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Related Concept Videos

Labeling DNA Probes03:31

Labeling DNA Probes

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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.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
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Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication
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Development of Single-Molecule Enzyme Activity Assay for Serine Hydrolases Using Activity-Based Protein Labeling

Seiya Ishii1, Mayano Minoda1, Tadahaya Mizuno1

  • 1Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, 113-0033, Japan.

Small Methods
|September 30, 2025
PubMed
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New single-molecule assays detect active enzymes across superfamilies using activity-based probes. This method identifies active granzyme B in blood, potentially indicating liver damage and immune activation.

Keywords:
chemical biologyenzymesenzymomicssingle‐molecule analysis

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Single-molecule enzyme activity assays offer insights into aberrant protein function at the proteoform level.
  • A key limitation is the restricted number of enzymes targetable by current assay methods.

Purpose of the Study:

  • To develop novel single-molecule enzyme activity assays capable of targeting a broader range of enzymes.
  • To utilize activity-based probes for superfamily-wide labeling of active enzymes.

Main Methods:

  • Development of single-molecule enzyme activity assays employing activity-based probes.
  • Proof-of-principle study using fluorophosphonate-based probes.
  • Detection of active serine hydrolases, including PSA and granzyme B, at the single-molecule level in blood.

Main Results:

  • Demonstrated the ability to detect active serine hydrolases at the single-molecule level in complex biological samples like blood.
  • Successfully identified active granzyme B in blood samples.

Conclusions:

  • Active granzyme B in blood shows potential as a biomarker for liver damage.
  • This biomarker is associated with immune cell activation, suggesting a link between liver injury and immune response.