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. 2023:2709:191-202.
doi: 10.1007/978-1-0716-3417-2_12.

Characterization of RNA Nanoparticles and Their Dynamic Properties Using Atomic Force Microscopy

Alexander J Lushnikov  1 , Yelixza I Avila  2 , Kirill A Afonin  2 , Alexey V Krasnoslobodtsev  3
Affiliations

Characterization of RNA Nanoparticles and Their Dynamic Properties Using Atomic Force Microscopy

Alexander J Lushnikov et al. Methods Mol Biol. 2023.

Abstract

The protocol described in this chapter allows for acquiring topography images of RNA-based nanoring structures and assessing their dynamic properties using atomic force microscopy (AFM) imaging. AFM is an indispensable tool for characterization of nucleic acid-based nanostructures with the exceptional capability of observing complexes in the range of a few nanometers. This method can visualize structural characteristics and evaluate differences between individual structurally different RNA nanorings. Due to the highly resolved AFM topography images, we introduce an approach that allows to distinguish the differences in the dynamic behavior of RNA nanoparticles not amenable to other experimental techniques. This protocol describes in detail the preparation procedures of RNA nanostructures, AFM imaging, and data analysis.

Keywords: Atomic force microscopy; Flexibility analysis; Mechanical stability; Mica surface modification; RNA nanoparticle; Topography imaging.

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Figures

Fig. 1
Fig. 1
Hexameric RNA nanostructure
Fig. 2
Fig. 2
Illustration of sample preparation procedure: (1) APS-modified mica, (2) deposit 2–5 μL of sample solution, (3) incubate for 2 min, (4) rinse with copious amount of ultrapure water, (5) mount on metal puck for imaging
Fig. 3
Fig. 3
(a) AFM topography image of RNA nanorings (600x300 nm2), no gap construct. Bottom panel shows individual nanorings extracted from the images for deformation factor analysis. (b) Deformation factor (DF) is calculated as a ratio of distances between NA strands (maxima in the cross section through the center of NA ring) for perpendicularly placed vertical and horizontal cross-sectional lines. (c) AFM topography image of RNA nanorings (600x300 nm2), 6 base gap construct. Bottom panel shows individual nanorings extracted from the images for deformation factor analysis. (d) Statistical histogram of DF values for “no gap” RNA nanoring. (e) Statistical histogram of DF values for “6 base gap” RNA nanoring
See this image and copyright information in PMC

References

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