Abstract
The structures of RNA:RNA complexes regulate many biological processes. Despite their importance, protein-free RNA:RNA complexes represent a tiny fraction of experimentally determined structures. Here, we describe a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach to structurally interrogate conformational changes in a model RNA:RNA complex. Using SAXS, we measured the solution structures of the individual RNAs and of the overall RNA:RNA complex. With SANS, we demonstrate, as a proof of principle, that isotope labeling and contrast matching (CM) can be combined to probe the bound state structure of an RNA within a selectively deuterated RNA:RNA complex. Furthermore, we show that experimental scattering data can validate and improve predicted AlphaFold 3 RNA:RNA complex structures to reflect its solution structure. Our work demonstrates that in silico modeling, SAXS, and CM-SANS can be used in concert to directly analyze conformational changes within RNAs when in complex, enhancing our understanding of RNA structure in functional assemblies.
| Original language | English |
|---|---|
| Pages (from-to) | 728-739.e4 |
| Journal | Structure |
| Volume | 33 |
| Issue number | 4 |
| DOIs | |
| State | Published - Apr 3 2025 |
Funding
We are grateful to the staff at BioCAT (beamline 18ID, APS, ANL) and Bio-SANS (CG-3, HFIR, ORNL). We are also grateful to Dr. Grzegorz Chojnowski for assistance using RNAMasonry with SANS data. This research was supported by the National Science Foundation (MCB-1942398, to S.C.K.), Oak Ridge Associated Universities (Ralph E. Powe Junior Faculty Enhancement award, to S.C.K.), National Institutes of Health Chemistry-Biology Interface training program (T32 GM132046, to A.M), and the Rackham Graduate School (Rackham Merit Fellowship, to A.M.). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. BioCAT was supported by grant P30 GM138395 from the National Institute of General Medical Sciences of the National Institutes of Health. This research used resources at the High Flux Isotope Reactor, a DOE Office of Science User Facility operated by the Oak Ridge National Laboratory. SANS studies were performed using the Bio-SANS instrument of the Center for Structural Molecular Biology (FWP ERKP291), a DOE Office of Biological and Environmental Research (OBER) Structural Biology Resource. The beam time was allocated to proposal numbers IPTS-30007.1 and IPTS-31445.1. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institute of General Medical Sciences or the National Institutes of Health. This manuscript has been authored by UT-Battelle, LLC, under contract no. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). We are grateful to the staff at BioCAT (beamline 18ID, APS, ANL) and Bio-SANS (CG-3, HFIR, ORNL). We are also grateful to Dr. Grzegorz Chojnowski for assistance using RNAMasonry with SANS data. This research was supported by the National Science Foundation ( MCB-1942398 , to S.C.K.), Oak Ridge Associated Universities (Ralph E. Powe Junior Faculty Enhancement award, to S.C.K.), National Institutes of Health Chemistry-Biology Interface training program (T32 GM132046 , to A.M), and the Rackham Graduate School ( Rackham Merit Fellowship , to A.M.). This research used resources of the Advanced Photon Sciences , a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357 . BioCAT was supported by grant P30 GM138395 from the National Institute of General Medical Sciences of the National Institutes of Health . This research used resources at the High Flux Isotope Reactor , a DOE Office of Science User Facility operated by the Oak Ridge National Laboratory . SANS studies were performed using the Bio-SANS instrument of the Center for Structural Molecular Biology (FWP ERKP291 ), a DOE Office of Biological and Environmental Research (OBER) Structural Biology Resource . The beam time was allocated to proposal numbers IPTS-30007.1 and IPTS-31445.1. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institute of General Medical Sciences or the National Institutes of Health. This manuscript has been authored by UT-Battelle, LLC, under contract no. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ).
Keywords
- HIV-1 dimerization initiation site
- RNA structure
- RNA structure modeling
- RNA:RNA complex
- integrative structural biology
- isotope labeling
- small-angle X-ray scattering
- small-angle neutron scattering