The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami.
The
HIV capsid is a multifunctional protein capsule that mediates
the delivery of the viral genetic material into the nucleus of the
target cell. Host cell proteins bind to a number of repeating binding
sites on the capsid to regulate steps in the replication cycle. Here,
we develop a fluorescence fluctuation spectroscopy method using self-assembled
capsid particles as the bait to screen for fluorescence-labeled capsid-binding
analytes (“prey” molecules) in solution. The assay capitalizes
on the property of the HIV capsid as a multivalent interaction platform,
facilitating high sensitivity detection of multiple prey molecules
that have accumulated onto capsids as spikes in fluorescence intensity
traces. By using a scanning stage, we reduced the measurement time
to 10 s without compromising on sensitivity, providing a rapid binding
assay for screening libraries of potential capsid interactors. The
assay can also identify interfaces for host molecule binding by using
capsids with defects in known interaction interfaces. Two-color coincidence
detection using the fluorescent capsid as the bait further allows
the quantification of binding levels and determination of binding
affinities. Overall, the assay provides new tools for the discovery
and characterization of molecules used by the HIV capsid to orchestrate
infection. The measurement principle can be extended for the development
of sensitive interaction assays, utilizing natural or synthetic multivalent
scaffolds as analyte-binding platforms.
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