DNA: details of the nanoconstructor

DNA-nanotechnology. A brief overview
Trusov L. A., "Nanometer"DNA, like other biological macromolecules (RNA, proteins), has a surprisingly strict organization at the nanoscale level.

This feature can be used as a powerful tool for designing nanostructures and nanodevices "from the bottom up". From the point of view of nanotechnology, an important quality of a DNA molecule is its ability to recognize and bind complementary bases, as well as the relative stability of the DNA double helix, and much less attention is paid to the genetic or other biological role of DNA. DNA as a carrier and regulator of genetic information is an area of interest, rather, biotechnology.

The source of DNA nanotechnology is the work of Nadrian Seeman (Figure 1), started about 30 years ago [1].

Then it seemed that with the help of properly selected DNA chains, it was possible to put together a figure of any complexity corresponding to any goals. In general, the reality did not deceive the expectations of scientists.

Two significant areas of DNA use in nanotechnology can be distinguished:

• DNA as a structural element for creating complex structures and
• DNA as a functional element in nanomachines.

The world of DNA seems logical, rigorous and almost mathematically correct. Two antiparallel DNA chains, twining one around the other, form a double helix (duplex). Opposite each nitrogenous base of one chain is a strictly defined nitrogenous base of another chain: adenine (A) opposite thymine (T), guanine (G) opposite cytosine (C) (Figure 2).

Figure 2. Complementary interactions of two DNA chains.
Nitrogenous bases form hydrogen bonds with each other,
moreover, there are two such connections in the A-T pair, and three in the G-C pair.

This interaction is called canonical, or Watson-Crick, after the scientists who suggested and subsequently proved the formation of complementary pairs (Francis Crick and James Watson, together with Maurice Wilkins, received the Nobel Prize in Physiology or Medicine in 1962 for their outstanding contribution to deciphering the structure of DNA). Neighboring base pairs are also connected by stacking interaction, which provides additional rigidity and stability of the DNA molecule. Under physiological conditions, the DNA double chain exists mainly in B-form with the following parameters: one turn of the helix consists of 10.4 pairs of nucleotides, has 3.4 nm in length and about 2.2 nm in diameter; the width of the large groove is 2.2 nm, the width of the small groove is 1.2 nm (Figure 3).

Figure 3. B-shape of the DNA double helix - the "classical" form of DNA
both in wildlife and in DNA nanotechnology.

The nucleotide composition of DNA practically does not affect the parameters of the double helix, which significantly unties the hands of researchers. After all, it turns out that the nucleotide sequence of DNA can be any that meets the goals of the researcher, and will not introduce distortions into the calculated structure.

In some cases, however, the composition of the DNA chain itself turns out to be quite important: for example, guanine-rich sites tend to form guanine quadruplexes stabilized by a metal ion instead of a double helix (Figure 4).

Figure 4. Guanine quadruplexes - structures,
formed not by two, but by four DNA chains.

Under conditions other than physiological: with a change in pH, ionic strength, the –B-form of DNA can pass into the A-form or, with a certain nucleotide composition, the Z-form, while the length of the double helix decreases or increases, the number of nucleotides per turn changes. A living cell is also a kind of nanotechnology; it uses different states of the DNA molecule to regulate the activity of genes, to make certain areas more accessible or inaccessible to enzymes, for replication and transcription. A nanotechnologist-scientist uses mechanisms honed by a cell and, using the full breadth of accumulated knowledge, constructs nanostructures or nanomachines for his own purposes.

Here it is necessary to make a small clarification to the very process of manufacturing and assembling these structures. After the idea has arisen, it is necessary to select the nucleotide composition of DNA chains in such a way that the maximum possible number of Watson-Crick interactions is realized in the conceived structure. Short oligonucleotides are usually synthesized chemically; modern technologies make it easy and cheap to synthesize oligonucleotides up to 160 bases long. Longer DNA fragments, from hundreds to several thousand bases, can be synthesized enzymatically by PCR. And then comes the moment of miracle – more precisely, the moment of self-assembly: after mixing all the necessary DNA fragments in the right proportions under the right conditions, the researcher waits until the complementary pairs find each other and form exactly the structure that he pictured in his imagination or in a graphic editor… Thus, the whole complexity of using DNA as a structural element consists in the birth of an idea. The selection of oligonucleotide sequences is carried out programmatically, synthesis is not a problem, and if these stages are passed without errors, self-assembly will take place without complications.

The problem may occur in an unexpected place. So, inspired by the possibilities open to him, Ned Seaman, back in the fall of 1980, proposed to assemble three-dimensional figures from double DNA chains: a cube, a truncated octahedron. However, it soon became clear that although the edges of the resulting structures have sufficient rigidity, the angles do not stand up to any criticism, and the resulting figures are far from perfect. Therefore, scientists moved from a three-dimensional domain to a two-dimensional one to begin with and developed strong structural motifs that could serve as building blocks for assembling extended two-dimensional fields. This is, first of all, the DX motif - two double helices with two intersections (four–connected nodes). It is worth noting that a similar structure is found in wildlife and is known as the Holliday structure. This is a very rare example of the existence of branched DNA structures in the natural environment. Halliday structures are formed at a certain life stage between homologous DNA duplexes. In this case, each of the four DNA chains forming this structure is complementary to two chains at once: chains from its own and from the neighboring duplex. Due to this, the intersection point is not strictly defined and can shift in one direction or another. Nanotechnologists do not rely on chance and select oligonucleotides so that the intersection point is clearly defined (Figure 5).

Figure 5. Halliday structure and a four-connected node.
(a) In the Holliday structure, each red DNA chain is complementary to any of the blue chains,
due to this, the middle of the intersection can shift in any direction;
(b) in a four-connected node, the blue chain is complementary to the red one, the orange one is light green
and so on, which leads to self-assembly of the only possible motive
with a fixed intersection point.

In DX-blocks, the central parts of the chains are complementary not to their pair, but to the chains from the neighboring duplex, due to which two intersections (nodes) are formed. Other elementary structural bricks are DX-blocks with an additional loop (DX-J), three-connected nodes, protruding "sticky ends". Combinations of these elements make it possible to design many nanoobjects. For example, DX blocks with alternating sticky ends are suitable for the synthesis of extended flat arrays (Figure 6, [2]) and hollow tubes [3].

Figure 6. DX and DX-J blocks with sticky ends (top),
from which you can assemble long flat ribbons (at the bottom).
The AFM image of such tapes is shown on the side.

Repeating motifs-intersections allow you to create a two-dimensional network (Figure 7) [4].

Figure 7. Structural unit (left)
and the AFM image of a two-dimensional network,
obtained from such elements (on the right)

A vivid illustration of the programmed assembly of DNA into certain patterns is the DNA origami method proposed by Paul Rothemund, when one long single-stranded (for example, viral) DNA is stacked in a certain way with the help of relatively short oligonucleotide DNA clips, which connect sections of long DNA and create a strictly defined pattern (Figure 8) [5].

Figure 8. Fun DNA origami: going to amaze the world with a new technology for assembling complex structures from DNA molecules,
Paul Rotmunt started with useless, but attractive shapes: stars, faces and triangles.

Nanometer wrote a lot about DNA origami and with great pleasure. Three-dimensional nano-caskets, equipped with a nano-lock that can be locked and unlocked, still amaze the imagination (Figure 9) [6].

Figure 9. Three-dimensional reconstruction of a nanoscale,
collected by DNA origami.

DNA arrays can be associated with organic and inorganic molecules or particles. Different purposes can be pursued here: DNA can be modified for the convenience of further manipulations (for example, thiol groups), to give the molecule additional functionality (dyes, proteins), for a geometrically specified arrangement of the elements of interest.

The use of DNA as a functional element in nanomachines is based, again, on complementary interaction, as well as on the ability of single-stranded DNA to displace one of the duplex chains, provided that the displacing chain forms more complementary interactions than the displaced one. This remarkable ability was demonstrated in 2000 by Bernard Yurke and his colleagues [7]. It is enough to leave a few nucleotides on one of the duplex chains (the protruding "sticky end"), and then when adding a single-stranded DNA that is completely complementary to a longer chain, this DNA will first bind to a free single-stranded fragment, and then displace the shorter chain from the duplex. Based on this property, molecular switches have been developed (Figure 10, [8]) and even logic circuits [9].

Figure 10. DNA device proposed by Ned Seaman and his colleagues.

In the position shown above and below, the device is "empty", these two forms can easily pass into each other. The blue "keys" are complementary, one to the red, the other to the green single-stranded section, thanks to which they communicate with the device and fix it in a "double parallel" position (on the right). At the same time, the "keys" have loose sticky ends, so they can be removed by adding the molecules shown in blue, which are completely complementary to the blue "keys". The device is "empty" again. Adding another pair of "keys", purple in the figure, leads to fixing the device in the "cross" position (on the left).

A similar principle applies when the chains are initially not completely complementary, but under certain conditions the places of mismatches can be stabilized. For example, two thymine residues located opposite each other do not form a complementary pair, but when mercury salts are added, a T-Hg2+-T complex is formed. In the absence of mercury ions, binding occurs preferably with another DNA chain in which adenine is located opposite the thymine residue (a canonical A-T pair is formed). Thus, a molecular machine sensitive to the presence of Hg2+ ions is obtained [10].

The ability of DNA to adopt an unusual conformation depending on conditions has also found its application. So, in 1999, Mao, Siman and colleagues published an article in Nature, where they showed that if you bind DX blocks of a special double-stranded DNA consisting of alternating residues of guanine and cytosine, then with the addition of Co(NH3)6+ salt, this binding DNA turns into a Z-form, thanks to which DX blocks they rotate relative to each other. When the salt is removed, the binding DNA reverts to the B-form (Figure 11) [11].

Figure 11. DNA is a nanoswitch based on the transition of the B-form of DNA to the Z-form.

Another unusual DNA conformation that has also found a place in nanotenology is the so–called i-form formed by four cytosine-rich DNA chains. This form is stable at low pH values and breaks down at pH>6.3. Based on this property, molecular machines sensitive to the acidity of the medium have been created: one of the DNA chains capable of folding into the i-form has been modified at the ends with a fluorescent dye and a fluorescence extinguisher (Figure 12).

Figure 12. The pH-dependent i-form of DNA allows you to design
molecular devices sensitive to the acidity of the medium.

A double helix (B-form) is formed in an alkaline medium, the fluorophore and the extinguisher are spatially separated and do not interact; fluorescence is observed. When the pH decreases, the i-form becomes preferred, the extinguisher approaches the fluorophore and radiation is not observed [12].

You can also read (and write) about specific interesting and elegant solutions to problems in the framework of DNA nanotechnology on the Nanometer website.

Literature:1. Seeman N. (1982). "Nucleic acid junctions and lattices".
Journal of Theoretical Biology 99 (2): 237.
2. Winfree E.; Liu F.; Wenzler L. A., Seeman N. C. (1998). "Design and self-assembly of two-dimensional DNA crystals". Nature 394 (6693): 529–544.
3. Sharma J., Chhabra R., Cheng A., Brownell J., Liu Y., Yan H. (2009). “Control of Self-Assembly of DNA Tubules Through Integration of Gold Nanoparticles”. Science 323 (5910): 112-116.
4. Yan H., Park S. H., Finkelstein G., Reif J. H., LaBean T. H. (2003) “DNA-templated self-assembly of protein arrays and highly conductive nanowires”. Science 301: 1882–1884.
5. Rothemund, P. W. K. (2006). "Folding DNA to create nanoscale shapes and patterns". Nature 440 (7082): 297–302.
6. Andersen E.S., Dong M., Nielsen M. M., Jahn K., Subramani R., Mamdouh W., Golas M. M., Sander B., Stark H., Oliveira C. L. P., Pedersen J. S., Birkedal V., Besenbacher F., Gothelf K. V., Kjems J. (2009). “Self-assembly of a nanoscale DNA box with a controllable lid”. Nature 459: 73-7.
7. Yurke B., Turberfield A. J., Mills A. P. Jr., Simmel F. C., Neumann J. L. (2000). “A DNA-fuelled molecular machine made of DNA”. Nature 406 (6796): 605-8.
8. Yan H., Zhang X., Shen Z., Seeman N. C. (2002). “A robust DNA mechanical device controlled by hybridization topology”. Nature 415(6867): 62-5.
9. Voelcker N. H., Guckian K. M., Saghatelian A., Ghadiri M. R. (2008). “Sequence-Addressable DNA Logic”. Small 4 (4): 427-431.
10. Wang Z-G., Elbaz J., Willner I. (2011). “DNA Machines: Bipedal Walker and Stepper”. Nano Lett. 11 (1): 304–309.
11. Mao C., Sun W., Shen Z., Seeman N. C. (1999). "A DNA Nanomechanical Device Based on the B-Z Transition". Nature 397 (6715): 144–146.
12. Liu D, Balasubramanian S. (2003). “A proton-fuelled DNA nanomachine”. Angew Chem Int Ed Engl. 42(46): 5734-6.

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