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Fluorescence Resonance Energy
       Transfer (FRET)
Fluorescence Resonance Energy
    Molecular Ruler with scale:
           10 to 100 Å
               What is FRET ?

• (FRET) is a distance-dependent interaction between
  the electronic excited states of two dye molecules
  in which excitation is transferred from a donor
  molecule to an acceptor molecule without emission
  of a photon.
• The efficiency of FRET is dependent on the
  inverse sixth power of the intermolecular
  separation, making it useful over distances
  comparable with the dimensions of
  biological macromolecules.
• FRET is an important technique for investigating a
  variety of biological phenomena that produce
  changes in molecular proximity.
• When FRET is used as a contrast mechanism,
  colocalization of proteins and other molecules can
  be imaged with spatial resolution beyond the limits
  of conventional optical microscopy.
Primary Conditions for FRET
• Donor and acceptor molecules must be in
  close proximity (typically 10–100 Å).
• The absorption spectrum of the acceptor
  must overlap the fluorescence emission
  spectrum of the donor.
Schematic representation of the
FRET spectral overlap integral
CFP (Cyan Fluorescence Protein)
• Blue fluorescence
• Excitation: 450-550 nm
• Emission: 500-600 nm
YFP (Yellow Fluorescence Protein)

• Yellow fluorescence
• Excitation: 350-500 nm
• Emission: 400-600 nm
Monitor interaction between two
Principle of the fluorogenic response to protease cleavage
exhibited by HIV protease substrate 1. Quenching of the
EDANS fluorophore (F) by distance-dependent resonance
energy transfer to the dabcyl quencher (Q) is eliminated upon
cleavage of the intervening peptide linker.
Molecular beacons.
        In the hairpin loop structure,
        the quencher (black circle)
        forms a nonfluorescent
        complex with the fluorophore
        (green circle). Upon
        hybridization of the molecular
        beacon to a complementary
        sequence, the fluorophore and
        quencher are separated,
        restoring the fluorescence.
       Wavelength-shifting molecular
• The molecular beacon has two
  fluorophores on one end — a
  "harvester" (green circle) and an
  "emitter" (red circle) — and a
  quencher on the other end (black
  circle). In the hairpin loop
  structure, the quencher forms a
  nonfluorescent complex with the
  harvester. Upon hybridization of
  the molecular beacon to a
  complementary sequence,
  quenching of the harvester
  fluorophore is relieved, and it
  transfers energy (via FRET) to
  the emitter, which emits
      Selected Applications of FRET
•   Structure and conformation of proteins
•   Spatial distribution and assembly of protein complexes
•   Receptor/ligand interactions
•   Immunoassays
•   Probing interactions of single molecules
•   Structure and conformation of nucleic acids
•   Real-time PCR assays and SNP detection
•   Detection of nucleic acid hybridization
•   Primer-extension assays for detecting mutations
•   Automated DNA sequencing
•   Distribution and transport of lipids
•   Membrane fusion assays
•   Membrane potential sensing
•   Fluorogenic protease substrates
•   Indicators for cyclic AMP and zinc

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