DNA microscopy images complex nanocages


Rebecca Pool

Friday, March 14, 2014 - 15:30

US researchers have used DNA-based super-resolution microscopy to image some of the largest and most complex structures ever constructed from DNA.

The self-assembling DNA cages have edge widths of 100nm and weigh only tens of megadaltons, paving the way to new drug delivery systems and high-sensitivity photonic sensors.

While DNA nanotechnology has produced myriad shape-controlled nanostructures from tetrahedra and cubes to nanoprisms and buckyballs, reliably fabricating more complex structures has proven difficult.

The five cage-shaped DNA polyhedra are by far the largest and sturdiest DNA cages yet. The largest, a hexagonal prism (right), is one-tenth the size of an average bacterium. [Yonggang Ke/Harvard's Wyss Institute]

However, Professor Peng Yin and colleagues from the Wyss Institute for Biologically Inspired Engineering, Harvard University, have developed a straightforward strategy for building complex polyhedra, based on a novel three-armed origami tile, called a DNA tripod.

The team programmed the DNA to fold into sturdy tripods, some sixty times larger than past tripod-like building blocks; these tripods then self-assembled into 3D polyhedra, including a pentagonal and hexagonal prism.

Crucially, the team was able to use so-called 3D DNA-PAINT super-resolution microscopy to image the structures.

As Yin highlights: "[This] offers a minimally invasive way to obtain true single molecule 3D images of DNA nanostructures in their native, 'hydrated' environment."

Pioneered by Yin, DNA-PAINT creates so-called 'imager strands' by tagging small pieces of DNA with a fluorescent dye.

Each of these fluorescently labelled oligonucleotides then transiently, and repeatedly, binds to a matching DNA strand attached to the target molecule, so the target appears to 'blink'. This allows researchers to obtain sub-diffraction resolution single molecule images of structures.

"In [standard] reconstruction microscopy, most molecules are switched to a fluorescent dark, OFF state, and only a few emit fluorescence - the ON state. Each molecule is then localised... by fitting its emissions to a 2D Gaussian function," explains Yin.

"In DNA-PAINT, the 'switching' between the ON- and OFF-states is facilitated by repetitive, transient binding of the imager strands to complementary 'docking strands'," he adds.

To create supersharp images of their cage-shaped DNA polyhedral, researchers used DNA-PAINT, a microscopy method that uses short strands of DNA (yellow) labelled with a fluorescent chemical (green) to bind and release partner strands on polyhedra corners, causing them to blink. The blinking corners reveal the shape of structures far too small to be seen with a conventional light microscope. [Harvard's Wyss Institute and Harvard Medical School]

As part of his self-assembling polyhedra work, Yin used optical astigmatism to extend DNA-PAINT to 3D imaging, applying the technique to obtain single-molecule images.

"To ensure all vertices of a polyhedron were imaged, each vertex was modified with about 18 docking strands in a symmetric arrangement," he adds.

Having produced high resolution 3D images of these single DNA molecules in native environments, the researchers now hope to design even more sophisticated polyhedra.

"Such structures could be used to template guest molecules for diverse applications, such as spatially arranging enzymes into efficient reaction cascades," says Yin.

The researcher is also confident that DNA-PAINT can complement present electron microscopy methods.

"Cryo-EM offers higher spatial resolution imaging of unlabelled structures, but DNA-PAINT is less technically involved to implement and obtains true single molecule images of individual structures, and preserves the multi-colour capability of fluorescent microscopy," he says.

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