Novel method could quantify gene expression in a single cell
David L. Shenkenberg
Gene expression can vary greatly among individual cells in the same tissue, especially in the case of cancer cells. Although technologies exist for quantifying gene expression in tissues, including DNA microarrays and reverse-transcription polymerase chain reaction (PCR), these technologies cannot quantify gene expression in a single cell.
DNA microarrays and reverse-transcription PCR measure gene expression by quantifying messenger RNA binding to labeled DNA probes. The probes often contain a fluorophore and a quencher, such that messenger RNA binding releases the quencher and triggers a fluorescence signal. Achieving an optically readable fluorescence signal requires using relatively large amounts of genetic material, which is one reason why these technologies remain limited to tissues.
It also is difficult to work with minuscule single-cell volumes. Although standard PCR amplifies genetic material, significant errors often occur with amplification, so the actual genetic material from a cell must be used. In contrast, atomic force microscopy (AFM) is compatible with such small volumes and with such small amounts of genetic material, and it is not limited by optics because it is not an optical technique.
AFM mechanically probes surfaces in the same way that a blind man uses his cane. The narrowness of the “cane” determines the resolution, and AFM probes can be made so narrow that they can resolve single molecules. A liquid-phase nanoarray developed by researchers from Arizona State University in Tempe enables gene expression profiling with AFM. Consequently, it could detect single messenger RNA molecules in a single cell.
The liquid-phase nanoarrays are similar to tiny microarrays placed in solution. However, instead of printing a microarray on a slide, the substrate is formed from nanoscale tiles made of DNA with surface probes for specific messenger RNA (Figure 1). Principal investigator Hao Yan said that, because conventional microarrays contain probes fixed to a solid surface rather than in solution, binding occurs more slowly, and probe placement is impossible to control. As such, the conventional technology is less precise than the novel liquid-phase nanoarrays.
Figure 1. Researchers engineered DNA into nanoscale rectangular tiles containing surface probes for messenger RNA. They mixed the nanotiles with messenger RNA in a test tube, pipetted the solution onto a mica surface and scanned the surface with AFM. This method has advantages over conventional microarrays and reverse-transcription PCR. Reprinted with permission of Science.
The DNA nanotiles are formed using the “DNA origami” technique developed by Paul W.K. Rothemund in the laboratory of Erik Winfree at California Institute of Technology in Pasadena. The technique involves mixing short single strands with one much longer single strand in test tubes and placing the tubes in a PCR heat cycler. In the heated tubes, the short strands act cooperatively to fold the longer strand into complex shapes determined by the sequence of bases and the classic Watson-Crick pairing rules. Cooling allows the longer strand to remain folded. The technique enables the creation of trillions of origami shapes with almost a perfect 100 percent yield. The shapes are viewable with AFM once the solutions containing the folded strands are pipetted onto a flat mica surface, which is conventionally used for AFM.
The Arizona researchers used the origami technique to fold the DNA into rectangular tiles. These nanometer-scale tiles served as passive scaffolds for holding small probe strands of DNA that bind to messenger RNA with complementary sequences. The rectangular tile shape allowed the AFM tip to run across the flat surface and detect the upraised probes with relative ease.
Each probe consists of two half-strands that protrude outward at opposite angles on the surface of the nano-tile. When a strand of messenger RNA from an unknown sample moves between the half-strands, the half-strands come together and sandwich the RNA strand between them. As a result, the three strands become stuck in a relatively upright position, whereas before, the two half-strands jutted outward and moved more freely.
Because the strands become locked in position, they become stiffer as detectable by AFM. In the AFM image, unbound probes appear as white dots on the nanotiles, and binding causes the white dots to come together to form a white line, corresponding to a height increase as the strands become more upright. The researchers arranged the dots and lines in four different ways so as to form “molecular bar codes” that enabled them to distinguish among the probes (Figure 2). The molecular bar codes are conceptually similar to the upraised dots used in the Braille language for the blind.
Figure 2. The probes on the surface of the nanotiles appear as white lines when bound to RNA and as white dots when unbound, comprising a “molecular bar code” that can be read to learn which genes are expressed in cells, possibly in a single cell.
Molecular bar codes
The first nanotile contained a single control probe, the second bore three irregularly arranged probes for the rag-1 gene, the third carried two diagonally arranged probes for c-myc, and the fourth had three probes for b-actin arranged in a V-shape. All three genes have been implicated in cancer. The researchers designed the probe sequences to correspond to important cancer genes because they expect that cancer cell gene expression analysis will be an important application of this technology, Yan said.
The researchers scanned the molecular bar codes in tapping mode with an atomic force microscope from Agilent Technologies and tips from Veeco Instruments Inc. of Woodbury, N.Y. In results detailed in the Jan. 11 issue of Science, they discovered that the probes on the edge of the nanotiles could bind more readily to the messenger RNA than could those in the center of the nanotiles, so they put all the probes on the edge to increase the binding efficiency, something that cannot be done with conventional microarrays. Binding to the probe occurred in less than 30 min. They also found that they could detect minuscule quantities of RNA by reading the bar codes, proving that the technique works in concept.
Yan said that the use of conventional pipettes prevented single-molecule precision and the use of single-cell volumes. To achieve higher precision, they must use a more precise device than a conventional pipette. He said that such a device is commercially available. Alternatively, they could use microfluidics, which could enable high-throughput detection.
The group had shown previously that it can place proteins on nanotiles using synthetic DNA. Yan said that this application will be especially important because, unlike genetic material, proteins cannot be amplified.
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