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

2. 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.

3. Signal-to-Noise Ratio Raman 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 -15 l)

4. 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

5. Confocal Detection Pinhole allows observation of light only from the focal point, thus reducing the observed volume.

6. 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

7. Example: k on, k off measurement Histogram of off & on time durations

8. 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-coated microsphere Example: Linear rate of transcription by RNA-Pol. measured in distance/nt per time under constant force: Note random pauses!

9. Principle of optical trap Intense 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

10. 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

11. Example 2: fun Tying a knot in an actin fiber, using optical tweezers.