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Labeling DNA Probes03:31

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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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Development of a DUB-selective fluorogenic substrate.

Lorina Gjonaj1, Aysegul Sapmaz1, Dennis Flierman1

  • 1Oncode Institute , Department of Cell and Chemical Biology , Leiden University Medical Center , Einthovenweg 20 , 2333 ZC , Leiden , The Netherlands .

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|February 29, 2020
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Summary
This summary is machine-generated.

Researchers developed a novel probe to specifically detect USP16 activity, a key enzyme in cell division and stem cell renewal. This tool enables precise monitoring of deubiquitinating enzymes (DUBs) for potential therapeutic applications.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Ubiquitination is a crucial post-translational modification regulating numerous cellular processes.
  • Deubiquitinating enzymes (DUBs) are increasingly recognized as therapeutic targets for various diseases.
  • USP16 is implicated in mitosis and stem cell self-renewal, making it a significant target of interest.

Purpose of the Study:

  • To design a highly selective activity-based probe (ABP) for the deubiquitinating enzyme USP16.
  • To overcome the limitations of traditional ubiquitin-based probes lacking target specificity.
  • To enable precise monitoring of USP16 activity in complex biological samples.

Main Methods:

  • Utilized structural modeling and mutational calculation predictions to engineer a selective Ub-based ABP.
  • Developed a novel fluorogenic substrate specifically processed by USP16.
  • Combined computational predictions with biochemical assay development.

Main Results:

  • Successfully designed and validated a Ub-based ABP with high selectivity for USP16.
  • Developed the first fluorogenic substrate exclusively processed by USP16.
  • Demonstrated the ability to monitor USP16 activity in complex biological contexts.

Conclusions:

  • The developed probe and substrate offer a selective tool for studying USP16.
  • This advancement facilitates research into USP16's role in cellular processes and disease.
  • Provides a foundation for developing targeted therapeutics modulating USP16 activity.