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Related Concept Videos

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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.
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Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
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Single-Molecule Analysis for RISC Assembly and Target Cleavage.

Hiroshi M Sasaki1,2, Hisashi Tadakuma3,4, Yukihide Tomari5,6

  • 1Intsitute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Methods in Molecular Biology (Clifton, N.J.)
|October 15, 2017
PubMed
Summary
This summary is machine-generated.

Single-molecule analysis using TIRF microscopy visualizes RNA-induced silencing complex (RISC) assembly dynamics. This method details chaperone-assisted fly Ago2-RISC formation and target RNA cleavage reactions.

Keywords:
ArgonauteRISCRNA interferenceSingle-molecule imagingSmall interfering RNATotal internal reflection fluorescence microscopy

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • The RNA-induced silencing complex (RISC) is crucial for gene regulation via RNA silencing.
  • Traditional biochemical methods have limitations in resolving intermediate states of RISC assembly.
  • Understanding RISC dynamics is key to elucidating RNA silencing mechanisms.

Purpose of the Study:

  • To develop a single-molecule assay for observing RISC assembly in real-time.
  • To characterize the dynamics of chaperone-assisted fly Ago2-RISC formation.
  • To analyze the target RNA cleavage activity of reconstituted RISC.

Main Methods:

  • Single-molecule analysis utilizing total internal reflection fluorescence (TIRF) microscopy.
  • In vitro reconstitution of Drosophila melanogaster Ago2-RISC.
  • Observation of individual RISC complexes during assembly and function.

Main Results:

  • Established a sensitive single-molecule system for fly Ago2-RISC assembly.
  • Visualized dynamic intermediate states during RISC formation.
  • Quantified the kinetics of target RNA binding and cleavage by individual RISC complexes.

Conclusions:

  • Single-molecule TIRF microscopy provides unprecedented insight into RISC assembly dynamics.
  • The developed system facilitates detailed mechanistic studies of RNA silencing.
  • This approach advances our understanding of gene regulation by small RNAs.