Scientists at Arizona State University’s Biodesign Institute have
developed the world’s first gene detection platform made up entirely
from self-assembled DNA nanostructures. The results, appearing in the
January 11 issue of the journal Science, could have broad implications
for gene chip technology and may also revolutionize the way in which
gene expression is analyzed in a single cell.
"We are starting with the most well-known structure in biology, DNA,
and applying it as a nano-scale building material, " said Hao Yan, a
member of the institute’s Center for Single Molecule Biophysics and an
assistant professor of chemistry and biochemistry in the College of
Liberal and Sciences.
Yan is a researcher in the fast-moving field known as structural DNA
nanotechnology — that assembles the molecule of life into a variety of
nanostructures with a broad range of applications from human health to
Yan led an interdisciplinary ASU team to develop a way to use
structural DNA nanotechnology to target the chemical messengers of
genes, called RNA.
The team included: lead author and chemistry and biochemistry
graduate student Yonggang Ke; assistant professor of chemistry and
biochemistry Yan Liu; Center for Single Molecule Biophysics director
and physics professor Stuart Lindsay; and associate professor in the
School of Life Sciences, Yung Chang.
"This is one of the first practical applications of a powerful
technology, that, till now, has mainly been the subject of research
demonstrations," said Lindsay. "The field of structural DNA
nanotechnology has recently seen much exciting progress from
constructing geometrical and topological nanostructures through tile
based DNA self-assembly initially demonstrated by Ned Seeman, Erik
Winfree and colleagues," said Yan.
A recent breakthrough of making spatially addressable DNA nanoarrays
came from Paul Rothemund’s work on scaffolded DNA origami, a method in
which a long, single-stranded viral DNA scaffold can be folded and
stapled by a large number of short synthetic "helper strands" into
nanostructures that display complex patterns.
"But the potential of structural DNA nanotechnology in biological
applications has been underestimated, and if we look at the process of
DNA self-assembly, you will be amazed that trillions of DNA
nanostructures can form simultaneously in a solution of few
microliters, and very importantly, they are biocompatible and water
soluble," said Yan.
DNA chip and microarray technology have become a multi-billion
dollar industry as scientists use it to examine thousands of genes at
the same time for mutations or uncovering clues to disease. However,
because DNA probes are pinned to the solid surface of the microarray
chips, it is relatively slow process for the targets to search and find
the probes. Also, it is hard to control the distances between the
probes with nanometer accuracy.
"In this work, we developed a water soluble nanoarray that can take
advantage of the DNA self-assembling process and also have benefits
that the macroscopic DNA microchip arrays do not have," said Yan. "The
arrays themselves are reagents, instead of solid surface chips."
To make the DNA origami RNA probes, Yan has taken advantage of the
basic DNA pairing rules in the DNA chemical alphabet ("A" can only form
a zipper-like chemical bond with "T" and "G" only pair with "C"). By
controlling the exact position and location of the chemical bases
within a synthetic replica of DNA, Yan programmed a single stranded
genomic DNA, M13, into nanotiles to contain the probes for specific
gene expression targets.
Yan refers to the self-assembled DNA nanoarrays as nucleic acid
probe tiles, which look like a nanosized postage stamp. In a single
step, the M13 scaffold system can churn out as many as 100 trillion of
the tiles with close to100 percent yield.
Yan’s team designed three different DNA probe tiles to detect three
different RNA genes along with a bar code index to tell the tiles apart
from each other. "Each probe can be distinguished by its own bar code,
so we mixed them together in one solution and we used this for
multiplex detection," said Yan. The group uses a powerful instrument,
atomic force microscopy (AFM), which allows the researchers to image
the tiles at the single molecule level.
On the surface of each DNA probe tile is a dangling single stranded
piece of DNA that can bind to the RNA target of interest. "Each probe
actually contains two half probes, so when the target RNA comes in, it
will hybridize to the half probes and turn the single stranded dangling
probes into a stiff structure," said Yan. "When it is stiffened, it
will be sensed by the atomic force microscope cantilever, and you can
see a bright line, which is a height increase. The result is a
mechanical, label-free detection."
The technology is able to detect minute quantities of RNA. "Since
the DNA-RNA hybridization has such a strong affinity, in principle, a
single molecule would be able to hybridize to the probe tile," said Yan.
Although there are still many technical hurdles yet to overcome, the
group is excited about the potential applications of the technology.
"What our approach provides is that the probe tiles are a water-soluble
reagent, so the sample volume can potentially be shrunk down to the
volume of a single cell level. Our ultimate goal is to detect RNA gene
expression at the single cell level."
The research was performed in the Biodesign Institute’s Center for
Single Molecule Biophysics, Center for Infectious Diseases and
Vaccinology, and ASU’s Department of Chemistry and Biochemistry,
Department of Physics and School of Life Sciences.
This research is partly supported by funding from NIH and from NSF,
U.S. Air Force Office of Scientific Research, and Office of Naval
Arizona State University. January 2008.