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<p>Fluorescent DNA probes commonly used for <i>in situ</i> imaging of cell surface glycans are “always-on” probes (AOPs), which produce high background noise and lack spatial specificity. Molecular dynamics simulations indicate that single-stranded DNA (ssDNA) can stably associate with the cell membrane, contributing to persistent background signals in AOPs. To overcome this challenge, a stepwise activated probe (SAP) is developed for <i>in situ</i> imaging of cell surface glycans. In SAP, the cell surface glycan is labeled by rolling circle amplification product through metabolic glycan labeling strategies. The product hybridizes a fluorophore and quencher-labeled ssDNA reporter to form a double strand producing primary enhancement of fluorescence signal. Nicking enzyme (Nb.BbvCI) cleaves the double strand and releases the quencher, further enhancing the signal. Nonspecific absorption of the reporter on the cellular membrane does not increase the fluorophore-quencher distance in SAP or trigger Nb.BbvCI cleavage. As a result, only the glycan sites are illuminated. SAP not only exhibits high imaging specificity but also greatly simplifies the imaging procedure by reducing the washing steps. SAP offers a considerable improvement in detection specificity and sensitivity by employing a two-step activation and amplification process, making it a powerful method for <i>in situ</i> fluorescence imaging.</p>

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Stepwise activated probe for cell surface glycan imaging

  • 期刊论文

  • 发表日期：

    2025年2月6日

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  • Chunyi Zhang,
  • Jing Chen,
  • Xiaoyao Bai,
  • Shengnan Fu,
  • Xin Su

摘要

Fluorescent DNA probes commonly used for in situ imaging of cell surface glycans are “always-on” probes (AOPs), which produce high background noise and lack spatial specificity. Molecular dynamics simulations indicate that single-stranded DNA (ssDNA) can stably associate with the cell membrane, contributing to persistent background signals in AOPs. To overcome this challenge, a stepwise activated probe (SAP) is developed for in situ imaging of cell surface glycans. In SAP, the cell surface glycan is labeled by rolling circle amplification product through metabolic glycan labeling strategies. The product hybridizes a fluorophore and quencher-labeled ssDNA reporter to form a double strand producing primary enhancement of fluorescence signal. Nicking enzyme (Nb.BbvCI) cleaves the double strand and releases the quencher, further enhancing the signal. Nonspecific absorption of the reporter on the cellular membrane does not increase the fluorophore-quencher distance in SAP or trigger Nb.BbvCI cleavage. As a result, only the glycan sites are illuminated. SAP not only exhibits high imaging specificity but also greatly simplifies the imaging procedure by reducing the washing steps. SAP offers a considerable improvement in detection specificity and sensitivity by employing a two-step activation and amplification process, making it a powerful method for in situ fluorescence imaging.