Abstract
Fluorescence resonance energy transfer (FRET) reporters are commonly used in the final stages of nucleic acid amplification tests to indicate the presence of nucleic acid targets, where fluorescence is restored by nucleases that cleave the FRET reporters. However, the need for dual labelling and purification during manufacturing contributes to the high cost of FRET reporters. Here we demonstrate a low-cost silver nanocluster reporter that does not rely on FRET as the on/off switching mechanism, but rather on a cluster transformation process that leads to fluorescence color change upon nuclease digestion. Notably, a 90 nm red shift in emission is observed upon reporter cleavage, a result unattainable by a simple donor-quencher FRET reporter. Electrospray ionization–mass spectrometry results suggest that the stoichiometric change of the silver nanoclusters from Ag13 (in the intact DNA host) to Ag10 (in the fragments) is probably responsible for the emission colour change observed after reporter digestion. Our results demonstrate that DNA-templated silver nanocluster probes can be versatile reporters for detecting nuclease activities and provide insights into the interactions between nucleases and metallo-DNA nanomaterials.
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Data availability
Source data are provided with this paper and available online at https://doi.org/10.5281/zenodo.10394146.
Code availability
The code designed for data collection and analysis of this study is available online at https://doi.org/10.5281/zenodo.10394146.
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Acknowledgements
This work was supported by the National Science Foundation grants (CBET2029266 to H.-C.Y. and J.S.B., and CBET2041340 to M.J.K.) and the National Institutes of Health grant (EY033106 to H.-C.Y.). The authors thank I. C. Santos for improving the ESI–MS analysis workflow on DNA/AgNCs, S. Kim for her assistance in the RNase A experiment and A. Yeh for his assistance in the revision.
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Contributions
S.H. and H.-C.Y. conceived the project and designed the experiments. S.H. designed the DNA sequences for Subak reporters and mutation tests to optimize the reporters. S.H. developed a gel-purification method to purify DNA/AgNCs. S.H. performed DNase I, Cas12a and RNase A experiments. A.T.L. and J.M. performed fluorescence measurements and developed a Python code for excitation–emission matrices analysis. S.H. and J.N.W. prepared the samples for MS measurements. J.N.W. performed MS measurements and data analysis with assistance from S.W.J.S. and J.S.B. J.S.B. supervised all MS experiments and data analysis. T.D.N., Y.-A.K. and Y.-I.C. collected the absorption data. Y.H. and A.-T.N. checked the buffer compatibility for DNase I digestion and in vitro CRISPR–Cas reaction. M.L.G. and M.J.K. optimized the AgNC synthesis. S.H. and H.-C.Y. wrote the article with input from all authors. H.-C.Y. supervised the project.
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Supplementary Tables 1–4, Equations 1 and 2, Figs. 1–29, Videos 1–4 and Source Data 1–5.
Supplementary Video 1
Fluorescence changes of gel-purified Subak-1 under UV excitation during DNase I digestion.
Supplementary Video 2
Fluorescence changes of gel-purified Subak-2 (II in Fig. 5) under UV excitation during DNase I digestion.
Supplementary Video 3
Visible colour changes of gel-purified Subak-1 during DNase I digestion.
Supplementary Video 4
Visible colour changes of gel-purified Subak-2 (II in Fig. 5) during DNase I digestion.
Source data
Source Data Fig. 2
Unprocessed gel.
Source Data Fig. 3
Unprocessed gel.
Source Data Fig. 5
Unprocessed gel.
Source Data Fig. 2
Statistical source data.
Source Data Fig. 6
Statistical source data.
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Hong, S., Walker, J.N., Luong, A.T. et al. A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion. Nat. Nanotechnol. (2024). https://doi.org/10.1038/s41565-024-01612-6
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DOI: https://doi.org/10.1038/s41565-024-01612-6