DNA nanotechnology: a nanomachine goes live.

Imagine a machine so small that it could fit inside a microscopic compartment inside a living cell and perform tasks such as cargo transport, gene regulation and catalysis. On page 325 of this issue, Yamuna Krishnan and colleagues at the National Centre for Biological Sciences in Bangalore report an important step towards this vision in the form of a DNA nanomachine that can probe the local pH inside a living cell1. This proof-of-principle demonstration confirms the great potential of nanodevices based on DNA and other nucleic acids for applications in cell biology and biomedical engineering. Researchers in DNA nanotechnology take advantage of the well-defined base pairing between the four bases found in nucleic acids — adenine always pairs with thymine and cytosine always pairs with guanine — to design short strands of DNA or RNA that self-assemble into specific twoand three-dimensional nanomachines that are capable of performing tasks on the nanometre scale2,3. Journal covers featuring DNA-based smiley faces and octahedra have long captured the imagination of scientists of all types4,5, and these static structures can be used as building blocks to make switches, motors and sensors that perform dynamic functions such as catalytic hybridization, triggered self-assembly and molecular computing6,7. It is especially important to have reliable sensors that can detect when a particular function needs to be turned on. Various nanoscale components with exotic names like the G-quadruplex and the Holliday junction undergo structural changes when they bind to ligands, and therefore can serve as sensor modules. The I-switch designed by Krishnan and colleagues is one such DNA-based sensor, responding to the pH of the local environment by dramatically changing shape. The I-switch contains two flexible strands of DNA that are rich in cytosine, with a third strand that holds them together. Under acidic conditions, the two flexible strands form an antiparallel folding motif known as an i-tetraplex, transforming the molecule from a linear or open conformation to a triangular or closed conformation (Fig. 1; refs 8, 9). To quantify the conformational change directly, a technique called fluorescence resonance energy transfer (FRET) is used as a molecular ruler to measure distance changes on the nanometre scale. Each end of the flexible strand is labelled with a fluorescent dye — a donor (D) and an acceptor (A) — and the ratio of fluorescence intensity (D/A) reflects the relative amounts of open and closed molecules in a given population. A FRETbased calibration curve shows that the device is sensitive to pHs in the range 5.5–6.8, which is relevant to physiology. Under repeated exposures to acids and bases, the I-switch shows robust and reversible transitions with a response time of 1–2 mins in vitro. Moreover, unlike many other DNA nanomachines, it can switch back and forth without a supply of additional DNA strands. What really sets this work apart from other DNA nanomachine studies is the demonstration of nearly real-time measurements inside the living cells called haemocytes. These cells are part of the immune system, and their function is to engulf substances (a process known as endocytosis) and encapsulate them inside cellular organelles known as endosomes (Fig. 1). The I-switch is engulfed by fruit-fly haemocytes and the fluorescence signals from inside the cell are followed over time. Proton pumps embedded within the membranes of the endosomes change the environment inside from neutral (pH ~7) to acidic (pH ~5), at which point the endosomes fuse with organelles known as a lysosomes.The increase in acidity inside the endosome — a process known as maturation — has a major role in triggering processes such as the intake and digestion of nutrients in cells. This pathway is also exploited by toxins and viruses, and is responsible for many diseases. I-switch molecules in endosomes appear as micrometre-size fluorescent spots inside cells. Krishnan and co-workers spatially and temporally record the D/A fluorescence signals from each of these spots and show the progressive shift towards lower D/A (acidic) values over the course of two hours. These results show a close agreement with previously reported values, confirming that the I-switch can function as a pH sensor in the cellular environment. Furthermore, they demonstrate that the nanomachine can be used to target a specific cellular location by coupling it to a protein called transferrin that hitches a ride into the cell during endocytosis. Krishnan and colleagues suggest that this demonstration shows the promise of DNA nanomachines for applications such as sensors, diagnostic markers and targeted therapies in living cells. For example, one can imagine designing nanodevices that first detect the messenger RNA associated with a disease, then release a regulatory DNA strand and subsequently trigger cell death. To realize such devices, however, additional technological breakthroughs are needed. The work of Krishnan and colleagues was possible owing to clever use of the endocytic pathway, but alternative methods of delivering nanomachines into other parts of the cell such as the cytoplasm or nucleus are also desired. The acidification of the endosome has been well characterized using pH-sensitive organic dyes, but the strength of DNA-based devices lies in the fact that they can be coupled to a number of other devices DNA NANOTECHNOLOGy