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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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Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
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Size-Dependent Target Engagement of Covalent Probes.

László Petri1, Ronen Gabizon2, György G Ferenczy1

  • 1Medicinal Chemistry Research Group and National Drug Discovery and Development Laboratory, HUN-REN Research Centre for Natural Sciences, 2 Magyar tudósok krt, Budapest 1117, Hungary.

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Protein labeling with covalent ligands is crucial for proteomics and targeted covalent inhibitor (TCI) development. Noncovalent affinity drives target engagement for lead compounds, while reactivity is key for fragment-based labeling of nucleophilic residues.

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Area of Science:

  • Chemical Biology
  • Proteomics
  • Drug Discovery

Background:

  • Covalent ligand labeling is increasingly used in proteomics and for developing targeted covalent inhibitors (TCIs).
  • Labeling efficiency is assessed by target covalent occupancy or biochemical activity.
  • Understanding the interplay between noncovalent affinity, covalent reactivity, and experimental conditions is vital.

Purpose of the Study:

  • To investigate the relationship between intrinsic complex formation parameters (noncovalent affinity, covalent reactivity) and observed labeling efficiency.
  • To analyze the influence of experimental conditions (incubation time, ligand concentration) on protein labeling outcomes.
  • To compare the suitability of different compound types (lead-like vs. fragment-sized) for TCI development and residue exploration.

Main Methods:

  • Theoretical investigation of labeling efficiency based on intrinsic binding and reactivity parameters.
  • Analysis of how incubation time and ligand concentration affect target engagement.
  • Comparative assessment of lead-like compounds and fragment-sized compounds in covalent labeling.

Main Results:

  • Target engagement is favorably driven by noncovalent recognition for lead-like compounds, aiding TCI optimization.
  • Fragment-sized compounds primarily rely on covalent reactivity for labeling due to limited noncovalent affinity.
  • Lead-like compounds offer detectable occupancy and fixed binding modes, suitable for TCI development.
  • Fragment-sized compounds are effective for exploring ligandable nucleophilic residues but less ideal for TCI starting points.

Conclusions:

  • Noncovalent affinity is key for efficient target engagement with lead-like compounds in covalent labeling strategies.
  • Covalent reactivity dominates labeling for fragment-sized compounds, making them valuable for residue identification.
  • Compound size dictates the optimal strategy: lead-like for TCI development, fragments for residue exploration.