Abstract
Sequence-specific recognition of double-stranded nucleic acids is essential for molecular diagnostics and in situ imaging. Clustered regularly interspaced short palindromic repeats and Cas systems rely on protospacer-adjacent motif (PAM)-dependent double-stranded DNA (dsDNA) recognition, limiting the range of targetable sequences and leading to undesired off-target effects. Using single-molecule fluorescence resonance energy transfer analysis, we discover the enzymatic activity of bacteriophage λ exonuclease (λExo). We show binding of 5′-phosphorylated single-stranded DNA (pDNA) to complementary regions on dsDNA and DNA–RNA duplexes, without the need for a PAM-like motif. Upon binding, the λExo–pDNA system catalytically digests the pDNA into nucleotides in the presence of Mg2+. This process is sensitive to mismatches within a wide range of the pDNA-binding region, resulting in exceptional sequence specificity and reduced off-target effects in various applications. The absence of a requirement for a specific motif such as a PAM sequence greatly broadens the range of targets. We demonstrate that the λExo–pDNA system is a versatile tool for molecular diagnostics, DNA computing and gene imaging applications.
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Data availability
Source data are provided with this paper.
Code availability
The code used in this study was described in a previous study51 and is available from GitHub (https://github.com/Su-Laboratory/smFRET).
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Acknowledgements
This work was supported by the State Key Research Development Program of China (2022YFC2603902), the National Natural Science Foundation of China (32271521, 22325801 and 31971361), the Natural Science Foundation of Beijing Municipality (5212013), the Fundamental Research Funds for the Central Universities (PT2406) and the Talent Cultivation of State Key Laboratory of Organic–Inorganic Composites, Beijing University of Chemical Technology (oic-2024020007).
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X.S. conceptualized and designed the study. S.F., J. Li, J.C., X.S., L.Z. and J. Liu designed, performed and analyzed the experiments. X.S., S.F. and H.L. wrote and revised the paper. All authors discussed the results and revised or commented on the paper.
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Fu, S., Li, J., Chen, J. et al. Bacteriophage λ exonuclease and a 5′-phosphorylated DNA guide allow PAM-independent targeting of double-stranded nucleic acids. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02388-9
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DOI: https://doi.org/10.1038/s41587-024-02388-9
