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

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.

Versatile kit of robust nanoshapes self-assembling from RNA and DNA modules.

DNA and RNA have emerged as a material for nanotechnology applications that take advantage of the nucleic acids' ability to encode folding and programmable self-assembly through mainly base pairing. The two types of nucleic acid have rarely been used in combination to enhance structural diversity or for partitioning of functional and architectural roles. Here, we report a design and screening strategy to integrate combinations of RNA motifs as architectural joints and DNA building blocks as functional modules for programmable self-assembly of a versatile toolkit of polygonal nucleic acid nanoshapes. Clean incorporation of diverse DNA modules with various topologies attest to the extraordinary robustness of the RNA-DNA hybrid framework. The design and screening strategy enables systematic development of RNA-DNA hybrid nanoshapes as programmable platforms for applications in molecular recognition, sensor and catalyst development as well as protein interaction studies.

Interaction of APOBEC3A with DNA assessed by atomic force microscopy.

The APOBEC3 family of DNA cytosine deaminases functions to block the spread of endogenous retroelements and retroviruses including HIV-1. Potency varies among family members depending on the type of parasitic substrate. APOBEC3A (A3A) is unique among the human enzymes in that it is expressed predominantly in myeloid lineage cell types, it is strongly induced by innate immune agonists such as type 1 interferon, and it has the capacity to accommodate both normal and 5-methyl cytosine nucleobases. Here we apply atomic force microscopy (AFM) to characterize the interaction between A3A and single- and double-stranded DNA using a hybrid DNA approach in which a single-stranded region is flanked by defined length duplexes. AFM image analyses reveal A3A binding to single-stranded DNA, and that this interaction becomes most evident (∼80% complex yield) at high protein-to-DNA ratios (at least 100∶1). A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM. These properties agree well with recent, biochemical, biophysical, and structural studies. However, these characteristics contrast with those of the related enzyme APOBEC3G, which in similar assays can exist as a monomer but tends to form oligomers in a concentration-dependent manner. These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G. The potential relationships between these properties and biological functions in innate immunity are discussed.