Journal article
Single-molecule imaging for unraveling the functional diversity of 10–23 DNAzymes
- Abstract:
- DNA-based enzymes, also known as DNAzymes, have opened new opportunities for signal generation and amplification in several fields including biosensing. However, biosensor performance can be hampered by heterogeneity in the catalytic activity of such DNAzymes, especially when relying on a limited number of molecules to generate signal. In this regard, single-molecule studies are essential to discern the behavior among such heterogeneous molecules otherwise masked by ensemble measurements. This work presents a novel methodology to study the 10–23 RNA-cleaving DNAzyme at the single-molecule level. By means of measuring the distance-sensitive efficiency of Förster Resonance Energy Transfer using alternating-laser excitation on a superresolution microscope, we determined the kinetics of individual DNAzymes in terms of substrate turnover, rates of different reaction steps, and changes in performance over time. Our results revealed that, despite high concentrations of the reaction cofactor (i.e., Mg2+), a maximum of only 70% of the DNAzymes are actively cleaving multiple substrate sequences; the DNAzyme molecules also showed a wide range of substrate turnover rates. Our findings shed new light on the functional diversity of DNAzymes and the importance of exploring sequence modifications to improve their catalytic performance. Ultimately, this work presents a technique to obtain time-dependent information, which could be easily implemented to study other types of enzymes or biomolecular interactions.
- Publication status:
- Published
- Peer review status:
- Peer reviewed
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- Files:
-
-
(Preview, Accepted manuscript, pdf, 772.3KB, Terms of use)
-
- Publisher copy:
- 10.1021/acs.analchem.5c01439
Authors
+ Wellcome Trust
More from this funder
- Funder identifier:
- https://ror.org/029chgv08
- Grant:
- 110164/Z/15/Z
+ Engineering and Physical Sciences Research Council
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- Funder identifier:
- https://ror.org/0439y7842
- Publisher:
- American Chemical Society
- Journal:
- Analytical Chemistry More from this journal
- Volume:
- 97
- Issue:
- 25
- Pages:
- 13300–13309
- Place of publication:
- United States
- Publication date:
- 2025-06-17
- Acceptance date:
- 2025-06-12
- DOI:
- EISSN:
-
1520-6882
- ISSN:
-
0003-2700
- Pmid:
-
40526377
- Language:
-
English
- Keywords:
- Pubs id:
-
2133046
- Local pid:
-
pubs:2133046
- Deposit date:
-
2025-08-02
Terms of use
- Copyright holder:
- Pagès et al.
- Copyright date:
- 2025
- Rights statement:
- © 2025 The Authors. Published by American Chemical Society.
- Notes:
- The author accepted manuscript (AAM) of this paper has been made available under the University of Oxford's Open Access Publications Policy, and a CC BY public copyright licence has been applied.
- Licence:
- CC Attribution (CC BY)
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