Methods: Single-Molecule Techniques Biochemistry 4000 Dr. Ute Kothe
Dec 21, 2015
Methods:Single-Molecule Techniques
Biochemistry 4000
Dr. Ute Kothe
Why single-molecule detection?
Hairpin Ribozyme
Standard ensemble measurements provide only an average of the measured property.
Single-molecule measurements reveal subpopulations with different properties.
Signal-to-Noise RatioRaman Scatter from Water:• Inelastic scattering of light at longer wavelength • Overlaps with light emitted by fluorophore
Use dyes with high quantum yield Measure 1 dye molecule in a small volume of solvent (1fl = 10-15l)
Total Internal Reflection
Above a certain angle, the light is totally internally reflected at an interface with low refractive index. But the incident beam can still interact with the sample at the interface because the intensity penetrates a short distance into the sample = evanescent field in limited volume.
Illumination through Prism
Confocal DetectionPinhole allows observation of light only from the focal point, thus reducing the observed volume.
Example: G protein activation
Intracellular Single Molecule:
YFP =yellow fluorescent protein
Visualization of RAs activation, i.e. of GTP binding to Ras
Using FRET between YFP And Bodipy-GTP
Example: kon, koff measurementHistogram of off & on time durations
Optical Tweezers• A bead is hold in place by an
optical trap (see below)
• Bead is linked to biomolecule
• Biomolecule is also linked to
coverslip
• Movement of coverslip relative
to optical trap generates force
on biomolecule
Force or distance can be
measured
Optical trap
Streptavidin-coatedmicrosphere
Example: Linear rate of transcription by RNA-Pol. measured in distance/nt per time under constant force: Note random pauses!
Principle of optical trapIntense beam of light (laser) is focussed into a very small region
Intense electromagnetic field will “capture” a small dielectric sphere
Intensity of light influence the force exerted on the bead if displaced from center
Example 1: measuring force
A feedback circuit, based on microscopic observation of the bead position, increases the laser power to keep the bead fixed as the force increases (and vice versa).
While the distance is increased (x axis), the force exerted on the microsphere is measured (y axis).
Stretching of a single chromatin fiber of whích one end is linked to the coverslip and the other end is linked to the microsphere
Example 2: fun
Tying a knot in an actin fiber, using optical tweezers.