5 research outputs found
Dimensions and Global Twist of Single-Layer DNA Origami Measured by Small-Angle X-ray Scattering
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
Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures
DNA nanotechnology provides methods for building custom membrane-interacting nanostructures with diverse functions, such as shaping membranes, tethering defined numbers of membrane proteins, and transmembrane nanopores. The modification of DNA nanostructures with hydrophobic groups, such as cholesterol, is required to facilitate membrane interactions. However, cholesterol-induced aggregation of DNA origami nanostructures remains a challenge. Aggregation can result in reduced assembly yield, defective structures, and the inhibition of membrane interaction. Here, we quantify the assembly yield of two cholesterol-modified DNA origami nanostructures: a 2D DNA origami tile (DOT) and a 3D DNA origami barrel (DOB), by gel electrophoresis. We found that the DOT assembly yield (relative to the no cholesterol control) could be maximised by reducing the number of cholesterols from 6 to 1 (2 ± 0.2% to 100 ± 2%), optimising the separation between adjacent cholesterols (64 ± 26% to 78 ± 30%), decreasing spacer length (38 ± 20% to 95 ± 5%), and using protective ssDNA 10T overhangs (38 ± 20% to 87 ± 6%). Two-step folding protocols for the DOB, where cholesterol strands are added in a second step, did not improve the yield. Detergent improved the yield of distal cholesterol configurations (26 ± 22% to 92 ± 12%), but samples re-aggregated after detergent removal (74 ± 3%). Finally, we confirmed functional membrane binding of the cholesterol-modified nanostructures. These findings provide fundamental guidelines to reducing the cholesterol-induced aggregation of membrane-interacting 2D and 3D DNA origami nanostructures, improving the yield of well-formed structures to facilitate future applications in nanomedicine and biophysics