Skip to main content
SpringerLink
Log in

High-Resolution Atomic Force Microscopy Imaging of RNA Molecules in Solution

  • Protocol
  • First Online:
RNA Structure and Dynamics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2568))

Abstract

Atomic force microscopy (AFM) is an important and versatile technique to investigate the structures and dynamics of biomolecules under physiologically relevant conditions at the single-molecule level. Recent progresses in high-resolution AFM imaging of nucleic acids have expanded this technique from simple characterization of double-stranded DNA or RNA to detailed analyses of the structure and dynamics of large functional RNAs with complex folds. Several technical developments, such as sharper probes and more stable instruments with novel imaging modes, AFM is capable of directly visualizing RNA conformational heterogeneity in solution in real time. Here, we introduce a comprehensive method for recording high-resolution images of RNA molecules, including sample preparation, instrument setup, data acquisition, and image processing.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Binnig G, Quate CF, Gerber C (1986) Atomic force microscope. Phys Rev Lett 56:930–933

    Article  CAS  Google Scholar 

  2. Ares P, Fuentes-Perez ME, Herrero-Galan E et al (2016) High resolution atomic force microscopy of double-stranded RNA. Nanoscale 8:11818–11826

    Article  CAS  Google Scholar 

  3. Kalle WH, Macville MV, Van De Corput MP et al (1996) Imaging of RNA in situ hybridization by atomic force microscopy. J Microsc 182:192–199

    Article  CAS  Google Scholar 

  4. Kuznetsov YG, Daijogo S, Zhou J et al (2005) Atomic force microscopy analysis of icosahedral virus RNA. J Mol Biol 347:41–52

    Article  CAS  Google Scholar 

  5. Kuznetsov YG, Dowell JJ, Gavira JA et al (2010) Biophysical and atomic force microscopy characterization of the RNA from satellite tobacco mosaic virus. Nucleic Acids Res 38:8284–8294

    Article  CAS  Google Scholar 

  6. Kuznetsov YG, Mcpherson A (2006) Atomic force microscopy investigation of Turnip Yellow Mosaic Virus capsid disruption and RNA extrusion. Virology 352:329–337

    Article  CAS  Google Scholar 

  7. Lymans’kyi OP (2007) Visualization of RNA transcripts with atomic force microscopy. Tsitol Genet 41:12–18

    PubMed  Google Scholar 

  8. Lyubchenko YL, Gall AA, Shlyakhtenko LS et al (1992) Atomic force microscopy imaging of double stranded DNA and RNA. J Biomol Struct Dyn 10:589–606

    Article  CAS  Google Scholar 

  9. Lyubchenko YL, Shlyakhtenko LS (1997) Visualization of supercoiled DNA with atomic force microscopy in situ. Proc Natl Acad Sci USA 94:496–501

    Article  CAS  Google Scholar 

  10. Lyubchenko YL, Shlyakhtenko LS, Ando T (2011) Imaging of nucleic acids with atomic force microscopy. Methods 54:274–283

    Article  CAS  Google Scholar 

  11. Ng JD, Kuznetsov YG, Malkin AJ et al (1997) Visualization of RNA crystal growth by atomic force microscopy. Nucleic Acids Res 25:2582–2588

    Article  CAS  Google Scholar 

  12. Pino S, Biasiucci M, Scardamaglia M et al (2011) Nonenzymatic ligation of an RNA oligonucleotide analyzed by atomic force microscopy. J Phys Chem B 115:6296–6303

    Article  CAS  Google Scholar 

  13. Hansma HG, Oroudjev E, Baudrey S et al (2003) TectoRNA and ‘kissing-loop’ RNA: atomic force microscopy of self-assembling RNA structures. J Microsc 212:273–279

    Article  CAS  Google Scholar 

  14. Henn A, Medalia O, Shi SP et al (2001) Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy. Proc Natl Acad Sci USA 98:5007–5012

    Article  CAS  Google Scholar 

  15. Kiselyova OI, Yaminsky IV, Karger EM et al (2001) Visualization by atomic force microscopy of tobacco mosaic virus movement protein-RNA complexes formed in vitro. J Gen Virol 82:1503–1508

    Article  CAS  Google Scholar 

  16. Limanskaia O, Limanskii AP (2008) Visualization of elongation complexes for t7 Rna polymerase by atomic force microscopy. Mol Biol (Mosk) 42:533–542

    Google Scholar 

  17. Lyubchenko YL, Shlyakhtenko LS (2009) AFM for analysis of structure and dynamics of DNA and protein-DNA complexes. Methods 47:206–213

    Article  CAS  Google Scholar 

  18. Smith BL, Gallie DR, Le H et al (1997) Visualization of poly(A)-binding protein complex formation with poly(A) RNA using atomic force microscopy. J Struct Biol 119:109–117

    Article  CAS  Google Scholar 

  19. Endo M, Tatsumi K, Terushima K et al (2012) Direct visualization of the movement of a single T7 RNA polymerase and transcription on a DNA nanostructure. Angew Chem Int Ed Engl 51:8778–8782

    Article  CAS  Google Scholar 

  20. Kueng A, Kranz C, Lugstein A et al (2003) Integrated AFM-SECM in tapping mode: simultaneous topographical and electrochemical imaging of enzyme activity. Angew Chem Int Ed Engl 42:3238–3240

    Article  CAS  Google Scholar 

  21. Trohalaki S (2012) Multifrequency force microscopy improves sensitivity and resolution over conventional AFM. MRS Bull 37:545–546

    Article  Google Scholar 

  22. Kocun M, Labuda A, Meinhold W et al (2017) Fast, high resolution, and wide modulus range nanomechanical mapping with bimodal tapping mode. ACS Nano 11:10097–10105

    Article  CAS  Google Scholar 

  23. Sokolov I, Dokukin ME (2018) Imaging of soft and biological samples using AFM ringing mode. Methods Mol Biol 1814:469–482

    Article  CAS  Google Scholar 

  24. Dzedzickis A, Bucinskas V, Virzonis D et al (2018) Modification of the AFM sensor by a precisely regulated air stream to increase imaging speed and accuracy in the contact mode. Sensors (Basel) 18:2694

    Article  Google Scholar 

  25. Koehler M, Fis A, Gruber HJ et al (2019) AFM-based force spectroscopy guided by recognition imaging: a new mode for mapping and studying interaction sites at low lateral density. Methods Protoc 2:6

    Article  CAS  Google Scholar 

  26. Kunicki P, Angelov T, Ivanov T et al (2019) Sensitivity improvement to active piezoresistive afm probes using focused ion beam processing. Sensors (Basel) 19:4429

    Article  CAS  Google Scholar 

  27. Heath GR, Kots E, Robertson JL et al (2021) Localization atomic force microscopy. Nature 594:385–390

    Article  CAS  Google Scholar 

  28. Dufrene YF, Ando T, Garcia R et al (2017) Imaging modes of atomic force microscopy for application in molecular and cell biology. Nat Nanotechnol 12:295–307

    Article  CAS  Google Scholar 

  29. Dufrene YF, Martinez-Martin D, Medalsy I et al (2013) Multiparametric imaging of biological systems by force-distance curve-based AFM. Nat Methods 10:847–854

    Article  CAS  Google Scholar 

  30. Garcia R, Proksch R (2013) Nanomechanical mapping of soft matter by bimodal force microscopy. Eur Polym J 49:1897–1906

    Article  CAS  Google Scholar 

  31. Heath GR, Scheuring S (2019) Advances in high-speed atomic force microscopy (HS-AFM) reveal dynamics of transmembrane channels and transporters. Curr Opin Struct Biol 57:93–102

    Article  CAS  Google Scholar 

  32. Ares P, Gomez-Herrero J, Moreno-Herrero F (2018) High-resolution atomic force microscopy imaging of nucleic acids. Methods Mol Biol 1814:3–17

    Article  CAS  Google Scholar 

  33. Ricci D, Braga PC (2004) Recognizing and avoiding artifacts in AFM imaging. Methods Mol Biol 242:25–37

    PubMed  Google Scholar 

  34. Golek F, Mazur P, Ryszka Z et al (2014) AFM image artifacts. Appl Surf Sci 304:11–19

    Article  CAS  Google Scholar 

  35. Canale C, Torre B, Ricci D et al (2011) Recognizing and avoiding artifacts in atomic force microscopy imaging. Methods Mol Biol 736:31–43

    Article  CAS  Google Scholar 

  36. Voigtlander B (2019) Artifacts in AFM. In: Atomic Force Microscopy. Nanosci Technol. Springer, Cham. https://doi.org/10.1007/978-3-030-13654-3_8

  37. Shlyakhtenko LS, Gall AA, Filonov A et al (2003) Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials. Ultramicroscopy 97:279–287

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research (Y-X. Wang).

Author information

Authors and Affiliations

  1. Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Jienyu Ding

Authors
  1. Jienyu Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jienyu Ding .

Editor information

Editors and Affiliations

  1. Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Jienyu Ding

  2. Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Jason R. Stagno

  3. Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Yun-Xing Wang

Rights and permissions

Reprints and permissions

Copyright information

© 2023 This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ding, J. (2023). High-Resolution Atomic Force Microscopy Imaging of RNA Molecules in Solution. In: Ding, J., Stagno, J.R., Wang, YX. (eds) RNA Structure and Dynamics. Methods in Molecular Biology, vol 2568. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2687-0_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2687-0_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2686-3

  • Online ISBN: 978-1-0716-2687-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation