Abstract
Characterization of the structure of long RNA molecules (>1 kb) is usually a time-consuming and tedious process. In this study, we have developed an imaging procedure for obtaining images of the extended secondary structures of long RNA molecules combined with automated MATLAB-based data processing algorithms for identification of the domain architecture of the molecules in these images. These algorithms include a molecule autoselection procedure based on height and area thresholding, a morphological thinning procedure to generate skeletons of the molecule in order to analyze the branched structure of the molecules, and a procedure to generate local volume profiles along the main chain of the molecule for identification of domains and prediction of the number of nucleotides comprising each domain. The single-molecule nature of this technique also allows for the identification of varying conformations of the molecule and assessment of the conformational flexibility of the identified domain organization.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Tellinghuisen, T.L., et al.: Studying hepatitis C virus: making the best of a bad virus. J. Virol. 81, 8853–8867 (2007). doi:10.1128/JVI.00753-07
Sagan, S.M., et al.: cis-Acting RNA elements in the hepatitis C virus RNA genome. Virus Res. 206, 90–98 (2015). doi:10.1016/j.virusres.2014.12.029
Gilmore, J.L., et al.: Nanoimaging of ssRNA: genome architecture of the Hepatitis C Virus revealed. J Nanomed. Nanotechnol. S5 (2014). doi:10.4172/2157-7439.S5-010
Brion, P., Westhof, E.: Hierarchy and dynamics of RNA folding. Ann. Rev. Biophys. Biomol. Struct. 26, 113–137 (1997). doi:10.1146/annurev.biophys.26.1.113
Tinoco Jr., I., Bustamante, C.: How RNA folds. J. Mol. Biol. 293, 271–281 (1999). doi:10.1006/jmbi.1999.3001
Wakita, T., et al.: Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11, 791–796 (2005). doi:10.1038/nm1268
Okuno, T., Hiruki, C.: Molecular biology and epidemiology of dianthoviruses. Adv. Virus Res. 87, 37–74 (2013). doi:10.1016/B978-0-12-407698-3.00002-8
Mauger, D.M., et al.: Functionally conserved architecture of hepatitis C virus RNA genomes. Proc. Nat. Acad. Sci. 112, 3692–3697 (2015). doi:10.1073/pnas.1416266112
MathWorks.: MATLAB and image processing toolbox release R2014b. (2014)
Gilmore, J.L., et al.: Single-molecule dynamics of the DNA-EcoRII protein complexes revealed with high-speed atomic force microscopy. Biochemistry 48, 10492–10498 (2009). doi:10.1021/bi9010368
Suzuki, Y., et al.: Visual analysis of concerted cleavage by type IIF restriction enzyme SfiI in subsecond time region. Biophys. J. 101, 2992–2998 (2011). doi:10.1016/j.bpj.2011.09.064
Acknowledgments
This work has been supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Molecular basis of host cell competency in virus infection” (#24115003) from MEXT Japan. We also thank Dr. James Hejna for access to the MATLAB software.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer International Publishing AG
About this paper
Cite this paper
Gilmore, J.L. et al. (2017). Structural Analysis of Long Single-Stranded RNA Molecules with Atomic Force Microscopy Imaging. In: Oral, A., Bahsi Oral, Z. (eds) 3rd International Multidisciplinary Microscopy and Microanalysis Congress (InterM). Springer Proceedings in Physics, vol 186. Springer, Cham. https://doi.org/10.1007/978-3-319-46601-9_1
Download citation
DOI: https://doi.org/10.1007/978-3-319-46601-9_1
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-46600-2
Online ISBN: 978-3-319-46601-9
eBook Packages: Physics and AstronomyPhysics and Astronomy (R0)