Methods: Single-Molecule Techniques Biochemistry 4000 Dr. Ute Kothe.

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.