Atomic force microscopy (AFM) has recently received increasing interest in molecular biology. This technique allows quick and reliable detection of biomolecules. However, studying RNA-protein complexes using AFM poses significant challenges. Here, we describe a simple and reliable method to visualize positively charged proteins bound to RNA that does not require metallic cations. This method allowed us to effectively detect and visualize Staufen-RNA complexes by height or logarithmic stiffness. The study of the mechanical properties is particularly important in the case of protein-coated RNA complexes, where RNA cannot be detected by height channel. In any case, it is necessary to compare AFM data with the data derived from other techniques like nuclear magnetic resonance, X-ray crystallography, cryogenic electron microscopy, and small-angle X-ray scattering. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Preparation and visualization of RNA-protein complex.

Central European Institute of Technology Masaryk University Brno Czech Republic

Czech Metrology Institute Brno Czech Republic

National Center of Biomolecular Research Brno Czech Republic

These authors contributed equally to this work

Zobrazit více v PubMed

Binnig, G. , Quate, C. F. , & Gerber, C. (1986). Atomic force microscope. Physical Review Letters, 56(9), 930–933. 10.1103/PhysRevLett.56.930 PubMed DOI

Chang, W. H. , Huang, S. H. , Lin, H. H. , Chung, S. C. , & Tu, I. P. (2021). Cryo‐EM analyses permit visualization of structural polymorphism of biological macromolecules. Frontiers in Bioinformatics, 1, 788308. 10.3389/fbinf.2021.788308 PubMed DOI PMC

DesGroseillers, L. , & Lemieux, N. (1996). Localization of a human double‐stranded RNA‐binding protein gene (STAU) to band 20q13.1 by fluorescence in situ hybridization. Genomics, 36(3), 527–529. 10.1006/geno.1996.0499 PubMed DOI

Ding, J. (2023). High‐resolution atomic force microscopy imaging of RNA molecules in solution. Methods in Molecular Biology, 2568, 133–145. 10.1007/978-1-0716-2687-0_9 PubMed DOI

Ding, J. , Lee, Y. T. , Bhandari, Y. , Schwieters, C. D. , Fan, L. , Yu, P. , Tarosov, S. G. , Stagno, J. R. , Ma, B. , Nussinov, R. , Rein, A. , Zhang, J. , & Wang, Y. X. (2023). Visualizing RNA conformational and architectural heterogeneity in solution. Nature Communications, 14(1), 714. 10.1038/s41467-023-36184-x PubMed DOI PMC

Gowravaram, M. , Schwarz, J. , Khilji, S. K. , Urlaub, H. , & Chakrabarti, S. (2019). Insights into the assembly and architecture of a Staufen‐mediated mRNA decay (SMD)‐competent mRNP. Nature Communications, 10(1), 5054. 10.1038/s41467-019-13080-x PubMed DOI PMC

Guaita, M. , Watters, S. C. , & Loerch, S. (2022). Recent advances and current trends in cryo‐electron microscopy. Current Opinion in Structural Biology, 77, 102484. 10.1016/j.sbi.2022.102484 PubMed DOI PMC

Heath, G. R. , Kots, E. , Robertson, J. L. , Lansky, S. , Khelashvili, G. , Weinstein, H. , & Scheuring, S. (2021). Localization atomic force microscopy. Nature, 594(7863), 385–390. 10.1038/s41586-021-03551-x PubMed DOI PMC

Kagra, D. , Jangra, R. , & Sharma, P. (2022). Exploring the nature of hydrogen bonding between RNA and proteins: A comprehensive analysis of RNA: Protein complexes. ChemPhysChem, 23(2), e202100731. 10.1002/cphc.202100731 PubMed DOI

LeBlanc, S. , Wilkins, H. , Li, Z. , Kaur, P. , Wang, H. , & Erie, D. A. (2017). Using atomic force microscopy to characterize the conformational properties of proteins and protein‐DNA complexes that carry out DNA repair. Methods in Enzymology, 592, 187–212. 10.1016/bs.mie.2017.04.004 PubMed DOI PMC

Müller, D. J. , Engel, A. , Carrascosa, J. L. , & Vélez, M. (1997). The bacteriophage phi29 head‐tail connector imaged at high resolution with the atomic force microscope in buffer solution. EMBO Journal, 16(10), 2547–2553. 10.1093/emboj/16.10.2547 PubMed DOI PMC

Nečas, D. , & Klapetek, P. (2012). Gwyddion: An open‐source software for SPM data analysis. Central European Journal of Physics, 10, 181–188. 10.2478/s11534-011-0096-2 DOI

Nwanochie, E. , & Uversky, V. N. (2019). Structure determination by single‐particle cryo‐electron microscopy: Only the sky (and Intrinsic Disorder) is the limit. International Journal of Molecular Sciences, 20(17), 4186. 10.3390/ijms20174186 PubMed DOI PMC

Park, E. , & Maquat, L. E. (2013). Staufen‐mediated mRNA decay. Wiley Interdisciplinary Reviews RNA, 4(4), 423–435. 10.1002/wrna.1168 PubMed DOI PMC

Pleshakova, T. O. , Bukharina, N. S. , Archakov, A. I. , & Ivanov, Y. D. (2018). Atomic force microscopy for protein detection and their physicoсhemical characterization. International Journal of Molecular Sciences, 19(4), 1142. 10.3390/ijms19041142 PubMed DOI PMC

Plevka, P. , Battisti, A. J. , Winkler, D. C. , Tars, K. , Holdaway, H. A. , Bator, C. M. , & Rossmann, M. G. (2012). Sample preparation induced artifacts in cryo‐electron tomographs. Microscopy and Microanalysis, 18(5), 1043–1048. 10.1017/S1431927612001298 PubMed DOI PMC

Rice, W. J. , Cheng, A. , Noble, A. J. , Eng, E. T. , Kim, L. Y. , Carragher, B. , & Potter, C. S. (2018). Routine determination of ice thickness for cryo‐EM grids. Journal of Structural Biology, 204(1), 38–44. 10.1016/j.jsb.2018.06.007 PubMed DOI PMC

Schön, P. (2018). Atomic force microscopy of RNA: State of the art and recent advancements. Seminars in Cell & Developmental Biology, 73, 209–219. 10.1016/j.semcdb.2017.08.040 PubMed DOI

Schön, P. , Luda, S. , & Lyubchenko, Y. L. (2013). Atomic force microscopy of RNA: Imaging and beyond. In Guo P. & Haque F. (Eds.), RNA nanotechnology and therapeutics (pp. 237–257). Boca Raton, FL: CRC Press (Taylor & Francis).

Shlyakhtenko, L. S. , Gall, A. A. , & Lyubchenko, Y. L. (2013). Mica functionalization for imaging of DNA and protein‐DNA complexes with atomic force microscopy. Methods in Molecular Biology, 931, 295–312. 10.1007/978-1-62703-056-4_14 PubMed DOI PMC

Sokolov, I. (2007). Atomic force microscopy in cancer cell research. In Nalwa H. S. & Webster T. (Eds.), Cancer nanotechnology (pp 1–17). Valencia, CA: American Scientific Publishers.

Vicidomini, G. , Bianchini, P. , & Diaspro, A. (2018). STED super‐resolved microscopy. Nature Methods, 15(3), 173–182. 10.1038/nmeth.4593 PubMed DOI

Xu, J. , Ma, H. , & Liu, Y. (2017). Stochastic Optical Reconstruction Microscopy (STORM). Current Protocols in Cytometry, 81, 12.46.1–12.46.27. 10.1002/cpcy.23 PubMed DOI PMC

Yadav, D. K. , Zigáčková, D. , Zlobina, M. , Klumpler, T. , Beaumont, C. , Kubíčková, M. , Vaňáčová, Š. , & Lukavsky, P. J. (2020). Staufen1 reads out structure and sequence features in ARF1 dsRNA for target recognition. Nucleic Acids Research, 48(4), 2091–2106. 10.1093/nar/gkz1163 PubMed DOI PMC

Zhang, K. , Julius, D. , & Cheng, Y. (2021). Structural snapshots of TRPV1 reveal mechanism of polymodal functionality. Cell, 184(20), 5138–5150. e12. 10.1016/j.cell.2021.08.012 PubMed DOI PMC

Zhang, K. H. , Zou, Y. J. , Shan, M. M. , Pan, Z. N. , Ju, J. Q. , Liu, J. C. , Ji, Y. M. , & Sun, S. C. (2024). Arf1 GTPase Regulates Golgi‐Dependent G2/M Transition and Spindle Organization in Oocyte Meiosis. Advance Science, 11(4), e2303009. 10.1002/advs.202303009 PubMed DOI PMC