RNA origami
Two groups of researchers have overcome the main obstacle to the use of ribonucleic acid nanostructures – the chemical instability of RNA and its rapid destruction in the body under the action of enzymes-RNase.
Nanostructures from RNA
Predicting the spatial structure for an arbitrary RNA molecule is a non–trivial task that has no general solution. Fortunately, nature has created many special cases of RNA molecules with amazing properties. Molecular biologists study these properties, and the task of nanotechnologists is to use them for the benefit of humanity.
This is exactly what a group of scientists from the United States of America did. Escherichia coli has an elegant replication control system based on the interaction of two RNA molecules – RNAI and RNAII. Each of these RNAs has a "hairpin" structural element consisting of a double-stranded stem and a single-stranded loop at the top of the stem. The sequence of nucleotides of the RNAI loop is complementary to the sequence of the RNAII loop, so that two RNA molecules can interact, forming one double–stranded from two single-stranded sections - the "kissing loops" structure (Figure 1). In this case, the molecules are arranged in such a way that the angle between the stems of the two hairpins is 120 °.
Scientists realized that with the help of such loops and kissing interactions, tiny hexagons can be constructed. The idea can be implemented in two ways: using "edges" with two different loops at the ends (one loop from RNAI, the other from RNAII) or two sets of edges with the same loops at the ends (Figure 2, variants SM and AB, respectively).
In Figure 3, you can see AFM images of the resulting nanoparticles: indeed, polygonal shapes were formed, mainly hexagons, but also four-, five-, seven-, octagons (for variant AB – as expected, only shapes with an even number of sides, for SM – including odd ones).
The scientists decided to go further and construct a "fully programmed" set of edges, from which hexagons would be guaranteed to be obtained, and not an arbitrary set of polyhedra. To do this, a unique kissing structure similar to RNAI/RNAII should be implemented at each vertex (Figure 4). But so many structures do not exist in nature… It doesn't matter! The scientists made several nucleotide substitutions in the existing RNAI/RNAII motif, and on the already tested system they looked at how these substitutions affect the ability of the "edges" to form polygons. Some replacements, unfortunately, were unsuccessful; from the remaining ones, however, it was possible to choose six sets that were not complementary to each other.
Now scientists are able to assemble nanogons with an edge length of only 6.5 nm – and, if you want six-, you want pentagons, and if you want, then squares. As further analysis showed, hexagons are the most advantageous shape, and if you also "stitch" the 3'-end with the 5'-end, then the resistance of the nanostructure to the action of nucleases that are present in blood plasma immediately increases.
Why would such nanoparticles be needed? In addition to being beautiful, they can be used for therapeutic purposes – to deliver short interfering RNAs (siRNAs) inside cells. These siRNAs can either be part of the "ribs" or form a separate stem perpendicular to the "rib" (Figure 5).
The scientists tested both variants and showed that in both cases the structure interacts with the Dicer protein in vitro. Dicer is a special intracellular protein that cuts siRNA from double–stranded RNA. So, we can expect that after the hexagons get into the cell, the natural enzyme Dicer will cut out the siRNA fragment from the RNA and launch a natural mechanism for suppressing the activity of those genes to which this siRNA is suitable.
A more complete pleasure from this work can be obtained by reading the text of the article “Self-Assembling RNA Nanorings Based on RNAI/II Inverse Kissing Complexes”, published in the journal Nano Letters.
Breakthrough in RNA nanotechnology: stable three-dimensional RNA nanostructures have been obtained
Scientists have been able to overcome a major obstacle to the use of RNA genetic material in nanotechnology – an area that develops machines thousands of times smaller than the thickness of a human hair, where DNA now dominates. Their results, which can accelerate the use of RNA nanotechnology in the field of medicine, are presented in the journal ACS Nano (Fabrication of Stable and RNase-Resistant RNA Nanoparticles Active in Gearing the Nanomotors for Viral DNA Packaging).
Professor of biomedical engineering at the University of Cincinnati, Ph.D. Peixuan Guo and his colleagues note that, having common chemical properties, DNA, a double-stranded genetic "life plan", and RNA, its single-stranded "cousin", can be used as building blocks for creating nanostructures and nanodevices. In some respects, RNA even has advantages over DNA. The field of DNA nanotechnology has been developing intensively for a long time. However, RNA nanotechnology, which is only 10 years old, is no less promising and can be successfully used in the treatment of cancer, as well as viral and genetic diseases. The slow progress of RNA nanotechnology is explained by the chemical instability of RNA and its destruction in the presence of enzymes.
Nanoscale motors like this one are made of a DNA shaft and six RNA screws –
can provide energy to tiny nanomachines,
each of which is thousands of times smaller than the thickness of a human hair.
"The RNase enzyme randomly cuts RNA into small fragments, doing it very efficiently – in just a few minutes," explains Professor Guo. "Given that RNase is present everywhere, obtaining RNA in the laboratory is an extremely difficult task."
By replacing one of the chemical groups in the macromolecule, Guo and his colleagues found a way to prevent the action of RNase and create stable three-dimensional configurations of RNA, significantly expanding the possibilities of its application in nanotechnology.
The scientists focused their attention on ribose rings, which together with phosphate groups make up the backbone of the nucleopolymer. By replacing one of the sections of the ribose ring, Guo and his colleagues made it impossible for it to bind to RNase, thus obtaining a destruction-resistant molecular structure.
"The interaction between RNase and RNA requires a matching structural conformation," explains Guo. "If the conformation of the RNA changes, the RNase cannot recognize the RNA and binding becomes problematic."
Having shown in earlier studies that such a change makes RNA stable in a double helix, scientists refused to study its potential in influencing the folding of RNA into a three-dimensional structure necessary in nanotechnology.
"We have established that the modified RNA can appropriately fold into a 3-D structure and perform its biological functions," says Guo.
Scientists have tested the ability of a three-dimensional RNA nanostructure to provide energy to a nanoscale biological motor of one of the bacteriophages – viruses infecting bacteria – working with the help of RNA molecules. The modified RNA showed excellent biological activity even in the presence of high concentrations of enzymes that usually destroy this polymer.
The data obtained confirm the feasibility of producing enzyme-resistant RNase, biologically active and stable RNAs for use in the field of nanotechnology.
Both DNA and RNA can serve as building blocks for the ever-growing production of nanostructures. The innovative approach proposed by Ned Seaman 30 years ago led to an explosion of information in the field of DNA nanotechnology. RNA molecules can be manipulated with the same simplicity as DNA, while obtaining non-canonical base pairing, universal functions and catalytic activity similar to protein. However, being afraid of the sensitivity of RNA to RNase, many scientists left RNA nanotechnology. We report the production of stable RNA nanoparticles resistant to decomposition by RNase. 2'-F (2'-fluorine)-RNA retains the ability to properly dimerize and biological activity in driving the phi29 bacteriophage nanomotor during viral DNA packaging and the formation of infectious viral particles. Our results demonstrate the feasibility of producing RNase-resistant, biologically active and stable RNA nanoparticles for use in nanotechnology. (Photo: pubs.acs.org )
"Since stable RNA molecules can be used to assemble various nanostructures, they are an ideal tool for delivering targeted therapeutic agents to cancer or virus–infected cells," says Professor Guo.
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24.01.2011