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DNAzyme-based faithful probing and pulldown to identify candidate biomarkers of low abundance

Nature Chemistry volume 16, pages 122–131 (2024)Cite this article

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Abstract

Biomarker discovery is essential for the understanding, diagnosis, targeted therapy and prognosis assessment of malignant diseases. However, it remains a huge challenge due to the lack of sensitive methods to identify disease-specific rare molecules. Here we present MORAC, molecular recognition based on affinity and catalysis, which enables the effective identification of candidate biomarkers with low abundance. MORAC relies on a class of DNAzymes, each cleaving a sole RNA linkage embedded in their DNA chain upon specifically sensing a complex system with no prior knowledge of the system’s molecular content. We show that signal amplification from catalysis ensures the DNAzymes high sensitivity (for target probing); meanwhile, a simple RNA-to-DNA mutation can shut down their RNA cleavage ability and turn them into a pure affinity tool (for target pulldown). Using MORAC, we identify previously unknown, low-abundance candidate biomarkers with clear clinical value, including apolipoprotein L6 in breast cancer and seryl-tRNA synthetase 1 in polyps preceding colon cancer.

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Fig. 1: The DNAzyme-based MORAC technique for discovery of potential biomarkers from disease samples.
Fig. 2: Identification of APOL6 as a candidate biomarker using MORAC on breast cancer cell lines.
Fig. 3: The tumour suppressor role of APOL6 in breast cancer.
Fig. 4: Identification of SARS1 as a candidate biomarker using MORAC on the tissue of polyps preceding colon cancer.
Fig. 5: Increase of SARS1 in colon LGIN and HGIN but not colon carcinoma.

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All data are available in the main text or the supplementary materials. Requests for resources and reagents should be directed to the corresponding authors. Source data are provided with this paper.

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Acknowledgements

We acknowledge funding by the National Key Research and Development Program of China (2020YFA0908901 to H.G.), the National Natural Science Foundation of China (82121002 and 91859104 to H.G.; 21991134 and T2188102 to C.F.), the Program of Shanghai Academic Research Leader (22XD1421500 to H.G.) and the New Cornerstone Science Foundation (to C.F.). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Qinqin Hu, Zongxuan Tong, Ayimukedisi Yalikong, Li-Ping Ge.

Authors and Affiliations

  1. Key Laboratory of Breast Cancer in Shanghai, Department of Breast Surgery, Institutes of Biomedical Sciences, Fudan University Shanghai Cancer Center, Shanghai Stomatological Hospital, and Endoscopy Center, Zhongshan Hospital, Fudan University, Shanghai, China

    Qinqin Hu, Zongxuan Tong, Ayimukedisi Yalikong, Li-Ping Ge, Qiang Shi, Xinyu Du, Pu Wang, Xi-Yu Liu, Wuqiang Zhan, Xia Gao, Di Sun, Tong Fu, Dan Ye, Yun-Shi Zhong, Yi-Zhou Jiang & Hongzhou Gu

  2. Department of Chemical Biology, School of Chemistry and Chemical Engineering, and School of Global Health, Shanghai Jiao Tong University, Shanghai, China

    Qinqin Hu, Chunhai Fan & Hongzhou Gu

  3. Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic Acids Chemistry and Nanomedicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

    Chunhai Fan

  4. New Cornerstone Science Laboratory, Frontiers Science Center for Transformative Molecules and National Center for Translational Medicine, Shanghai Jiao Tong University, Shanghai, China

    Chunhai Fan

  5. Zhangjiang Laboratory, Shanghai, China

    Chunhai Fan

  6. Department of Digestive Disease, Huashan Hospital, Fudan University, Shanghai, China

    Jie Liu

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Contributions

Q.H., Z.T., A.Y. and L.-P.G. conducted the selection, target identification and experimental validation of the target. Q.S., X.D., P.W., W.Z., X.G., D.S. and T.F. conducted the experimental validation of the target. X.-Y.L. and A.Y. collected clinical specimens. D.Y., J.L. and C.F. analysed the data. Y.-S.Z. and Y.-Z.J. supervised the project. H.G. developed the initial concept, supervised the project, interpreted the data and wrote the manuscript. All authors participated in the discussions and reviewed and approved the manuscript.

Corresponding authors

Correspondence to Yun-Shi Zhong, Yi-Zhou Jiang or Hongzhou Gu.

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Competing interests

Three Chinese patents have been filed, with a status of initiative for examination as to substance. Q.H., Z.T., L.-P.G., X.-Y.L., T.F., Y.-Z.J. and H.G. declare the following competing interests: patent no. CN202110364792.0 was applied for by Fudan University Shanghai Cancer Center, covering the identified DNAzyme probes that selectively sense MDA-MB-231 cells in this study; and patent no. CN202110361337.5 was also applied for by Fudan University Shanghai Cancer Center, covering the method to identify the potential biomarker (APOL6) of breast cancer based on the DNAzyme probes in this study. Z.T., A.Y., Q.S., D.S., Y.-S.Z. and H.G. declare the following competing interests: patent no. CN202111198079.X was applied for by Zhongshan Hospital, Fudan University, covering the findings of the DNAzyme probes that selectively sense LGIN/HGIN specimens and the probes’ utility in biomarker discovery in this study. X.D., P.W., W.Z., X.G., D.Y., C.F. and J.L. declare no competing interests.

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Nature Chemistry thanks Feng Li, Chao Liang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Figs. 1–26, Materials and Methods and unprocessed gels and micrographs for Supplementary Figs. 3–6, 8, 10, 11, 14, 16, 18–21, 23 and 26.

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Supplementary Data 1

Supplementary Tables 1–4.

Supplementary Video 1

Vesicle-like movement of APOL6–Emerald in MDA-MB-231 cells.

Supplementary Data 2

Statistical source data for Supplementary Figs. 2, 4, 5, 7, 15–17, 21, 22, 24 and 26.

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Unprocessed gels and western blots.

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Unprocessed micrographs.

Source Data Fig. 4

Unprocessed gels and micrographs.

Source Data Fig. 5

Unprocessed micrographs.

Source Data Figs. 2–5

Statistical source data for Figs. 2–5.

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Hu, Q., Tong, Z., Yalikong, A. et al. DNAzyme-based faithful probing and pulldown to identify candidate biomarkers of low abundance. Nat. Chem. 16, 122–131 (2024). https://doi.org/10.1038/s41557-023-01328-5

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