New methodology

We have introduced a number of new methods, many dealing with the application of segmental labeling of proteins (7) and RNA (8) to try to increase the size limit of protein-RNA complexes that can be analyzed by NMR. In recent years, the Allain lab has integrated additional structural biology approaches in order to solve RNPs of larger sizes by combining liquid-state NMR with Electron Paramagnetic Resonance (EPR, 6 and 9), MS (10) and solid-state NMR (11), respectively. Most relevant to this proposal, we have introduced a new technique named CLIR-MS (10) as an accurate and efficient “footprinting” technique to map protein-RNA contacts at single nucleotide and single amino-acid resolution and to determine their structure by a fast hybrid structural approach.

7. Skrisovska L, Allain FHT. (2008). “Improved segmental isotope labeling methods for the NMR study of multidomain or large proteins: application to the RRMs of Npl3p and hnRNP L.” J Mol Biol. 375(1):151-64.

8. Duss O, Maris C, von Schroetter C, Allain FH. (2010). “A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage.” Nucleic Acids Res. Nov;38(20):e188.

9. Duss O, Yulikov M, Jeschke G, Allain FHT. (2014) “EPR-aided approach for solution structure determination of large RNAs or protein-RNA complexes.” Nat Commun.;5:3669.

10. Dorn G, Leitner A, Boudet J, Campagne S, von Schroetter C, Moursy A, Aebersold R, Allain F.H.-T. (2017) "Structural modeling of protein-RNA complexes using crosslinking of segmentally isotope-labeled RNA and MS/MS." Nat. Methods. 14(5):487-490.

11- Boudet J, Devillier J.C, Wiegand T., Salmon L., Meier B.H, Lipps G., Allain F.H.T (2019) “A small helical bundle prepares primer synthesis by binding two nucleotides that enhance sequence-specific recognition of the DNA template.” Cell, 176(1-2):154-166.