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Peptide-Based Turn-On Fluorescent Probes for Highly Specific Detection of Survivin Protein in the Cancer Cells

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posted on 2024-05-02, 05:13 authored by Takeshi Fuchigami, Tomoe Nakayama, Yusuke Miyanari, Iori Nozaki, Natsumi Ishikawa, Ayako Tagawa, Sakura Yoshida, Masayuki Munekane, Morio Nakayama, Kazuma Ogawa
Survivin is highly expressed in most human cancers, making it a promising target for cancer diagnosis and treatment. In this study, we developed peptide probes consisting of Bor65–75, a high-affinity survivin-binding peptide, and a survivin protein segment using peptide linkers as survivin-sensitive fluorescent probes (SSFPs). All conjugates were attached to 5(6)-carboxyfluorescein (FAM) at the C-terminal as a fluorophore and to 4­((4­(dimethylamino)­phenyl)­azo)­benzoic acid (DABCYL) at the N-terminal as a quencher. Fluorescence (or Förster) resonance energy transfer (FRET) quenching via intramolecular binding of Bor65–75 with survivin protein segment could be diminished by the approach of survivin to SSFPs, which dissociate Bor65–75 from SSPF and increased the distance between FAM and DABCYL. A binding assay using recombinant human survivin protein (rSurvivin) demonstrated moderate to high affinity of SSFPs for survivin (dissociation constants (Kd) = 121–1740 nM). Although the SSFPs (0.5 μM) had almost no fluorescence under baseline conditions, a dose-dependent increase in fluorescence intensity was observed in the presence of rSurvivin (0.1–2.0 μM). In particular, the proline-rich SSFP (SSFP5) showed the highest (2.7-fold) fluorescence induction at 2.0 μM survivin compared to the signals in the absence of survivin. Confocal fluorescence imaging demonstrated that SSFP5 exhibited clear fluorescence signals in survivin-positive MDA-MB-231 cells, whereas no marked fluorescence signals were observed in survivin-negative MCF-10A cells. Collectively, these results suggest that SSFPs can be used as survivin-specific FRET imaging probes.

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