A revolution in the field of nanotechnology! An Inserm researcher[1] in collaboration with Harvard University has succeeded in creating 3D shapes of unprecedented sophistication, thanks to the four DNA bases A, T, C and G. In practice, these researchers can create nanoscopic (10-9 m) objects from 30,000 DNA sequences that fold and self-assemble like LEGO® bricks. In time, this will make it possible to manufacture new tools adapted to the size of our cells. These results have been published in Nature.

Nanotechnology represents a rapidly expanding scientific field, particularly when it comes to creating materials with increasingly specific properties. This is the case of carbon nanotubes, for example, which are light but solid and possess very high thermal and electrical conductivity. However, a slightly less well-known field of research exists: that of DNA-based nanotechnology,  whose objective is to model living matter to use as a therapeutic tool on a scale compatible with that of a human cell. However, this technology, which was developed in 2012 and is called DNA LEGO® bricks, encountered challenges in programming DNA sequences sufficient to create increasingly complex objects.

In this paper published in Nature, the researchers have reached a turning point. Their objects, manufactured according to the LEGO® brick method, use one million DNA bases, a size comparable to the genome of a bacterium, whereas until then the objects comprised only one thousand bases.

So, how does it work?

The method uses bricks, like LEGOs®, each comprising 52 DNA bases. One of the properties of DNA is based on the fact that the nucleobases of a DNA strand (A, T, C and G) can interact with those of another strand by always pairing in the same way: A with T, and C with G. Like LEGOs®, these units all have the same general shape but the internal order of the 52 bases determines which bricks will be able to interlock with which and at what level.

Next comes the choice of shape, which is either designed or selected from a database of 3D forms (cube, teddy bear, rabbit, Möbius strip, etc.). Then, each “voxel” ^[2] of the design is translated into DNA bricks using software developed by the researchers, called Nanobricks. “Nanobricks ‘codes’ the DNA by specifying in advance the order of the 52 bases of each brick that will subsequently be used. This step determines how the 30,000 initial sequences will fit together to produce one final 3D structure,” explains Gaëtan Bellot, Inserm researcher and co-author of this paper.

Once the IT procedures are complete, the 30,000 sequences are synthesized in a laboratory and mixed in a tube. The 30,000 DNA sequences are then completely destructured by means of a denaturing step performed at 80°C. The mixture is then gradually cooled to 25°C at a rate of 0.5°C/hour, which is when the self-assembly takes place. The molecules spontaneously fold and adopt a final shape in accordance with the 3D model selected. In this paper, the researchers produced 13 different objects.

To make objects from 30,000 sequences, they had to increase the diversity of the DNA brick sequences. By exploring various brick sizes, the research teams were able to define an optimal brick size (52 bases) making it possible both to maintain a 3D geometry similar to that of a LEGO brick and increase the diversity of individual bricks to 67 million.

This means that it is possible to obtain increasingly sophisticated objects with a greater diversity of individual bricks. The researchers have succeeded in creating objects with cavities. This level of precision is necessary if we are to design useful and effective tools. “With a key, you can open a car. With a DNA tool you can, for example, build a capsule into which you can place a medicine. And if this object has cavities, you can create a biological chain reaction depending on the products present in each cavity. Taking inspiration from life, this approach will enable the reproduction, on a nanometer scale, of solutions and inventions that occured after millions of years of evolution,” explains  Bellot.

This method offers two advantages. The first is that unlike industrial assembly processes, such as car production lines, this technology compresses all the stages into one. Imagine placing a car’s various components in the presence of each other for them to spontaneously self-assemble! The second resides in its rapidity: 30,000 components self-assemble within several hours into an object duplicated a billion times inside the same tube.

Unlike carbon nanotubes, DNA nanotechnologies are biocompatible and can be rapidly eliminated from the human body and the environment.  Nevertheless, even if the DNA molecules used are synthetic and as such not biologically active, potential interaction with the DNA present in living organisms cannot be ruled out.

[1] From the Institute for Functional Genomics (Inserm/CNRS/Université de Montpellier)

[2] Voxel, a contraction of the words “volume” and “element”, is a 3D pixel.

Transposable elements, also known as “jumping genes” are DNA fragments that can move or copy themselves from one location to another on the chromosomes. They have invaded the genomes of most living organisms, from bacteria to humans, via the plants. When they jump, they bring about complex modifications in genes near which or in which they insert themselves, and can thereby alter or abolish their function. This phenomenon contributes to the evolution and adaptation of species.

However, in the shorter term, “jumping genes” can have harmful effects. In humans, the only currently active transposon family, the LINE-1 type retrotransposons, causes new cases of genetic diseases, such as haemophilia and muscular dystrophy. This is why their activity is normally under tight control. However, in nearly half of epithelial cancers, they manage to escape the many cellular defence mechanisms that protect our DNA, and jump actively, contributing to the emergence and progression of cancers. Moreover, in studies they are often used as tumour biomarkers for diagnostic or prognostic purposes.

(c) Fotolia

One of the main problems raised by the study of “jumping genes” is related to their extremely repetitive nature. Our DNA contains thousands of copies of them, almost identical to one another, and every individual contains hundreds of copies that are not listed in the reference map of the human genome. Furthermore, until now, it had been impossible to know whether activation of “jumping genes” results from a general disruption, leading to massive mobilisation of all copies, or whether, on the contrary, only a small number of them manage to escape the protective controls. Through a new approach, published in the journal eLife, involving high-throughput sequencing, genomics, epigenomics and bioinformatics, the team led by Gaël Cristofari, Inserm Research Fellow, and his collaborators at Unit 1081, “Institute for Research on Cancer and Aging, Nice (IRCAN),” has succeeded in measuring the activity of the “jumping genes” in normal and cancer cells with unprecedented resolution.

According to their results, only a small number of copies are really guilty: those located in permissive regions of our chromosomes. And these regions vary depending on the type of cell. Furthermore, all these active copies are not present in all individuals!

“The important concept here is that the small group of LINE-1 transposons that escapes control is different from one cell type to another: in certain cancers, one group is important, in another type of cancer, it will be another group of copies. This observation suggests that behind each LINE-1 group, there is a mechanism and signals specific for a particular type of organ or tissue,” explains Gaël Cristofari.

These results provide a better understanding of how new mutations may emerge, they suggest the existence of genetic factors behind this phenomenon, and they provide new data for the rational use of LINE-1 retrotransposons as biomarkers in oncology, by focusing on the active copies in a given type of cell.

This work was made possible by financial support from ARC French Foundation for Cancer Research, Fondation pour la Recherche Médicale, Cancéropôle PACA, the European Research Council, the French National Research Agency (ANR) (Labex Signalife), and the Research Group on Mobile Genetic Elements (CNRS, GDR 3546).

Chemical substances that are safe for humans when taken in isolation can become harmful when they are combined. Three research teams bringing together researchers from Inserm and CNRS[1] in Montpellier have elucidated in vitro a molecular mechanism that could contribute to the phenomenon known as the “cocktail effect.” This study is published in the journal Nature Communications.

[1] Centre for Structural Biochemistry (CBS) (CNRS UMR5048 – Inserm U1054) at the Cancer Research Institute (Inserm U1194), and the Functional Genomics Institute (CNRS UMR5203 – Inserm U661)

Every day we are exposed to many exogenous compounds such as environmental pollutants, drugs or substances in our diet. Some of these molecules, known as endocrine disruptors, are strongly suspected of interacting inappropriately with regulatory proteins in our cells, and inducing numerous physiological or metabolic disorders (cancers, obesity, diabetes, etc.). Moreover, the combination of these molecules in complex mixtures with which we are in routine contact might exacerbate their toxicity.

In an article to be published in Nature Communications, researchers have unveiled a mechanism that might contribute to this effect of mixing, for which no rational explanation has been offered until now. They show that some oestrogens such as ethinyloestradiol (one of the active ingredients of contraceptive pills) and organochlorine pesticides such as trans-nonachlor, although very weakly active on their own, have the ability to bind simultaneously to a receptor located in the cell nucleus, and to activate it synergistically. 

Analyses at molecular level indicate that the two compounds bind cooperatively to the receptor, i.e. binding of the first molecule promotes binding of the second. This cooperativity is due to strong interactions at the level of the receptor binding site, so that the binary mixture induces a toxic effect at substantially lower concentrations than the individual molecules.

Separately, ethinyloestradiol (EE2) and trans-nonachlor (TNC) bind to the xenobiotic receptor (PXR) only at high concentrations, and are weak activators of this receptor. When they are used together, the two compounds mutually stabilise each other in the binding pocket of the receptor. The “supramolecular ligand” thus created has increased affinity for PXR, so that it can induce a toxic effect at doses at which each compound is inactive individually. © Vanessa Delfosse, William Bourguet 

Another step forward has just been taken in the area of synthetic biology. Research teams from Inserm and CNRS (French National Centre for Scientific Research) Montpellier, in association with Montpellier Regional University Hospital and Stanford University, have transformed bacteria into “secret agents” that can give warning of a disease based solely on the presence of characteristic molecules in the urine or blood. To perform this feat, the researchers inserted the equivalent of a computer programme into the DNA of the bacterial cells. The bacteria thus programmed detect the abnormal presence of glucose in the urine of diabetic patients. This work, published in the journal Science Translational Medicine, is the first step in the use of programmable cells for medical diagnosis.

Bacteria have a bad reputation, and are often considered to be our enemies, causing many diseases such as tuberculosis or cholera. However, they can also be allies, as witnessed by the growing numbers of research studies on our bacterial flora, or microbiota, which plays a key role in the working of the body. Since the advent of biotechnology, researchers have modified bacteria to produce therapeutic drugs or antibiotics. In this novel study, they have actually become a diagnostic tool.

Medical diagnosis is a major challenge for the early detection and subsequent monitoring of diseases. “In vitro” diagnosis is based on the presence in physiological fluids (blood and urine, for example) of molecules characteristic for a particular disease. Because of its noninvasiveness and ease of use, in vitro diagnosis is of great interest. However, in vitro tests are sometimes complex, and require sophisticated technologies that are often available only in hospitals.

This is where biological systems come into play. Living cells are real nano-machines that can detect and process many signals and respond to them. They are therefore obvious candidates for the development of powerful new diagnostic tests. However, they have to be provided with the appropriate “programme” for them to successfully accomplish the required tasks.

To do this, Jérôme Bonnet’s team in Montpellier’s Centre for Structural Biochemistry (CBS) had the idea of using concepts from synthetic biology[1] derived from electronics to construct genetic systems making it possible to “programme” living cells like a computer.

The transcriptor: the cornerstone of genetic programming

The transistor is the central component of modern electronic systems. It acts both as a switch and as a signal amplifier. In informatics, by combining several transistors, it is possible to construct “logic gates,” i.e. systems that respond to different signal combinations according to a predetermined logic. For example, a dual input “AND” logic gate will produce a signal only if two input signals are present. All calculations completed by the electronic instruments we use every day, such as smartphones, rely on the use of transistors and logic gates.

During his postdoctoral fellowship at Stanford University in the United States, Jérôme Bonnet invented a genetic transistor, the transcriptor.

The insertion of one or more transcriptors into bacteria transforms them into microscopic calculators. The electrical signals used in electronics are replaced by molecular signals that control gene expression. It is thus now possible to implant simple genetic “programmes” into living cells in response to different combinations of molecules[2].

In this new work, the teams led by Jérôme Bonnet (CBS, Inserm U1054, CNRS UMR5048, Montpellier University), Franck Molina (SysDiag, CNRS FRE 3690), in association with Professor Eric Renard (Montpellier Regional University Hospital) and Drew Endy (Stanford University), applied this new technology to the detection of disease signals in clinical samples.

Clinical samples are complex environments, in which it is difficult to detect signals. The authors used the transcriptor’s amplification abilities to detect disease markers, even if present in very small amounts. They also succeeded in storing the results of the test in the bacterial DNA for several months.

The cells thus acquire the ability to perform different functions based on the presence of several markers, opening the way to more accurate diagnostic tests that rely on detection of molecular “signatures” using different markers.

Figure 1: Principle of the use of modified bacteria for medical diagnosis. ©J. Bonnet/ Inserm.

“We have standardised our method, and confirmed the robustness of our synthetic bacterial systems in clinical samples. We have also developed a rapid technique for connecting the transcriptor to new detection systems. All this should make it easier to reuse our system,” says Alexis Courbet, a postgraduate student and first author of the article.

As a proof of concept, the authors connected the genetic transistor to a bacterial system that responds to glucose, and detected the abnormal presence of glucose in the urine of diabetic patients.

“We have deposited the genetic components used in this work in the public domain to allow their unrestricted reuse by other public or private researchers,[3]” says Jérôme Bonnet.

“Our work is presently focused on the engineering of artificial genetic systems that can be modified on demand to detect different molecular disease markers,” he adds. In future, this work might also be applied to engineering the microbial flora in order to treat various diseases, especially intestinal diseases.

This work received financial support from Inserm, CNRS, the Stanford-France Center for Interdisciplinary Studies, and Stanford University. Jérôme Bonnet is a recipient of the Atip-Avenir programme award, and is supported by the Bettencourt-Schueller Foundation.