Monday, 15th of December at 14:00, we will have the pleasure to listen to Dr. Sonja Schmid, from the University of Basel, Switzerland and Prof. Felix Ritort from the University of Barcelona, Spain. Sonja will talk about Dye-Cycling, allowing hour-long single-molecule FRET observations and overcoming photo-bleaching for biological studies. Felix will talk about RNA folding landscapes in low-temperature conditions using single-RNA force spectroscopy.
reaking the photobleaching limit in single-molecule FRET by DyeCycling., Benjamin Vermeer1,3, Dong Hoon Shin2,4, Fabian Zundel, Sabina Caneva2, Sonja Schmid1,3*
1 Department of Chemistry, University of Basel, Mattenstrasse 22, Building 1096, 4058 Basel, Switzerland.
2 Swiss Nanoscience Institute, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
3 Department of Precision and Microsystems Engineering, Delft University of Technology, Mekelweg 2, 2628 CD Delft, The Netherlands.
4 Current address: Department of Electronics and Information Engineering, Korea University, Sejong 30019, Republic of Korea.
*correspondence:
Paradoxically, single-molecule FRET studies rely on ensemble averaging during data analysis, because early photo-bleaching prohibits the recording of sufficient information from single molecules. As a result, the FRET-based study of inter- and intra-molecular heterogeneity in biomolecular function – a specific hallmark of single-molecule techniques – is hardly possible, preventing insights into dynamic disorder, the effects of post-translational and other modifications, of rare but decisive states, etc. Here, we demonstrate hour-long single-molecule FRET observations using DyeCycling in zero-mode waveguides, which circumvents photobleaching through reversible fluorophore binding. We detect the conformational dynamics of single molecules over four orders of magnitude in time (milliseconds-hour), enabling us to directly observe slow kinetic regime changes within individual molecules that were intractable previously. Moreover, we demonstrate the versatility of DyeCycling with DNA and protein molecules. Together, these advances establish DyeCycling/FRET as a powerful new approach that vastly expands the information gain of single-molecule FRET, enabling the study of important biological questions that were previously inaccessible.
Universal Biochemistry of RNA in the Cold, Felix Ritort1, 2, 3
1Small Biosystems Lab, Condensed Matter Physics Department, University of Barcelona, Carrer Marti i Franques 1, 08028 Barcelona (Spain)
2 Institut de Nanociència i Nanotecnologia (IN2UB), 08028 Barcelona (Spain)
3Reial AcadÈmia de Ciencies i Arts de Barcelona, La Rambla 115, 08002 Barcelona (Spain)
RNA impacts biological diversity and life. The main difference with DNA is the presence of the ribose 2’OH hydroxyl group, which enables RNA to hydrogen-bond
with nucleotides and water and to catalyze reactions mediated by metal ions. Recent single-RNA pulling experiments in our lab with a temperature-jump optical trap in the temperature range 5-50ºC found that RNA hairpins invariably form alternative misfolded structures below a glass transition temperature TG~20ºC where the heat capacity of the folded hairpin abruptly drops. Moreover, maximum stability is found at TS»5ºC where the water density is maximum, and cold denaturation transition is predicted to occur at TC» -50ºC. The prominent role of the ribose-water clathrate in the cold suggests that TG, TS and TC are universal temperatures weakly modulated by sequence. The promiscuous sequence-independent ribose-water interactions mediated by the 2’OH hydroxyl group portend an altered RNA biochemistry at low temperatures, with consequences for novel RNA functionalities and catalytic activities at freezing and subfreezing temperatures, for RNA function in present-day cold-loving psychrophilic organisms, and potentially for RNA evolution.
Reference: P. Rissone, A. Severino, I. Pastor and F. Ritort, Universal cold phase transitions in RNA,
Proceedings of the National Academy of Sciences121 (34) e2408313121 (2024)
