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Related Concept Videos

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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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Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...
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Related Experiment Video

Updated: Sep 10, 2025

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes
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Fluorescence Light-Up of G4 DNA Structures Using Azlactone-Based Probes.

Annyesha Biswas1, Nitesh Ayare1, Y Dilnawaj1

  • 1Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India.

Biochemistry
|August 20, 2025
PubMed
Summary
This summary is machine-generated.

New azlactone-based fluorescent probes (AZL1-3) effectively detect G-quadruplex (G4) DNA structures, including those in c-MYC and c-KIT1. These probes show potential for bioimaging and diagnostics.

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Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors
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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Chemical Biology

Background:

  • G-rich sequences in DNA and RNA form G-quadruplex (G4) structures.
  • G4 structures play crucial roles in various biological processes.
  • Understanding G4 topology, location, and function is vital in cellular and cell-free environments.

Purpose of the Study:

  • To develop novel small-molecule fluorescent probes for G4 structure detection.
  • To investigate the sensing capabilities of azlactone-based probes (AZL1-3) for specific G4 topologies.
  • To evaluate the cellular localization and potential applications of these probes.

Main Methods:

  • Synthesis of three azlactone-based fluorescent probes (AZL1-3).
  • Fluorescence spectroscopy to quantify probe binding and light-up response with G4 DNAs (c-MYC, c-KIT1, HRCC).
  • Cellular assays using HeLa cells to assess probe localization and cytotoxicity.

Main Results:

  • AZL1-3 probes exhibited significant fluorescence light-up (65-135-fold) for parallel G4 topologies of c-MYC, c-KIT1, and mitochondrial HRCC G4 DNA.
  • The lead probe, AZL1, demonstrated a 2:1 binding stoichiometry with c-KIT1 G4 DNA, interacting with G-quartets.
  • Limited cytotoxicity was observed, with probes showing cytoplasmic localization in HeLa cells, including colocalization with lipid droplets.

Conclusions:

  • Azlactone-based probes are effective tools for sensing G-quadruplex structures in cell-free conditions.
  • The developed probes show promise for potential bioimaging and diagnostic applications.
  • Further engineering of these probes could enhance their utility in cellular G4 detection.