Researchers at the University of Illinois at Urbana-Champaign and at Howard Hughes Medical Institute, both in Urbana, have the beat — at least on the nanometer scale. They have constructed a DNA-based nanomechanical device that displays an adjustable ticking rate, somewhat like a metronome. The rate can be changed by adjusting ion concentration or by altering a set of DNA-based deactivating and activating switches. The latter may hold the key to a single-molecule sensor that can detect small sequence differences in target DNA. esearchers have developed a stochastic nanoscale metronome using DNA constructs that flip between two conformations (A and D), modifying the time spent in each state by attaching single-strand DNA to the construct. They modified the metronomic cycling rate by adding a deactivator (E) and an activator (F). Courtesy of Taekjip Ha. “We showed that even a single basepair difference can be detected by the device,” said Taekjip Ha, an associate professor of physics at the university and an investigator at the institute. He led the team that fabricated the nano-metronome. The genesis of the device was the group’s research on four-way Holliday junctions. These DNA constructs are tetrahedral, with strands that can be exchanged, creating a structure that naturally “ticks” from one conformation to another. In building the nanometronome, the scientists combined this function with zipping and unzipping via single-strand DNA. Measuring conformations The group started by selecting a junction where the lifetimes of the two conformers were about equal. It added two single-stranded overhangs on the end of the sequence: In one conformation, these overhangs were far from other structures and had little impact; in the other, they fell near other parts of the structure and, in effect, created a sticky force that kept the junction in that conformation longer. To determine the state of the metronome, the researchers used a total internal reflection fluorescence setup — comprising an Olympus inverted microscope equipped with a frequency-doubled Nd:YAG 532-nm laser supplied by CrystaLaser of Reno, Nev. — to study Förster resonance energy transfer in the system. The distance between the acceptor and donor molecules went from far to near and back again as the junction flipped between conformations. When this happened, the fluorescence of the two changed, leading to an observable fluctuation. The investigators used total internal reflection fluorescence microscopy because it reduces background fluorescence and provides excellent signal to noise in single-molecule fluorescence measurements, according to Ha. Because the imaging had to be done at high speed and low light levels, the researchers also used an electron-multiplying CCD camera from Andor Technology of Belfast, UK. Ha noted that a traditional metronome ticks regularly and periodically, but that the nanoscale version did not change state at a given frequency. Instead, it flipped between states at a variable rate. The investigators controlled that rate by two means. The first was by altering the concentration of doubly charged magnesium ions, which were critical for the occurrence of conformational transitions. Changing the concentration directly also affected the speed of the changes. The second was by introducing a short, single-stranded deactivator/activator. The deactivator would bind to one of the free overhangs, rendering it inactive and thereby decreasing the tendency for the junction to prefer a particular conformation. Subsequent addition of an activator strand removed the deactivator and restored the metronome’s rate. In tests, the researchers could see a difference in the rate if the overhangs differed by as little as one base pair, indicating that the technique potentially could be used as a single-molecule DNA sensor, which Ha noted is the subject of ongoing research. “We are designing constructs that can detect single-strand DNA in solution, and it should work at the research/publication level,” he said. Nano Letters, March 8, 2006, pp. 496-500.