Nanopores, DNA, and the bacteria game

Sunday 28th November 2010
Credit: ‘Image courtesy Cees Dekker lab TU Delft / Tremani’) Artistic rendering of the formation of hybrid pores by the directed insertion of the biological protein pore alpha hemolysin (pink) into solid-state nanopores (holes in the green bottom layer). An applied electric field drives a double-stranded DNA molecule (blue, left) into the hole, which subsequently drags the pink hemolysin protein into position. Once assembled, these hybrid nanopores can be used to pull single-strand DNA (blue, center) through, for analysis and sequencing

Cees Dekker and his researchers at Delft University of Technology working with Oxford University researchers have announce a new type of nanopore device that could help in developing fast and cheap genetic analysis. In the today's journal "Nature Nanotechnology" they report on a new method that combines man-made and biological materials to result in a tiny hole on a chip, which is able to measure and analyze single DNA molecules.

Last year, Delft University of Technology developed a technique that can measure both the charge and diameter of a single molecule for the first time. It employs solid-state nanopores, that clearly distinguish between molecules of DNA with a protein coating. and those without, that could be useful for DNA sequencing and detecting markers for genetically inherited diseases.

“The first mapping of the human genome - where the content of the human DNA was read off (‘sequenced’) - completed in 2003 cost an estimated $3bn. Imagine if that cost could drop to a level of a few €100, where every one could have their own personal genome sequenced.

"That would allow doctors to  diagnose diseases and treat them before any symptoms arise,” (right) says Professor Cees Dekker, Kavli Institute of Nanoscience at Delft.

One promising device is the nanopore: a minute hole that can be used to ‘read’ information from a single molecule of DNA as it threads through the hole.

New research by Dekker’s group in collaboration with (left) Prof. Hagan Bayley of Oxford University, now demonstrates a new, much more robust type of nanopore device, that combines biological and artificial building blocks.

Fragile
 lipid support

Dekker says: "Nanopores are already used for DNA analysis by inserting naturally occurring, pore-forming proteins into a liquid-like membrane made of lipids. DNA molecules can be pulled individually through the pore by applying an electrical voltage across it, and analyzed in much the same way that music is read from an old cassette tape as it is threaded through a player.

"One aspect that makes this biological technology especially difficult, however, is the reliance on the fragile lipid support layer. This new hybrid approach is much more robust and suitable to integrate nanopores into devices.
Putting proteins onto a silicon chip


The new research, performed chiefly by lead author (right) Dr. Adam Hall at TU Delft, now demonstrates a simple method to implant the pore-forming proteins into a robust layer in a silicon chip.

Essentially, an individual protein is attached to a larger piece of DNA, which is then pulled through a pre-made opening in a silicon nitride membrane.

When the DNA molecule threads through the hole, it pulls the pore-forming protein behind it, eventually lodging it in the opening and creating a strong, chip-based system that is tailor-made for arrays and device applications.

The researchers have shown that the hybrid device is fully functional and can be used to detect DNA molecules

Nanofabrication techniques are allowing researchers to build precisely defined landscapes on a chip, in order to study the adaptation and evolution of bacteria.

Pushing the bio-nanosicience boundaries


(Above: Nanofabrication techniques allow researchers to build precisely defined landscapes on a chip, in order to study the adaptation and evolution of bacteria.)

Earlier this month a prestigious ERC Advanced Grant for exceptional scientists who submit ambitious, groundbreaking research proposals, was awarded to Prof Dekker (Left) with Mildred Dresselhaud, MIT Emeritus Institute Professor of Physics and Electrical Engineering) who will use the allocated €2.5m for research in bio-nanoscience.

“We want to use the power of nanofabrication, a skill that is particularly advanced at TU Delft, to find out more about big biological questions,  as the precise working of processes within cells.”

Galapagos Islands for bacteria 

In the first area of the research, Dekker will look at bacteria. 
“Nanofabrication techniques allow us to build precisely defined landscapes on a chip, in order to study adaptation and evolution of bacteria. We are actually creating a kind of miniature Galapagos Islands for bacteria.

"Some of them will cross over to a different island, others won’t. By varying the environmental factors and properties of the bacteria, we can gain more insight into how bacteria adapt. We can directly observe evolution in space and time.”

“A particularly interesting aspect of this research is the fact that it brings in elements of game theory. For example, some bacteria aim for cooperation, while others are ‘cheaters’, which benefit from the work of their fellows. We can manage those properties too, and study them under controlled circumstances.”

The exotic

(Left nanopore complex)
In the connections between the islands, bacteria move through narrow channels. Within these nanochannels, they are flattened completely, eventually emerging from the other side in all kinds of amazing shapes.

The research suggests that there may be many more bacteria present in narrow spaces than previously thought, which in turn has consequences for e.g. membrane filters and medical equipment.

Notably, the bacteria continue to grow and divide at normal speed within the channels. Dekker now wants to investigate further how cell division works in these exotic flat bacteria.
Nanopores

In research with nanopores (the other part of the research) the Dekker group will manufacture tiny holes (of only a couple of nanometers) with electron bundles through which DNA molecules can move and be tracked and screened. Eventually it may be possible to read the detailed genetic code of DNA molecules by entirely new means, and observe which genes are ‘on’ or ‘off’.

Gatekeepers

Finally, Dekker will try to mimic the construction of biological pores. He will focus on the minuscule holes in the membrane of the cell nucleus.

“In those holes there are certain proteins which function as a kind of gatekeeper to the cell nucleus.

They determine which molecules are allowed out or in. But exactly how they do that is still a mystery. By mimicking holes with nanofabrication and coating them with these gatekeeper proteins, we hope to discover more about this important mechanism.”

The research will lead to increased knowledge concerning some diseases and gene therapy, DNA, gatekeepers and domain of bacteria.

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