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

Fabio D. Steffen, Mokrane Khier, Danny Kowerko, Richard A. Cunha, Richard Börner, and Roland K.O. Sigel, Nat. Commun, 2020, 11, 104.
doi: 10.1038/s41467-019-13683-4

Susann Zelger-Paulus, Mélodie C.A.S. Hadzic, Roland K.O. Sigel, Richard Börner, RNA Spectroscopy. Methods in Molecular Biology, 2020, 2113, 1-16.
ISSN: 1064-3745
doi: 10.1007/978-1-0716-0278-2_1

Meng Zhao, Richard Börner, Roland K.O. Sigel, Eva Freisinger, RNA Chaperones. Methods in Molecular Biology, 2020, 2106, 253-270.
ISSN: 1064-3745
doi: 10.1007/978-1-0716-0231-7_16

Fabio D. Steffen, Richard Börner, Eva Freisinger, and Roland K.O. Sigel, CHIMIA, 2019, 73, 257-261.
doi: 10.2533/chimia.2019.257

Meng Zhao, Fabio D. Steffen, Richard Börner, Roland K.O. Sigel, Eva Freisinger, Nucleic Acids Res., 2018, 46, e13.
doi: 10.1093/nar/gkx1100

Bishnu P. Paudel, Erica Fiorini, Richard Börner, Roland K.O. Sigel, and David S. Rueda, Proc. Natl. Acad. Sci. USA, 2018, 115, 11917-11922.
doi: 10.1073/pnas.1806685115

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

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

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. USA, 2015, 112 (11), 3403–3408.
doi: 10.1073/pnas.1322759112

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