posted on 2021-06-17, 13:36authored byChristopher M. Green, William L. Hughes, Elton Graugnard, Wan Kuang
To bring real-world applications
of DNA nanostructures to fruition,
advanced microscopy techniques are needed to shed light on factors
limiting the availability of addressable sites. Correlative microscopy,
where two or more microscopies are combined to characterize the same
sample, is an approach to overcome the limitations of individual techniques,
yet it has seen limited use for DNA nanotechnology. We have developed
an accessible strategy for high resolution, correlative DNA-based
points accumulation for imaging in nanoscale topography (DNA-PAINT)
super-resolution and atomic force microscopy (AFM) of DNA nanostructures,
enabled by a simple and robust method to selectively bind DNA origami
to cover glass. Using this technique, we examined addressable “docking”
sites on DNA origami to distinguish between two defect scenarios–structurally
incorporated but inactive docking sites, and unincorporated docking
sites. We found that over 75% of defective docking sites were incorporated
but inactive, suggesting unincorporated strands played a minor role
in limiting the availability of addressable sites. We further explored
the effects of strand purification, UV irradiation, and photooxidation
on availability, providing insight on potential sources of defects
and pathways toward improving the fidelity of DNA nanostructures.