Structural analysis of a DNA tetrahedron by electron cryomicroscopy

Graduate School of Frontire Biosciences, Osaka university* Deparatment of Physics, University of Oxford, United Kingdom**
○Takayuki Kato* Russell Goodman** Richard Berry** Andrew Turberfild** Keiichi Namba*

The bottom-up approach to making nano-scale structures can mass-produce various nano-structures if self-assembly can be utilized and therefore is potentially more advantageous than the top-down approach. DNA is an ideal material for nanofabrication of rigid structures because it can be designed by base pairing of specific sequences and is relatively inexpensive and simple to execute.
We synthesized four single-stranded DNAs that are designed to form a stable tetrahedral structure. Stoichiometric mixtures of these four DNAs were heated to 95°C for 2 minutes and then cooled to 4°C over 30 seconds to let them form base pairs. The DNA tetrahedra thus produced were run on a 6% PAGE gel, and an appropriate band was cut out and eluted using the crush and soak method. Then purified tetrahedra were examined by electron cryomicroscopy (cryo-EM). Simple selection of particle images proved impossible because of the low contrast due to the small size of the objects (10nm). We therefore merged two images of the same area recorded at different defocus values by using a Wiener filter. This resulted in higher contrast, and single particle image analysis was successfully carried out to reconstitute the structure of the DNA tetrahedron at 20 Å resolution. The structure was close to what was designed, having an edge length of 7 nm that correlates well with the 20 bp length. We believe that this is the smallest structure revealed by cryo-EM and single particle image analysis, and this fast and simple method would be valuable in 3D nanofabrication.