Dr. Richard Börner, Sigel lab - subgroupleader smFRET,
Department of Chemistry, University Zürich

Understanding RNA folding: crowding, metal ions and smFRET

As we use carbocyanine dyes as FRET pairs to study RNA folding on the single molecule level, we characterized their photophysical properties in the realm of RNA. Our findings motivate to include dye-RNA interactions in the accessible volume (AV) calculations as dye model to improve structure predictions based on smFRET constrains (1).

The formation of tertiary contacts among nascent secondary structure elements is an integral part of RNA folding. As a model system, we use an obligate tertiary contact common to all classes of group II intron ribozymes: the exon and intron binding site 1 (EBS1/(d)IBS1) with known NMR structure, and study the metal ion dependent tertiary contact formation. A known feature of such contacts is their kinetic heterogeneity (2). By means of smFRET in combination with global hidden Markov modeling we resolve the heterogenous unbinding rates. Further, we use molecular dynamics (MD) simulation to study the structural origin of observed differences between the RNA-RNA and RNA-DNA contacts (3).

RNAs require a high magnesium(II) concentration to show folding and function in vitro. In contrast, in vivo conditions are characterized by a highly crowded cellular environment and a much lower ion concentration. We combined bulk activity assays, smFRET experiments and NMR to study RNA folding in a crowded environment screening PEG volume fraction (%) and molecular weight (MW). Strikingly, our study unveiled an optimal pore size in terms of the ribozyme’s catalytic activity (4).

(1) Steffen et al. Phys. Chem. Chem. Phys. (2016) 18(42):29045–29055.
(2) Börner et al. Coord. Chem. Rev. (2016) 327-328:123–142. Kowerko et al. Proc. Nat. Acad. Sci. U.S.A. (2015) 112(11):3403–3408.
(3) Steffen and Börner et al. (2019) submitted.
(4) Börner et al. Proc. Natl. Acad. Sci. U.S.A. (2018) 115(47) 11917-11922.

Host: Associate Professor Victoria Birkedal, iNANO & Department of Chemistry