Fig. 1. Top: scheme of rotation of the rotor (shown in red) DNA-motor (ie the transition from B-form to Z-form) when the concentration of Mg2 + and reverse transition with decreasing ion concentration. Below: images obtained by atomic force microscopy, and confirming that the transition occurred. Figure out of the article in the Journal of American Chemical Society
In recent years, DNA has become a popular material for nanomodelirovaniya: using DNA origami technique (see DNA origami) can create two-dimensional design of the required form (see creator DNA origami technique Paul Rotmunda Folding DNA to create nanoscale shapes and patterns / /Nature.
2006. V. 440. P. 297-302), and the recently developed technique of DNA building blocks that resemble parts designer LEGO, can receive the bulk structures of arbitrary shape (see note nanostructures from DNA can be collected on the basis of the designer "Lego", "Elements", 04/12/2012) . While allowing the designers of artificial DNA is not limited to the role of passive building blocks: already synthesized DNA molecules that have catalytic functions (see: S. Santoro, G. Joyce, 1997. A general purpose RNA-cleaving DNA enzyme), and even set up primitive nanodevices based on them (see: Kyle Lund et al., 2010. Molecular Robots Guided by Prescriptive Landscapes). And recently managed to get a functional structure similar to ion channel (see German biotechnologists created from the DNA of artificial ion channels, "Elements", 11/20/2012).
A group of Japanese scientists from Tokyo and Kyoto DNA used as building material for the "body" and the moving part ("rotor") engine, and stored in the conformation of the DNA molecule the energy spent on the motor.
DNA consists of a chain (polymer) of nucleotides consisting of phosphoric acid, deoxyribose and a nitrogenous base. Nitrogenous base can be one of four types: adenine, guanine, cytosine or thymine (they are given in Latin letters A, G, C and T, respectively). Since nucleotides differ only nitrogenous bases, for simplicity use only those letters, sometimes with the prefix «d» before the letter (dG, dC, and so forth) to understand was that it is a DNA rather than RNA (then would console «r»).
Depending on the environmental conditions and the nucleotide composition of the double helix of the DNA molecule can be a particular conformation– One of the possible spatial forms that arise when the relative orientation of the individual parts of the molecule as a result of rotation of atoms or groups of atoms about single bonds, bending bonds, etc. (Fig. 2). B-form DNA is a pravozakruchennuyu spiral, and the Z-form – the left-twisted. Left-twisted helix with long coils can be produced only from the chains with the formula d (CG)n, that is composed of regularly alternating cytosine and guaninsoderzhaschih nucleotides.
In living cells, the vast majority of the DNA molecules is in the B-form. The potential to transform into Z-shape parts that contain only G and C. Such a transition can be stimulated by increasing the content of ions in solution or certain proteins, stabilizing Z-form DNA. In the transition of a molecule from B-to Z-form DNA double helix turns, and then wrapped in the opposite direction, turning from a left-twisted pravozakruchennoy.
The idea is to use the rotary motion for nanomotor was implemented in 1999 by a group of scientists from the United States (see: Mao et al., 1999. A nanomechanical device based on the B-Z transition of DNA). Then received construction consisted of double-central portion of the nucleotides C and G, are able to change conformation and rigid side groups that continue the thread of the central area (they can rotate relative to each other when the conformation of the central part), Fig. 3. Side groups on the motor mount two molecules of fluorophores in which the method of FRET determined whether turned of the engine apart (increased if the distance between the fluorophores), that is, whether there was a BZ-transition.
By fluorescent or fersterovskogo, resonance energy transfer (FRET) is the efficiency of energy transfer between two fluorescent molecules (fluorophores) depends on the distance between them, the greater the distance, the less energy is transferred. Thus, by measuring the fluorescence, we can determine the slightest change in the distance between the micro-objects.
However, if scientists could only make sure that the rotation occurs. About the dynamics of the process (how many revolutions do the rotor, in which direction they rotate, how long does one turn) nothing definite to say it was impossible.
The answer to these questions would help to monitor in real time. But for such tiny objects are not easy to organize. Even with a microscope with a suitable resolution, that is at a level where you can see the individual molecular structures can be difficult to prepare a sample as to be able to focus on microscopic objects studied. This can be achieved by attaching objects to the substrate, but in the case nanomotors important not impede their work.
Japanese scientists have been able to cope with these problems. By fixing motors in frames made of DNA ("body"), attached to the substrate, they can not diffuse into the solution, and they can be focused microscope. At the same time, the frame does not interfere with the motor, and for his work came to observe.
First, the method of DNA origami they collected the frame (Fig. 4). Each frame secured capable of rotating structure of DNA (the rotor), and control non-rotating design of the same structure (it is called the "stator"), but with random nucleotides in place of a chain of G and C. If you change the content of ions in a solution of the test design should not rotate because it was not simple sequences that can move in a Z-shape.
The rotor design, created by their American counterparts, they are a little improved: used methylated nucleotides, d (5meCG)6,
instead of the usual, and the methylated C and G-nucleotides clearly alternated (ie after each C was necessarily G), for even greater stability Z-shape and facilitated the transition of DNA from B-form to Z-form. For the "start" of the motor used an increased concentration of Mg2 + (And not Na+, as the authors of articles in 1999), since the ions Mg2 + improve the scope of the attachment of DNA origami to the substrate.
To make it easier to follow the rotation of the rotor, the authors of the new work will be hooked on the rotor structure of DNA – "check" – in which you can keep track of how he turned the rotor. The start position (when the concentration of salt in the solution is low) check box and the rotor , and the "stator" (nonrotating control) downward.
In fact, the control box is also sometimes turned the other way, just by random rotation of the design on its axis. So the only difference between the check box on the stator-control of the check box on the rotor – is that the position of the check box in the control does not change when the concentration of Mg2 +. Regardless of the concentration of Mg2 +, 70-75% in the control flags are turned down. With rotor as the situation is different: when the concentration of Mg2 + 5 mmol / l 70% of the rotor has an option, turned down, as well as in control, but if you gradually increase the concentration of Mg2 +, the percentage of the check boxes to turn up, will increase. At a concentration of 25 mmol / L Mg2 +
only 30% of the rotors will have flags, turned down, and about 70% – the boxes turned up (the opposite situation compared to the control).
Images were obtained by high-speed atomic force microscopy. This method produces images with a resolution of a few nanometers. For example, if such resolution is visible check of DNA length of 15 nm and a width of 8 nm (1 nm = 10-9m), compared with the average diameter of the cells of the animal or plant is the order of 100 microns (1 micron = 10-6m).
"High Speed" microscopy in this case means that a picture can be obtained in five seconds. To carry out the recording in this mode, the authors picked an average concentration of salts Mg2 + (10 mmol / l), in which BZ is happening, but at a slow rate, suitable for the available time resolution.
As a result, researchers have obtained a series of micrographs (Fig. 5). They show that at the beginning of shooting box on the rotor was in the "down", and then began to turn, reaching the top position (rotate 180 °) in 30 seconds, then the rotor has made a half-turn and found himself in the "down" to 40 seconds of shooting. At 85 seconds of the rotor was already back up the box, and on the 120th – again a flag down. That is, the researchers were able to trace the two rotations of the rotor, the first rotation is two times faster than the second (40 and 80 seconds, respectively). Checkbox on the "stator" throughout the scene was turned down.
This corresponds to the theoretical calculation, which predicts a 128 ° rotation for each pair of nucleotides in changing the conformation of parts. Because of this construction was 6 pairs, one might expect to see a 2.1 turn. Stator when the salt content remained almost stationary, which is also in line with the expectations of the researchers.
Once accomplished BZ transition, the motor can be reset by removing the excess salt from the solution – adding connections linking ions (eg, EDTA), or simply diluted solution, which contains the motors. An important advantage of this nanomachine – independence from foreign energy sources because the energy required to operate the engine, stored in the conformation of the DNA itself.
Authors was the first to make a direct observation of the work of artificial nanomachines and simultaneously for the first time to see the BZ-transition in a DNA molecule in real time. This work also provides a good example of the technique of DNA origami structures for well-defined shape – rigid frame, suitable to the size of the motor and help monitor the performance of individual nanodevices. These new approaches to visualize molecular-scale events will make the nano a little closer and clearer.
Source: Arivazhagan Rajendran, Masayuki Endo, Kumi Hidaka, and Hiroshi Sugiyama. Direct and Real-Time Observation of Rotary Movement of a DNA Nanomechanical Device / / Journal of American Chemical Society.2013. V. 135 (3). P. 1117-1123.
Chengde Mao, Weiqiong Sun, Zhiyong Shen & Nadrian C. Seeman. A nanomechanical device based on the B-Z transition of DNA / / Nature. 14 January 1999. V. 397. P. 144-146.