Tag Archives: Synthetic biology

Using the origami technique to design RNA nanostructures

Researchers from Aarhus University and Berkeley Laboratory have designed RNA molecules, that folds into nanoscale rectangles, cylinders, and satellites, and have studied their 3D structure and dynamics with advanced nanotechnological methods. In an article in the journal Nature Nanotechnology, the researchers describe their work and how it has led to the discovery of rules and mechanisms for RNA folding that will make it possible to build more ideal and functional RNA particles for use in RNA-based medicine.

The RNA molecule is commonly recognized as messenger between DNA and protein, but it can also be folded into intricate molecular machines. An example of a naturally occurring RNA machine is the ribosome, that functions as a protein factory in all cells. Inspired by natural RNA machines, researchers at the Interdisciplinary Nanoscience Center (iNANO) have developed a method called “RNA origami”, which makes it possible to design artificial RNA nanostructures that fold from a single stand of RNA. The method is inspired by the Japanese paper folding art, origami, where a single piece of paper can be folded into a given shape, such as a paper bird.

Frozen folds provide new insight

The research paper in Nature Nanotechnology describes how the RNA origami technique was used to design RNA nanostructures, that were characterized by cryo-electron microscopy (cryo-EM) at the Danish National cryo-EM Facility EMBION. Cryo-EM is a method for determining the 3D structure of biomolecules, which works by freezing the sample so quickly that water does not have time to form ice crystals, which means that frozen biomolecules can be observed more clearly with the electron microscope. Images of many thousands of molecules can be converted on the computer into a 3D map, that is used to build an atomic model of the molecule. The cryo-EM investigations provided valuable insight into the detailed structure of the RNA origamis, which allowed optimization of the design process and resulted in more ideal shapes.

With precise feedback from cryo-EM, we now have the opportunity to fine-tune our molecular designs and construct increasingly intricate nanostructures.”

Ebbe Sloth Andersen, Associate Professor at iNANO, Aarhus University

Discovery of a slow folding trap

Cryo-EM images of an RNA cylinder sample turned out to contain two very different shapes, and by freezing the sample at different times it was evident that a transition between the two shapes was taking place. Using the technique of small-angle X-ray scattering (SAXS), where the samples are not frozen, the researchers were able to observe this transition in real time and found that the folding transition occurred after approx. 10 hours. The researchers had discovered a so-called “folding trap” where the RNA gets trapped during transcription and only later gets released (see video).

“It was quite a surprise to discover an RNA molecule that refolds this slow since folding typically takes place in less than a second” tells Jan Skov Pedersen, Professor at Department of Chemistry and iNANO, Aarhus University.

“We hope to be able to exploit similar mechanisms to activate RNA therapeutics at the right time and place in the patient”, explains Ewan McRae, the first author of the study, who is now starting his own research group at the “Centre for RNA Therapeutics” at the Houston Methodist Research Institute in Texas, USA.

Construction of a nanosatellite from RNA

To demonstrate the formation of complex shapes, the researchers combined RNA rectangles and cylinders to create a multi-domain “nanosatellite” shape, inspired by the Hubble Space Telescope.

“I designed the nanosatellite as a symbol of how RNA design allows us to explore folding space (possibility space of folding) and intracellular space, since the nanosatellite can be expressed in cells”, says Cody Geary, assistant professor at iNANO, who originally developed the RNA-origami method.

However, the satellite proved difficult to characterize by cryo-EM due to its flexible properties, so the sample was sent to a laboratory in the USA, where they specialize in determining the 3D structure of individual particles by electron tomography, the so-called IPET-method.

“The RNA satellite was a big challenge! But by using our IPET method, we were able to characterize the 3D shape of individual particles and thus determine the positions of the dynamic solar panels on the nanosatellite”, says Gary Ren from the Molecular Foundry at Lawrence Berkeley National Laboratory, California, USA.

The future of RNA medicine

The investigation of the RNA origamis contributes to improving the rational design of RNA molecules for use in medicine and synthetic biology. A new interdisciplinary consortium, COFOLD, supported by the Novo Nordisk Foundation, will continue the investigations of RNA folding processes by involving researchers from computer science, chemistry, molecular biology, and microbiology to design, simulate and measure folding at higher time resolution.

“With the RNA design problem partially solved, the road is now open to creating functional RNA nanostructures that can be used for RNA-based medicine, or act as RNA regulatory elements to reprogram cells”, predicts Ebbe Sloth Andersen.

Journal reference:

McRae, E.K.S., et al. (2023) Structure, folding and flexibility of co-transcriptional RNA origami. Nature Nanotechnology. doi.org/10.1038/s41565-023-01321-6.

Mobility is an important ability for many organisms, who often have to swim, walk, or otherwise move around …

Mobility is an important ability for many organisms, who often have to swim, walk, or otherwise move around to look for food or shelter. Even single-celled organisms and individual cells have to be able to move to the right places to survive. But we don’t know much about how motility came about during evolution. Researchers recently explored motility in very tiny organism. In this study, they noted that movement by certain parts of the cell, including proteins in the cytoskeleton like actin or tubulin, and the cellular fuel ATP may have been major contributors to the origins of cellular movement.

With the expression of only a few additional proteins, the small, synthetic bacteria formed helices that allow the microbe to swim by spiraling, making them the mobile lifeforms with the smallest genomes./ Credit: Makoto Miyata, Osaka Metropolitan University

There are also very tiny microbes that move around in different ways. Some called Spiroplasma have a helical shape and can swim around by switching the direction of their helix.

In a new study reported in Science Advances, scientists built on the synthetic organism created by the J. Craig Venter Institute (JCVI) in 2016, which was engineered based on a type of Spiroplasma bacteria. In this study, the researchers enabled this synthetic organism to move around by adding only seven genes. These genes encode for proteins that are thought to be directly related to the switching of helical rotation that allows the microbe to swim. Additional work showed that only two of the newly added genes were absolutely necessary for swimming.

However, the study authors also acknowledged that some of the other genes that were in the original minimal genome, such as those related to cell division, for example, may be contributing to motility in some way that has not yet been revealed.

“Studying the world’s smallest bacterium with the smallest functional motor apparatus could be used to develop movement for cell-mimicking microrobots or protein-based motors,” said senior study author Professor Miyata of the Graduate School of Science at Osaka Metropolitan University. “Our swimming syn3 can be said to be the ‘smallest mobile lifeform’ with the ability to move on its own. The results of this research are expected to advance how we understand the evolution and origins of cell motility.”

Sources: Osaka Metropolitan University, Science Advances

Carmen Leitch

Genetically engineered bacteria make living materials for self-repairing walls and cleaning up pollution

With just an incubator and some broth, researchers can grow reusable filters made of bacteria to clean up polluted water, detect chemicals in the environment and protect surfaces from rust and mold.

I am a synthetic biologist who studies engineered living materials – substances made from living cells that have a variety of functions. In my recently published research, I programmed bacteria to form living materials that can not only be modified for different applications, but are also quick and easy to produce.

Like human cells, bacteria contain DNA that provides the instructions to build proteins. Bacterial DNA can be modified to instruct the cell to build new proteins, including ones that don’t exist in nature. Researchers can even control exactly where these proteins will be located within the cell.

Because engineered living materials are made of living cells, they can be genetically engineered to perform a broad variety of functions, almost like programming a cellphone with different apps. For example, researchers can turn bacteria into sensors for environmental pollutants by modifying them to change color in the presence of certain molecules. Researchers have also used bacteria to create limestone particles, the chemical used to make Styrofoam and living photovoltaics, among others.

A primary challenge for engineered living materials has been figuring out how to induce them to produce a matrix, or substances surrounding the cell, that allows researchers to control the physical properties of the final material, such as its viscosity, elasticity and stiffness. To address this, my team and I created a system to encode this matrix in the bacteria’s DNA.

We modified the DNA of the bacteria Caulobacter crescentus so that the bacterial cells would produce on their surfaces a matrix made of large amounts of elastic proteins. These elastic proteins have the ability to bind to each other and form hydrogels, a type of material that can retain large amounts of water.

When two genetically modified bacterial cells come in close proximity, these proteins come together and keep the cells attached to each other. By surrounding each cell with this sticky, elastic material, bacterial cells will cluster together to form a living slime.

Furthermore, we can modify the elastic proteins to change the properties of the final material. For example, we could turn bacteria into hard construction materials that have the ability to self-repair in the event of damage. Alternatively, we could turn bacteria into soft materials that could be used as fillers in products.

Usually, creating multifunctional materials is extremely difficult, due in part to very expensive processing costs. Like a tree growing from a seed, living materials, on the other hand, grow from cells that have minimal nutrient and energy requirements. Their biodegradability and minimal production requirements allow for sustainable and economical production.

The technology to make living materials is unsophisticated and cheap. It only takes a shaking incubator, proteins and sugars to grow a multifunctional, high-performing material from bacteria. The incubator is just a metal or plastic box that keeps the temperature at about 98.6 degrees Fahrenheit (37 Celsius), which is much lower than a conventional home oven, and shakes the cells at speeds slower than a blender.

Transforming bacteria into living materials is also a quick process. My team and I were able to grow our bacterial living materials in about 24 hours. This is pretty fast compared to the manufacturing process of other materials, including living materials like wood that can take years to produce.

Moreover, our living bacterial slime is easy to transport and store. It can survive in a jar at room temperature for up to three weeks and placed back into a fresh medium to regrow. This could lower the cost of future technology based on these materials.

Lastly, engineered living materials are an environmentally friendly technology. Because they are made of living cells, they are biocompatible, or nontoxic, and biodegradable, or naturally decomposable.

There are still some aspects of our bacterial living material that need to be clarified. For example, we don’t know exactly how the proteins on the bacterial cell surface interact with each other, or how strongly they bind to each other. We also don’t know exactly how many protein molecules are required to keep cells together.

Answering these questions will enable us to further customize living materials with desired qualities for different functions.

Next, I’m planning to explore growing different types of bacteria as living materials to expand the applications they can be used for. Some types of bacteria are better than others for different purposes. For example, some bacteria survive best in specific environments, such as the human body, soil or fresh water. Some, on the other hand, can adapt to different external conditions, like varying temperature, acidity and salinity.

By having many types of bacteria to choose from, researchers can further customize the materials they can create.

Sara Molinari

The Conversation