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Self-Assembling DNA Tiles – Deciphering the Biology of Individual Cells: December 19, 2008

Although all cells in a body harbor the same genes, in any given cell, some genes are silent and some are expressed (i.e., the genetic code is converted into gene products—RNA and proteins). A particular mix of gene expression products gives cells individual identity. Uncovering molecular blueprints of single cells is one of the most difficult challenges in modern biomedical research. Current technologies only allow assessment of average gene expression from millions of cells, and unfortunately, averaging the expression from a large pool of cells can mask the differences in gene expression among single cells.

Above: An enlargement of a rectangular DNA origami tile is shown with barcode (in red) made of protruding DNA loops in the top left corner and a line of single-stranded DNA overhangs called probes (in green) positioned along the right edge. Below: An atomic force microscope (AFM) cantilever (in purple) scans the surfaces of DNA tiles. When an RNA target gene binds to a probe, the AFM cantilever readily detects the stiff DNA-RNA duplex that forms (seen as bright line).

To address this issue, an Arizona State University research team led by Stuart Lindsay, Professor of Physics and Chemistry and Biochemistry; Hao Yan, Professor of Chemistry and Biochemistry; and assisted by Yung Chang, Associate Professor of Immunology, designed a self-assembling DNA nanodevice for measuring the products of gene expression in small samples.

Building DNA Nanostructures

DNA, often called the building block of life, is increasingly being explored as a material for building artificial nanostructures. From an engineering perspective, DNA is a desirable material, thanks to a unique blend of chemical and physical stability, stiffness, and flexibility. In addition, DNA can be synthesized quickly and economically, and its dimensions are ideally suited for building structures on a small scale.

In a DNA double helix, two single strands are held together through bonds between components of the DNA backbone called bases (e.g., base A binds to base T and base C to base G). This principle of base pairing is utilized in the “DNA origami” technique, where numerous short, single DNA strands act like staples to fold a long single DNA strand called a scaffold. By programming the sequence of the staples to bind at specific locations, a scaffold can be folded into virtually any shape.

Lindsay and Yan’s team created a rectangular tile consisting of a viral DNA and more than 200 short helper strands. Some of the short strands carried a single-stranded DNA overhang or probe. Each probe contained a unique sequence that acted as a molecular glue to capture mRNA, the chemical messenger between the genes and proteins. When the probe captured the mRNA, a stiff V-shaped DNA-RNA double helix was formed. The local change in stiffness was readily sensed by an atomic force microscope scanning the tile surface. This basic tile can be modified to obtain a comprehensive gene expression profile by detecting many genes at the same time. “It is a one-pot assembly process. If you use a larger viral DNA sequence or larger single-stranded DNA sequence, you can make a larger tile. You can also make individual tiles in separate tubes and then combine them to make a super tile that contains more addressable probes,” explains Yan.

DNA Tiles versus Microarrays

A DNA tile with arrays of probes serves simultaneously as a binding surface and a chemical reagent. Because the reactions take place in solution, the hurdles of binding biomolecules onto solid surfaces, common in microarray technology, are avoided. “Compared to the microarray system where you know the average density of the array but you really don’t know the exact spacing between neighboring probes, here you can control the geometry and arrange the probes specifically. Because this array is water-soluble, it can be shrunk to small volumes. The ultimate goal would be to make this compatible with the volume of a single cell. The microarray is hard to scale down to that level,” says Yan.

A, Top: Each DNA tile contains a line of probes along its right edge to capture one of three target genes: Rag-1, C-myc, and ß-actin. Tiles are identifiable by a barcode (in red) in the top left corner. Middle: The atomic force microscope (AFM) can distinguish each tile by its barcode (seen as bright spots). Bottom: When a mixture of targets is combined with the four types of tiles, probes capture their specific targets, and the line on the edge of the tile lights up.

One can conduct mRNA profiling of single cells because even the smallest amounts of nucleic acids can be amplified in a test tube to obtain enough material for analysis. In contrast, proteins cannot be amplified outside of a living cell, and, according to Lindsay, “we are stuck with looking, in the very best case, at hundreds of thousands of cells using mass spectroscopy. In contrast,” he says, “I see this [DNA tile] technology as opening the possibility of doing array-like measurements at a single-cell level. There is probably a large cell-to-cell variation in the expression of proteins with a low copy number. By looking at individuals in a population rather than population average, you might get better answers to questions like, ‘In a population of cells all subject to the same environmental stress, why was it that cell X became tumorigenic?’”

Potential Applications of DNA Tiles

As with any dramatically new technology, cautions Lindsay, there will be obstacles to overcome before it finds application in the clinic, but the possibilities are remarkable. Lindsay’s and Yan’s labs are brimming with ideas.

Using aptamers – synthetic molecules designed to capture molecular targets with very high selectivity – attached to a DNA tile, one could capture proteins bearing various chemical groups (modifications) on their surface. These modifications play a pivotal role in cellular signaling, but they are difficult to detect with existing techniques, especially in small samples. “We can recognize with huge specificity one very small change in an amino acid residue in a large protein. By combining the chemical expertise with the self-assembly expertise, we think we can make some very radical new diagnostic products,” adds Lindsay.

In addition, one could use DNA scaffolds and aptamers to create artificial organelles (i.e., specialized subunits of cells, such as the cell’s powerhouse – mitochondrion). Constructing simple biological systems will allow researchers to gain a better understanding of their inner workings.

Another project revolves around making a very sophisticated nanoscale machine that does more than simple drugs do. Artificial nanochannels with multiple probes could be used to connect immune cells with cancer cells and enhance their ability to kill tumors.

Drs. Yan, Lindsay, and Chang are currently pursuing these goals. “This technology provides a new way of thinking. All the living systems are self-assembled and water-soluble. Why not just use the concept of self-assembly and make it compatible to whatever already exists in living systems?” wonders Yan. “It’s almost kitchen countertop nanotechnology; it’s very simple. Although we are not sure what the first commercial applications will be, there will undoubtedly be applications,” adds Lindsay.

References

Lin C, Liu Y, Rinker S, Yan H. DNA tile based self-assembly: building complex nanoarchitectures. Chemphyschem. 2006 Aug 11;7(8):1641–7.

Chhabra R, Sharma J, Ke Y, Liu Y, Rinker S, Lindsay S, Yan H. Spatially addressable multiprotein nanoarrays templated by aptamer-tagged DNA nanoarchitectures. J Am Chem Soc. 2007 Aug 29;129(34):10304–5.

Ke Y, Lindsay S, Chang Y, Liu Y, Yan H. Self-assembled water-soluble nucleic acid probe tiles for label-free RNA hybridization assays. Science. 2008 Jan 11;319(5860):180–3.

Rinker S, Ke Y, Liu Y, Yan H. Self-assembled DNA nanostructures for distance- dependent multivalent ligand-protein binding. Nat Nanotechnol. 2008 Jul;3(7):418–22.

Stuart Lindsay Hao Yan