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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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A Protocol for Analyzing Hepatitis C Virus Replication
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Hepatitis C Virus Helicase Binding Activity Monitored through Site-Specific Labeling Using an Expanded Genetic Code.

Christopher J Ablenas1,2, Yasser Gidi3, Megan H Powdrill2

  • 1Department of Biochemistry , McGill University , Montreal , Quebec H3G1Y6 , Canada.

ACS Infectious Diseases
|October 24, 2019
PubMed
Summary
This summary is machine-generated.

Hepatitis C virus NS3 helicase (NS3h) unwinding was studied using site-specific fluorescent labeling. Single-molecule FRET revealed NS3h binding dynamics on DNA, providing a new tool to monitor enzyme translocation during unwinding.

Keywords:
DNA helicaseRNA helicasefluorescence resonance energy transferhepatitis C virus (HCV)single-molecule biophysicsunnatural amino acids

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Virology

Background:

  • The hepatitis C virus nonstructural protein 3 helicase (NS3h) is a key enzyme for viral replication and a model for studying helicase mechanisms.
  • Previous research suggests NS3h utilizes an ATP-dependent, inchworm-like mechanism for nucleic acid unwinding.
  • Understanding NS3h's translocation dynamics is crucial for developing antiviral therapies.

Purpose of the Study:

  • To develop a site-specifically fluorescently labeled NS3h for single-molecule Förster resonance energy transfer (smFRET) studies.
  • To investigate the binding interactions of NS3h with different double-stranded DNA (dsDNA) substrates.
  • To establish a novel method for monitoring NS3h enzyme dynamics during unwinding.

Main Methods:

  • Screening for optimal unnatural amino acid incorporation sites in NS3h for fluorescent labeling.
  • Expression and purification of the site-specifically labeled NS3h recombinant protein.
  • Single-molecule FRET experiments to analyze NS3h binding to dsDNA with varying single-stranded (ssDNA) overhangs.

Main Results:

  • A single, functional site for unnatural amino acid incorporation and fluorescent labeling of NS3h was identified.
  • Incorporation of the unnatural amino acid did not impede NS3h unwinding activity.
  • smFRET data revealed distinct binding signatures for NS3h on short (6-7 base) versus long (24 base) ssDNA overhangs, indicating differential exploration of space.

Conclusions:

  • Site-specific fluorescent labeling of NS3h enables detailed single-molecule analysis of its DNA binding and translocation.
  • The developed NS3h construct is a valuable tool for future investigations into helicase dynamics.
  • This approach offers new insights into the mechanism of viral helicase unwinding at the single-molecule level.