High-sensitivity, protein-independent detection of dsDNA sequences
: Yan, Jiaqi; Bhadane, Rajendra; Xu, Wentao; Ran, Meixin; Ma, Xiaochao; Li, Yuanqiang; Jahnke, Kevin; Ma, Xiaodong; Salo-Ahen, Outi M. H.; Kostiainen, Mauri A.; Weitz, David A.; Zhang, Hongbo
Publisher: Proceedings of the National Academy of Sciences
: 2026
Proceedings of the National Academy of Sciences of the United States of America
: e2515765123
: 123
: 6
: 0027-8424
: 1091-6490
DOI: https://doi.org/10.1073/pnas.2515765123
: https://doi.org/10.1073/pnas.2515765123
: https://research.utu.fi/converis/portal/detail/Publication/515634781
Current methodologies for detecting the sequence of double-stranded DNA (dsDNA) require amplifying and denaturing the target into single-stranded DNA (ssDNA) to enable sequence detection through Watson-Crick base pairing. However, these approaches are limited by the risks of nonspecific amplification, reliance on complex, temperature-sensitive protein enzymes, and harsh reaction conditions, such as in strong base or acidic environments. Here, we introduce a dsDNA detection platform that integrates a peptide nucleic acid (PNA) as the dsDNA denaturation agent, with multicomponent deoxyribozyme as the ssDNA detection tool, in a droplet-based system. This protein- and amplification-free method offers single-nucleotide resolution, detects down to a single dsDNA molecule, and delivers results within 1 h at room temperature. This work introduces a conceptually unique approach, that may be useful for both diagnostics and therapeutics.
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We thank the Zhejiang Provincial Natural Science Foundation of China [BD24H180001 (H.Z.)], the Research Project [Grant No. 347897 (H.Z.)], Solution for Health Profile [Grant No. 336355 (H.Z.)], InFLAMES Flagship [Grant No. 337531 (H.Z.)], and “Printed Intelligence Infrastructure” (PII-FIRI) (H.Z.)” from Research Council of Finland; NIH/University of Pittsburgh [Grant No. R01AI153156 (D.A.W)]; and the Tor, Joe, and Pentti Borg Memorial Fund (O.S). J.Y. thanks the Finnish Culture Foundation Post Doc Pool grant. K.J. thanks the Alexander von Humboldt Foundation for financial support. We acknowledge the Academy of Finland Centers of Excellence Program (2022–2029) in Life-Inspired Hybrid Materials (LIBER) 346110 (M.A.K). We thank the Center for Nanoscale Systems (CNS) in Harvard University. We thank Electron Microscopy Laboratory, Institute of Biomedicine, University of Turku, and Biocenter Finland. Also, the Biocenter Finland Bioinformatics, CSC IT Center for Science, and Prof. Mark Johnson and Dr. Jukka Lehtonen are gratefully acknowledged for the excellent computational infrastructure at the Åbo Akademi University.
