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Related Concept Videos

Maxam-Gilbert Sequencing01:05

Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The...

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Assessment of DNase Activity by Ratiometric Fluorescence Resonance Energy Transfer
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Continuous fluorescence-based method for assessing dicer cleavage efficiency reveals 3' overhang nucleotide

Jonathan P DiNitto1, Leyu Wang, Joe C Wu

  • 1Pfizer Research Technology Center, Cambridge, MA, USA. jonathan.dinitto@biogenidec.com

Biotechniques
|June 24, 2010
PubMed
Summary

A new assay rapidly evaluates Dicer enzyme processing of double-stranded RNA (dsRNA) substrates. This method identified a preference for purine/purine 3' overhangs in Dicer substrate RNAs (D-siRNAs), aiding therapeutic development.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • RNA Interference

Background:

  • Dicer enzyme is crucial for gene silencing via the RNA-induced silencing complex (RISC).
  • Chemically modified RNA molecules are being developed as Dicer substrates for nucleic acid therapeutics.
  • Efficient evaluation of Dicer substrate processing is essential for therapeutic development.

Purpose of the Study:

  • To develop a rapid, quantitative assay for assessing Dicer substrate processing.
  • To evaluate the impact of RNA sequence and overhang structures on Dicer processing.
  • To facilitate the study of modified nucleic acids and regulatory factors affecting Dicer.

Main Methods:

  • A fluorescence quencher-based assay using a Cy5-Iowa Black RQ labeled dsRNA probe.
  • Heterologous competition assays to assess unlabeled test substrates without electrophoresis or radiolabeling.
  • Evaluation of 196 unlabeled 27-mer Dicer substrates with varying overhangs using purified Dicer enzyme.

Main Results:

  • Dicer showed no sequence preference in the main double-stranded region of substrates.
  • A preference for Dicer substrate RNAs (D-siRNAs) with purine/purine 3' overhangs was observed.
  • The assay effectively evaluated Dicer processing of various substrates and identified sequence preferences.

Conclusions:

  • The developed assay is a valuable tool for rapid Dicer substrate evaluation.
  • Dicer processing is influenced by specific overhang structures, particularly purine/purine 3' overhangs.
  • This method aids in the development of RNA-based therapeutics and the study of Dicer function.