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Labeling DNA Probes03:31

Labeling DNA Probes

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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Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation
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Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Published on: January 16, 2019

Red fluorescent scaffold for highly sensitive protease activity probes.

Yu Kushida1, Kenjiro Hanaoka, Toru Komatsu

  • 1Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Tokyo 113 0033, Japan.

Bioorganic & Medicinal Chemistry Letters
|May 22, 2012
PubMed
Summary

Researchers created a new red fluorescent dye, 2Me SiR600, for highly sensitive protease detection. Probes made with this dye show significant fluorescence increases upon enzyme activity, enabling precise activity monitoring.

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Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation
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Published on: January 16, 2019

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity
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Published on: August 4, 2009

Detection of Protease Activity by Fluorescent Peptide Zymography
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Detection of Protease Activity by Fluorescent Peptide Zymography

Published on: January 20, 2019

Area of Science:

  • Organic Chemistry
  • Biochemistry
  • Fluorescence Spectroscopy

Background:

  • Rhodamine dyes are widely used in biological imaging.
  • Developing novel fluorescent probes with high signal-to-noise ratios is crucial for sensitive biological detection.
  • Protease activity is a key indicator in various biological processes and disease states.

Purpose of the Study:

  • To develop a novel red fluorescent dye, 2Me SiR600, as a scaffold for protease activity probes.
  • To demonstrate the utility of 2Me SiR600 by creating probes for caspase-3 and leucine aminopeptidase.
  • To achieve extremely high signal-to-noise ratios in protease detection.

Main Methods:

  • Synthesis of the novel red fluorescent dye 2Me SiR600 by replacing an oxygen atom with a silicon atom in Rhodamine Green.
  • Design and synthesis of specific protease activity probes (Z-DEVD-SiR600 for caspase-3, Leu-SiR600 for leucine aminopeptidase).
  • Spectroscopic analysis to measure absorption and fluorescence changes upon enzymatic cleavage.

Main Results:

  • The novel dye 2Me SiR600 exhibits a maximum emission wavelength of 613 nm.
  • Enzymatic cleavage of the caspase-3 probe (Z-DEVD-SiR600) by caspase-3 resulted in a significant 93 nm bathochromic shift.
  • A remarkable 432-fold fluorescence enhancement was observed upon caspase-3-mediated cleavage, indicating high sensitivity.

Conclusions:

  • 2Me SiR600 is a novel red fluorescent dye suitable for developing highly sensitive protease activity probes.
  • The developed probes demonstrate excellent performance with substantial signal amplification upon target protease activity.
  • This new scaffold offers a promising platform for precise and sensitive detection of protease activity in biological systems.