Oral Presentation 23rd International Society of Magnetic Resonance Conference 2023

Combining orientation-selective PELDOR and 19F Mims ENDOR to investigate the conformational dynamics of dsRNA (#63)

Maximilian Gauger 1 , Anna-Lena J. Halbritter (Segler) 2 , Lukas S. Stelzl 3 , Marcel Heinz 4 , Gerhard Hummer 1 4 , Snorri T. Sigurdsson 5 , Thomas F. Prisner 1
  1. Goethe University, Frankfurt, Germany
  2. Ludwig-Maximilians-University Munich, Munich, Germany
  3. Gutenberg University, Mainz, Germany
  4. Max-Planck-Institute of Biophysics, Frankfurt, Germany
  5. University of Iceland, Reykjavik, Iceland

Differences in helix dynamics, specifically the internal twist-, stretch- and bending-motions, have been postulated for dsDNA and dsRNA molecules based on MD simulations1 and AFM methods2. The dynamics of dsDNA molecules have already been investigated using EPR spectroscopy. For these studies two rigid cytidine-analogue paramagnetic spin labels (Ç)3 were incorporated into a 20-mer dsDNA helix. Such rigid spin labels yield very precise distance and structural restraints from orientation-selective PELDOR/DEER experiments.4 In the past, it was possible to determine the twist-stretch coupling of dsDNA from such experiments and quantitatively compare the experimental data to MD simulations.5

In the project, which is presented here, a set of doubly Çm spin labelled 20-mer dsRNA molecules was investigated. We acquired orientation-selective PELDOR data at different frequency bands (X-, Q-, G-band) where differing degrees of orientation-selection and structural restraints are observable. These data encode for spin label distances and orientations which are dependent on the global conformational dynamics of the dsRNA they are attached to. Following recent developments,6 we performed 19F Mims ENDOR experiments on three singly Çm and singly Fluorine labelled dsRNA samples. From this we gain insights into the exact positioning of the Çm spin label in the local helix geometry.

In a quantitative comparison to MD simulations we found that state-of-the-art force fields can describe the conformational ensemble present in our experiments. This means that we can use our experimental PELDOR and simulated MD data to derive a comparison between the helical dynamics of dsDNA and dsRNA.

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  2. Lipferta, J.; Skinnera, G.M.; Keegstraa, J.M.; Hensgensa, T.; Jagera, T.; Dulina, D.; Köber, M.; Yu, Z.; Donkers, S.P.; Chou, F.C.; Das, R.; Dekker, N.H.; Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (43), 15408–15413
  3. Barhate, N.; Cekan, P.; Massey, A. P.; Sigurdsson, S. T.; Angew. Chemie Int. Ed. 2007, 46 (15), 2655–2658
  4. Marko, A.; Denysenkov, V.; Margraf, D.; Cekan, P.; Schiemann, O.; Sigurdsson, S. T.; Prisner, T. F.; J. Am. Chem. Soc. 2011, 133 (34), 13375–13379
  5. Stelzl, L. S.; Erlenbach, N.; Heinz, M.; Prisner, T. F.; Hummer, G.; J. Am. Chem. Soc. 2017, 139 (34), 11674-11677
  6. Meyer, A.; Dechert, S.; Dey, S.; Höbartner, C.; Bennati, M.; Angew. Chem. Int. Ed. 2020, 59, 373-379