Fluorescence Spectroscopy

Single-Molecule Förster Resonance Energy Transfer (smFRET)

fluorspec
fluorspec

In 1948, Theodor Förster predicted that an excited donor fluorophore may transfer its energy onto a spectrally overlapping acceptor, given that the two dyes are in close proximity (2-10 nm). This energy transfer follows a strong distance dependence of 1/r6 which makes FRET suitable to probe the dynamics of ribozyme catalysis or to detect conformational changes in metabolite-sensing riboswitches. Interrogating biomolecular processes on the single-molecule level has two key advantages over ensemble approaches: (i) subensemble heterogeneities are unraveled that would otherwise be averaged out. (ii) kinetics can be obtained from equilibrium experiments without the need to synchronize the molecules. We have implemented three-color based smFRET in a total internal reflection fluorescence (TIRF) microscope that uses stroboscopic alternating laser excitation (sALEX) to monitor dozens of RNA molecules in parallel with time-resolutions down to 1 ms.

Time-Correlated Single Photon Counting (TCSPC)

Fluorophore photophysics are known to affect FRET in various ways. Environment-sensitive probes like carbocyanines change their fluorescent lifetime, quantum yield and dynamic anisotropy according to the propensity of photo-induced cis-trans isomerization. Time-correlated single photon counting (TCSPC) measurements are routinely carried out in our lab to account for such photophysical effects that are generally referred to as RNA-induced fluorescent enhancement (RIFE).

Literature

[1] Lucia Cardo, Karunatilaka KS, David Rueda, Roland K.O. Sigel, In: Hartig JS, editor. Ribozymes. Methods and protocols. Totowa, NJ: Humana Press. 2012, 848, 227 - 251.
doi:10.1007/978-1-61779-545-9_15

[2] Danny Kowerko, Sebastian L. B. König, Miriam Skilandat, Daniela Kruschel, Mélodie C. A. S. Hadzic, Lucia Cardo, Roland K. O. Sigel*, Proc. Natl. Acad. Sci. U. S. A., 2015, 112 (11), 3403–3408.
doi:10.1073/pnas.1322759112

[3] Fabio D. Steffen, Roland K. O. Sigel, Richard Börner*, Phys. Chem. Chem. Phys., 2016, 18, 29045 - 29055.
doi:10.1039/c6cp04277e

[4] David Egloff, Igor A. Oleinich, Sebastian L. B. König, Roland K. O. Sigel, Eva Freisinger*, ACS Chem. Biol., 2016, 11, 2558-2567.
doi:10.1021/acschembio.6b00343

[5] Meng Zhao, Fabio D. Steffen, Richard Börner, Roland K.O. Sigel*, Eva Freisinger*, Nucleic Acids Res., 2017, published online.
doi:10.1093/nar/gkx1100