SingleMolecule Views of Natures NanoMachines

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Single-Molecule Views of Natures Nano-Machines

• Taekjip Ha
• Physics Department
• University of Illinois
• Urbana-Champaign

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Biology at the single molecule level

• Why study biological systems?
• Biological systems use components at the 1100 nm
  scale the scale of future nano-electronics
• Billions of years of engineering time have been
  used for the bugs (e.g. bacteria) to work out
  the bugs in biological systems
• Biological sensors often reach the physical
  limits of sensitivity (ears, eyes,)

Ultimate reverse engineering!
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Linear Motors
Actin filament, microtubules
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Single Molecule Techniques
FIONA
FRET
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Kinesin walking on microtubule
Two heads alternate -gt Hand-over-hand
Milligan, Vale et al.
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Myosin V (Kinesin) Hand-over-hand or Inchworm?
Hand-over-hand Head (foot) takes 74 (16.6) nm
steps
37 (8)nm
1 ATP c.m. 37 (8) nm
1 ATP c.m. 37 (8)nm
By measuring head-step size, can differentiate
models
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Experimental Setup Imaging Single Molecules
Total Internal Reflection Microscopy
Myosin V on actin Center of mass step size 37 nm
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Diffraction limited spot
Width of l/2 ? 250 nm
With enough photons (signal to noise) Center
can be determined to ? 1.5 nm.
Center represents (under appropriate conditions)
position of dye.
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74 nm stepping of a head
Myosin V walks!
Yildiz, Forkey, McKinney, Ha Goldman and Selvin
(2003)
Can we do it for DNA motors?
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0.34 nm
It has not escaped our notice that the specific
pairing suggests a possible copying mechanism
for the genetic material. -Watson Crick
(Nature, April 25th, 1953)
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Helicase unwinds and moves along nucleic acids
motor protein
Also uses ATP hydrolysis
Helicase
Human Genome contains more than 100 helicases
DNA replication
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Measuring conformational dynamics using
single-molecule FRET
Acceptor (Cy5)
Donor (Cy3)
Ha et al, PNAS (1996)
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Single stranded DNA translocation via FRET
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Experimental Scheme

• 100s of molecules
• 30 ms time resolution

Ha et al, Nature 2002
Saito K, Tokunaga M, Iwane AH, Yanagida T , J. of
Micr. 1997
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ssDNA translocation by wild type Rep
acceptor
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donor
(dT)50
5
biotin
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Blockade induced repetition of DNA translocation
acceptor
3
T50
5
biotin
Molecular Sisyphus!
Any biological significance?
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Stalled replication fork
Leading strand synthesis
Okazaki fragment
Lagging strand synthesis
Template DNA
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Summary
FIONA and FRET for Motor Studies
FRET
FIONA

• Blockade-induced repetition of ssDNA
  translocation by a DNA motor (molecular sisyphus)
• Potential novel in vivo function of a Rep
  helicase monomer in replication restart
  (molecular floss)

• One nanometer localization of single fluorescent
  molecule
• Myosin V walks hand-over-hand

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Future Directions

• Combination with Force/Manipulations
• Single molecule in live cells (Quantum dots)
• Fuse with nanotechnology

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Marriage between FRET and optical trap
coverslip
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Future Directions

• Combination with Force/Manipulations
• Single molecule in live cells (Quantum dots)
• Fuse with nanotechnology

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Suppression of quantum dot blinking
Hohng and Ha, J.Am.Chem.Soc. (2004)
Qdot diffusion on a membrane
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Future Directions

• Combination with Force/Manipulations
• Single molecule in live cells (Quantum dots)
• Fuse with nanotechnology

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DNA transport
Diez et al, Nano Letters (2003)
Microtube (Red) slides over kinesin surface and
carries DNA (Green)