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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
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An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
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A fluorescent probe to simultaneously detect both O-GlcNAcase and phosphatase.

Jihyeon Boo1, Jongwon Lee1, Young-Hyun Kim1

  • 1Department of Chemistry, Yonsei University, Seoul, Republic of Korea.

Frontiers in Chemistry
|March 20, 2023
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Summary

Researchers developed a novel fluorescent probe, βGlcNAc-CM-Rhod-P, to simultaneously detect the activities of O-GlcNAcase (OGA) and phosphatase. This tool enables differential monitoring of crucial protein posttranslational modifications in cellular environments.

Keywords:
O-GlcNAcasecoumarinenzymefluorescent probephosphataserhodol

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Chemical Biology

Background:

  • Protein O-GlcNAcylation and phosphorylation are dynamic posttranslational modifications with significant physiological and pathological roles.
  • Crosstalk between these modifications influences numerous cellular processes.
  • Simultaneous detection of O-GlcNAcase (OGA) and phosphatase activities is crucial for understanding cellular regulation.

Purpose of the Study:

  • To design and synthesize a novel bifunctional fluorescent probe, βGlcNAc-CM-Rhod-P, for the simultaneous detection of OGA and phosphatase activities.
  • To characterize the probe's response to enzymatic cleavage by OGA and phosphatase.
  • To demonstrate the probe's utility in detecting enzyme activities in cell lysates and imaging in cells.

Main Methods:

  • Synthesis of the bifunctional fluorescent probe βGlcNAc-CM-Rhod-P, incorporating OGA and phosphatase substrates.
  • Enzymatic assays using purified OGA and phosphatase to validate probe response.
  • Spectroscopic analysis to confirm distinct fluorescence signals from liberated coumarin (CM) and rhodol (Rhod) moieties.
  • Application of the probe for activity detection in cell lysates and fluorescence imaging in live cells.

Main Results:

  • The probe βGlcNAc-CM-Rhod-P successfully generated distinct fluorescence signals upon sequential or simultaneous enzymatic cleavage by OGA and phosphatase.
  • OGA activity led to the release of CM, while phosphatase activity released Rhod, with minimal spectral interference due to a 100 nm emission maxima difference.
  • The probe enabled sensitive detection and fluorescent imaging of OGA and phosphatase activities within cellular contexts.

Conclusions:

  • βGlcNAc-CM-Rhod-P serves as an effective chemical tool for the simultaneous and differential assessment of OGA and phosphatase activities.
  • This probe facilitates a deeper understanding of the interplay between O-GlcNAcylation and phosphorylation in biological systems.
  • The developed probe holds potential for broader applications in studying cellular signaling pathways regulated by these key enzymes.