Protein induced fluorescence enhancement (PIFE) for probing protein-nucleic acid interactions.

Experimental setup of single molecule measurements

Both the prism-type and objective-type total internal reflection fluorescence (TIRF) microscope can be used for single molecule PIFE measurements (smPIFE) ( Fig. 1A ). The setup for smPIFE is identical to the TIRF microscope used for single molecule FRET measurements, with the exception that the dichroic beam-splitter is unnecessary. A detailed guide on building the TIRF setup has been previously published. 1 Similar to other single molecule fluorescence measurements, quartz and coverslip slides are passivated with mPEG (methoxypolyethylene glycol) to prevent non-specific binding of molecules to the surface ( Fig. 1B ). Approximately 1–2% of biotinylated-PEG is included in mPEG to coat the microscope slides. Once prepared, the slides can be stored at −20 or −80 °C for over one month. At the time of the measurement, the slide is assembled to create flow channels to which all aqueous sample solutions can be applied ( Fig. 1C ). NeutrAvidin is added to the biotin-PEG surface to prepare for “seeding” of fluorescent molecules. Next, fluorescently labeled and biotinylated DNA or RNA (50–100 pM) substrates are applied to the biotin–NeutrAvidin surface. This procedure should yield 300–600 fluorescent molecules in one field of view. 2 For PIFE imaging and histogram analysis, it is crucial to achieve an even illuminated surface that renders one sharply peaked intensity profile so that the intensity change induced by protein binding can be clearly distinguished ( Fig. 1D ). For the same reason, one needs to cautiously monitor the intensity of DNA-only or RNA-only signals before any protein is added. If the individual traces will be normalized to the protein-unbound intensity, the intensity distribution on the imaging surface does not need to be uniform.