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Supramolecular Nanostamping

A new six-step technique could make DNA analysis as routine as a blood test.

You may hold the cure for cancer. We all may. Some believe it’s written in our genes and locked away in our cells. There are about 50 trillion cells in a human body; in each cell, as many as 25,000 different genes hold the formula – written in DNA – for every cell’s function, whether it’s a muscle cell, nerve cell, or a blood cell. Figure out which genes make the cell work, and you can recognize malfunctions that might lead to diseases such as cancer. Even better, you can potentially fix them.

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One of the best decoding tools at the disposal of geneticists and biologists is a microarray, a silicon or glass chip about 1.5 centimeters square printed with a grid of microscopic dots – tens, even hundreds, of thousands of dots, each one a different segment of synthetic DNA. Just as a pregnancy test will change color to indicate the presence of a particular hormone, the DNA spots on a microarray will glow to indicate the presence of specific genes. Researchers use microarrays to test samples of real DNA or RNA, to identify healthy as well as mutated genes, and to determine which genes are active in a cell. Although scientists can essentially perform thousands of experiments simultaneously on a single microarray, one device can cost upwards of $500 – and it can only be used once.

Microarrays cost a lot today because the conventional method for manufacturing them is highly complex. Typically, a computer-controlled robot uses techniques borrowed from photolithography to create strands of synthetic DNA by linking nucleotides in the appropriate sequences. This method usually involves 70 to 80 steps, so it can take up to a week to produce a single microarray.

But now a new technique, invented by a team of researchers led by assistant professor Francesco Stellacci in MIT’s Department of Materials Science and Engineering, could shorten microarray production time to mere hours and make DNA analysis as inexpensive and common as a blood test.

The technique, dubbed supramolecular nanostamping, begins with a microarray that has been manufactured by conventional means. But Stellacci, capitalizing on DNA’s natural ability to replicate, has devised a simple system for copying the original microarray (known as the master) in just six steps – instead of the typical 70 or 80 – to produce another microarray. The copy so closely resembles the master that it can be used to produce other arrays, exponentially increasing the production rate.

“The beauty of this process is that it can be scaled up,” says Stellacci. Currently, it takes him three and a half hours to produce one microarray. But he anticipates that in a manufacturing setting, it would be possible to produce hundreds in the same amount of time. That could significantly reduce production costs and make DNA microarrays accessible not only to laboratory geneticists but also to health-care providers, fundamentally changing the way doctors diagnose and treat diseases. For example, instead of running a series of blood tests to determine what ails you, a doctor could analyze hundreds of your genes in one step.

Unzipping DNA
Stellacci, who studied materials science in Italy at the Politecnico di Milano, came to MIT in 2002 with the idea of replicating the printing process that naturally occurs in cells. When a cell divides, it first needs to replicate its DNA. With its double-helix design, DNA is like a long, twisted zipper. During replication, an enzyme unzips the DNA, separating it into two strands. Next, free-floating nucleotides – the DNA letters A, C, T, and G – match up with their complements in the separated strands, yielding two identical copies of the original double helix. The cell then divides into two “daughter” cells, and each gets its own copy of the DNA.

What Stellacci and his MIT team – including doctoral candidate Arum Amy Yu, Professor Henry Smith, and electrical-engineering graduate student Tim Savas – have done is harness this replication process as a manufacturing technique. Working out of two labs run by Stellacci in Building 13 on the third floor, the researchers started with master microarrays spotted with single strands of DNA and a solution containing those strands’ complements. The arrays were provided by chemical-engineering professor Anthony Guiseppi-Elie and doctoral candidate G. Scott Taylor, both from Virginia Commonwealth University in Richmond, VA. On each array were 16 dots, each containing a larger number of single-stranded DNA molecules aligned in upright positions, standing in rows like soldiers. The complementary DNA in the solution had been chemically modified so that one end of each strand contained an extra chemical group that likes to stick to surfaces such as gold or silicon.

In the first of three steps, Yu, who hopes to graduate in 2006 with a PhD in materials science, immersed a master microarray in the solution. The complementary strands automatically attached to the strands in the master, forming complete double-stranded DNA, with the sticky ends facing up. Next, Yu gently laid a piece of gold on top of the rows of upright molecules, so that the sticky ends bound to it. Last, she heated the genetic sandwich to 80 degreesC, which caused the DNA to unzip. When Yu pulled away the piece of gold, she had a surface spotted with single strands of DNA that were the mirror images of those on the master. She repeated the three steps using the mirror image and was able to produce a mirror image of it as well – that is, a rough copy of the master.

“The idea of rapid replication is very attractive. It lowers your cost. If you could reproduce a master with very little work, that’s ideal,” says Byron Gates, an expert in surface chemistry and an assistant professor at Simon Fraser University in Burnaby, British Columbia.

Making Copies
The technique sounds promising, but there are a couple of kinks the team needs to work out. For instance, only 75 percent of the DNA molecules in one of the master’s dots are transferred to the gold. But the good news, says Stellacci, is that subsequent copies maintained the 75 percent resolution. His goal is to achieve 100 percent transfer, but he believes that in the meantime, a microarray with extra dots could yield copies with the desired number.

Another problem has to do with pressing the gold surface against the surface of the master. “When you get down to the scale of nanometers, those surfaces are not perfect. Atoms stick out of the surface, so you won’t be able to get perfect contact,” says Taylor.

Stellacci says his team is developing a prototype with more dots. “We have proven 16 [dots], and the extension to 100 seems trivial,” he says. However, Stellacci concedes that the feat will take “some serious engineering.”

Since 2003, Stellacci has received grant money from MIT’s Deshpande Center for Technological Innovation, which funds early-stage research. If successful, supramolecular nanostamping could be applied to the manufacture of inorganic devices, too. DNA strands could be used, for example, to assemble tiny particles of metal into molecule-sized wires or single-electron transistors.

“They’re on the border of a number of different fields,” says Gates, “and that’s a beautiful place to be.”

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