Precise mechanical manipulation of individual long DNA molecules

Electron micrographs demonstrating Aeon
Biowares' patented Molecular Threading technology.
Left: DNA molecules threaded onto an electron microscopy
grid with an amorphous carbon surface; right:
DNA molecules threaded onto a graphene
coated grid (credit: Aeon Biowares and KurzweilAI)

Teams of researchers from Harvard University and Halcyon Molecular, Inc. have disclosed “Molecular Threading,” the first technology to allow single DNA molecules to be drawn from solution and precisely manipulated, allowing for faster, cheaper, more accurate DNA sequencing.

This novel technology pulls single high-molecular weight DNA molecules from solution into air and then places them onto any surface. Halcyon Molecular developed the processes and the intellectual property is now owned by Palo Alto-based biotechnology firm Aeon Biowares.


“Molecular Threading offers a unique means of reaching from the macroscopic world into the world of large molecules with unprecedented exactitude,” says Dr. Chris Melville, CEO of Aeon Biowares and former Director of Chemistry at Halcyon Molecular. …

Additional details from the Aeon Biowares news release: “Molecular Threading, News of the First Public Disclosure“

Molecular Threading was invented by brothers Michael and William Andregg, the co-founders of Halcyon Molecular, Inc. Both co-authors on the current paper [open access: Molecular Threading: Mechanical Extraction, Stretching and Placement of DNA Molecules from a Liquid-Air Interface], the Andregg brothers were previously the subjects of a major profile in the national UK-based newspaper The Independent (“Silicon Valley: The anatomy of a cutting-edge start-up“, Sunday 14 August 2011).

The invention was spurred by the Andregg brothers’ quest for faster, cheaper, and more accurate DNA sequencing technologies. In particular, they needed a way to place DNA molecules onto surfaces in a more controlled manner than current techniques allow. As they tried many different techniques, including methods that are contrasted in the paper, they made an elegant discovery. A simple glass needle tip pulled from a Bunsen flame and coated with a hydrophobic polymer could stretch individual DNA molecules from water into air and place them onto a surface. Furthermore, due to the tension between the needle and the liquid, the DNA molecule is stretched in a geometrically predictable and reproducible way. The movie linked to here from the paper’s Supplementary Information shows how a bundle of DNA molecules remain normal to an air-water interface when stretched into air by the needle: DNA Thread Normal to Droplet Surface.

By attaching the needle to a piezo-positioner, they were soon able to make arrays of parallel stretched molecules as shown in the electron micrographs above. The image on the left shows DNA molecules threaded onto an electron microscopy grid with an amorphous carbon surface, while the image on the right shows DNA molecules threaded onto a graphene coated grid.

Molecular Threading is the enabling technology that allowed researchers for the first time to know the exact physical location of the DNA backbone. This together with the reproducible stretching meant that images that reveal the positions of the DNA bases could be used to determine the nucleotide sequence. This soon attracted the interest of Founders Fund partners Luke Nosek, Peter Thiel and Elon Musk, whose investments allowed the team to exploit Molecular Threading for DNA sequencing by electron microscopy.

Further characterization of the invention was performed by a collaboration of researchers working at Harvard University in the lab of George Church and by several researchers at Halcyon Molecular, some of whom are now working at Aeon Biowares. This includes Halcyon founding team member Kent Kemmish, now founder and CTO at Aeon Biowares.

“This is the first time anyone has ever pulled single high-molecule weight DNA molecules—or any macromolecules for that matter—out of solution and positioned them in a controlled way,” says Kemmish. “Though still in pre-commercial development, it is arguably one of the most advanced nanotechnologies in existence today.”

This advance is a very important nanotechnology that is especially important for DNA sequencing, and thus for personalized medicine, a major component of future medical technology. Much genomic DNA is riddled with many copies of repeated sequences. Current sequencing methods can only read a few hundred nucleotides at a time, so that determining the unique sequences flanking each copy of a repeated sequence can be very difficult. Precise manipulation of long DNA molecules opens the way to reading much longer sequences than can be done with current technology. The disclosure publication speculates “Applications beyond sequencing include nanofabrication, such as aperiodic templates for organic or inorganic materials using DNA as an organizing scaffold, or precision-patterned DNA nanowire arrays.” Organizing complex arrays of catalytic properties could be a step toward building complex molecular machine systems, perhaps as a step toward atomically precise manufacturing.

—James Lewis, PhD

Source: http://www.foresight.org/nanodot/?p=5805

Capturing living cells in micropyramids

A whole field of pyramids, but at microscale, each pyramid containing a living cell. 3D micro and nanofabrication technology is yielding surprising new applications. In cell studies, for instance, the pyramids’ open sides enable cell interaction to be studied.

Many cell studies are carried out in a two-dimensional setting – not a natural situation, as the cells organize themselves differently from in the body, for instance. If you give them free play in a 3D setting, however, this is a better approximation of the natural situation, while you can still keep them within an array. This is how the ‘open-sided pyramids’ fabricated at the MESA+ Institute for Nanotechnology’s NanoLab work.

Material left in the tip

The technology used to achieve this, known as ‘corner lithography’, was in fact discovered by chance. If you bring together a number of planes of, say, silicon at a sharp angle, you can apply a film of another material to those planes. If you then remove that film, not everything is gone; a small amount of material remains in the tip. And that tiny residue of material provides the base for forming a minuscule pyramid, which can then be used as a tip for an atomic force microscope, for instance. The technology also offers the possibility of writing fluids at nanoscale: here the space above the pyramid is filled with material, part of the pyramid is etched away, leaving a ‘pen’ with a hole the same size as the base of the pyramid.

Fabrication of a whole array of open-sided pyramids on a membrane

Capturing cells

In collaboration with the UT’s MIRA Institute for Biomechanical Technology and Technical Medicine the researchers investigated whether the pyramids, attached to a membrane, can act as tiny cages. This was found to work fine with microspheres, so the scientists went on to experiment with chondrocytes, the cells that produce cartilage. Capillary fluid flow causes the cells to drop into the cages of their own accord through a hole in the underside. Compounds and protein-like deposits were soon seen forming between cells in nearby pyramids. Changes in cell phenotype can therefore be studied better than in a flat plane, as this is the right way to grow cells. This yields a promising tool for research into such things as tissue regeneration.

Microspheres captured in the micropyramids

Living cells, chondrocytes captured in micropyramids. They are already starting to form compounds.

The researchers expect there to be a whole range of ways of developing this technology: the ribs of the pyramids could be made hollow, for example, creating a fluid channel, or fluid channels could be fabricated between the pyramids so as to enable the cells to receive nutrition, for example.

3D Nanofabrication of Fluidic Components by Corner Lithography by Erwin Berenschot, Narges Barouni, Bart Schurink, Joost van Honschoten†, Remco Sanders, Roman Truckenmuller, Henri Jansen, Miko Elwenspoek, Aart van Apeldoorn and Niels Tas is to be published in December as an inside cover article in the journal Small. The digital version is already available on request.

http://www.utwente.nl/en/archive/2012/11/capturing_living_cells_in_micropyramids.doc/