generally require heating elements that supply high temperatures such as 95 °C in polymerase chain reaction and 60–69 °C in loop-mediated isothermal amplification, the recently developed CRISPR-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can operate at 37 °C or a similar ambient temperature. This unique advantage may be translated into highly energy-efficient or equipment-free molecular diagnostic systems with unrestricted deployability. SHERLOCK is characterized by ultra-high sensitivity when performed in a traditional two-step format. For RNA sensing, the first step combines reverse transcription with recombinase polymerase amplification, while the second step consists of T7 transcription and CRISPR-Cas13a detection. The sensitivity drops dramatically, however, when all these components are combined into a single reaction mixture, and it largely remains an unmet need in the field to establish a high-performance one-pot SHERLOCK assay. An underlying challenge, conceivably, is the extremely complex nature of a one-pot formulation, crowding a large number of reaction types using at least eight enzymes/proteins. Although previous work has made substantial improvements by serving individual enzymes/reactions with accommodating conditions, we reason that the interactions among different enzymatic reactions could be another layer of complicating factors. In this study, we seek optimization strategies by which inter-enzymatic interference may be eliminated or reduced and cooperation created or enhanced. Several such strategies are identified for SARS-CoV-2 detection, each leading to a significantly improved reaction profile with faster and stronger signal amplification. Designed based on common molecular biology principles, these strategies are expected to be customizable and generalizable with various buffer conditions or pathogen types, thus holding broad applicability for integration into future development of one-pot diagnostics in the form of a highly coordinated multi-enzyme reaction system.
dc.identifier.citation
Li, H., Kielich, D. M., Liu, G., Smith, G., Bello, A., Strong, J. E., & Pickering, B. S. (2023). Strategies to improve multi-enzyme compatibility and coordination in one-pot SHERLOCK. Analytical Chemistry, 95(28), 10522–10531. https://doi.org/10.1021/acs.analchem.2c05032
dc.identifier.doi
https://doi.org/10.1021/acs.analchem.2c05032
dc.identifier.issn
1520-6882
dc.identifier.uri
https://open-science.canada.ca/handle/123456789/3484
dc.language.iso
en
dc.publisher - en
ACS Publications
dc.rights - en
Creative Commons Attribution 4.0 International (CC BY 4.0)
dc.rights - fr
Creative Commons Attribution 4.0 International (CC BY 4.0)
dc.rights.uri - en
https://creativecommons.org/licenses/by/4.0/
dc.rights.uri - fr
https://creativecommons.org/licenses/by/4.0/deed.fr
dc.subject - en
Genetics
Coronaviruses
dc.subject - fr
Génétique
Coronavirus
dc.subject.en - en
Genetics
Coronaviruses
dc.subject.fr - fr
Génétique
Coronavirus
dc.title - en
Strategies to improve multi-enzyme compatibility and coordination in one-pot SHERLOCK
dc.type - en
Article
dc.type - fr
Article
local.article.journalissue
28
local.article.journaltitle - en
Analytical Chemistry
local.article.journalvolume
95
local.pagination
10522-10531
local.peerreview - en
Yes
local.peerreview - fr
Oui
Download(s)

Original bundle

Now showing 1 - 1 of 1

Thumbnail image

Name: StrategiesImproveMultiEnzymeCompatibility_2023.pdf

Size: 1.61 MB

Format: PDF

Download file

Collection(s)

Page details

Date modified: