New software automates the trickiest parts of 'RNA origami' inside cells

By Tara DiMaio
May 10, 2021
An impossibly small bit of engineering and "shaping" has now been automated. Above, a single strand of ribonucleic acid. (Shutterstock)

An impossibly small bit of engineering and "shaping" has now been automated. Above, a single strand of ribonucleic acid. (Shutterstock)

Scientists can now automate the most challenging parts of designing shapes from RNA at the nanolevel, a method known as RNA origami. The inventors of that technique have released new software that substantially accelerates and improves the process and could one day yield custom structures for use in personalized medicine.

Their RNA Origami Automated Design software, or ROAD, speeds up the complicated process of building RNA nanoscaffolds, which is time- and labor-intensive. RNA scaffolds are artificially created molecules that can organize proteins and enzymes at a cellular level. The quick and efficient design of RNA origami could offer unique advantages for doctors and scientists in nanomedicine and synthetic biology, according to the study introducing ROAD, published Monday in Nature Chemistry.

"As a molecular biologist studying RNA structure, it blew my mind that DNA (and possibly RNA) could be shaped at will and by human creativity," study co-author Ebbe S. Andersen told Fastinform. Andersen leads a biomolecular design lab in Denmark that spearheaded the creation of RNA origami. Two of his co-authors, Cody Geary and Paul W. K. Rothemund, joined forces to develop the RNA origami method at Andersen's lab. Rothemund's name may be familiar as the inventor of DNA origami, or from two TED Talks he gave on playing with genes and DNA folding that together have been viewed nearly 1.5 million times. 

"I became fascinated with RNA because of its ability to fold into intricate functional shapes," Geary said. RNA nanostructures are unique in that they fold at the growing end of the RNA strand while simultaneously being copied from DNA genes at the other end. The pattern, known as cotranscriptional folding, means that RNA can be genetically expressed in cells. "So if you can learn how to master RNA folding, then you can make some pretty compelling machines," Rothemund explained.

One such shape is a ribosome, which Rothemund noted is "arguably the most important machine in the cell." This microscopic machine links together amino acids to assemble proteins based on information from messenger RNA, or mRNA — a molecule that has recently received a lot of press, thanks to the Pfizer and Moderna mRNA coronavirus vaccines. Human mRNA was also shown to trigger a change in a protein in the Ebola virus, potentially offering a new way to combat the deadly disease.

Thanks to an earlier invention from Andersen, Rothemund and Geary, scientists can now create different shapes of RNA from a single strand in a framework called RNA origami. "By reverse-engineering the folding process of RNA, we are better able to build new RNA structures from the bottom up, like an architect does," Geary noted. Geary likened the assembly process to self-building furniture; "Imagine an Ikea furniture set that can read the instructions and put all the parts together on its own, it is really incredible!" But this process is not always easy or fast.

Previously, some models of RNA origami structures had to be drawn by hand. Part of the new computer software automates this 3D modeling, which allows the team to design more sophisticated patterns and larger structures. ROAD can build origami models at the same time that it spots possible folding barriers. As a result, the ROAD designs RNA structures that are optimized — and nucleotides that are longer.

"Cody did an amazing job in solving some of the core challenges in developing RNA design algorithms," Andersen noted. The first part of the software, called RNAbuild, translates blueprint drawings into models, so scientists can get feedback on the geometrical shapes of the RNA origami as they construct the model. The second program, RNApath, scans for possible topological barriers during folding, so a designer can choose a route with fewer obstacles.

"Thirdly, and most importantly, Cody developed the Revolvr sequence-design software that solves the problem of designing RNA sequences for highly pseudoknotted RNA structures — a problem that has been thought to be impossible," Andersen said. RNA pseudoknots are key in origami design because they allow a single strand of RNA to fold back on itself. Both Andersen and Geary noted that ROAD uses an unconventional design to solve complex problems. "The program runs almost totally backward, solving the most difficult sequence-level constraints only at the very end of the design, and I think that is essentially the secret to making it work well," Geary explained.

"I was surprised and pleased at how Cody's software gave results (RNA origami designs) in minutes that could not be achieved in days or weeks with existing software," Rothemund noted. With the ROAD software, the team created 32 different designs that were up to 2,360 nucleotides long. To put that number in perspective, the team reached a limit of 450 nucleotides for cotranscriptionally folded RNA origami in 2014.

The new computer software also speeds up the complex process of constructing custom RNA scaffolds. ROAD produced five designs that could scaffold proteins and seven that could structure molecules at a particular distance from each other. That distancing serves as an indicator for the scaffold's precision. "These RNA structures have the potential to be very useful in the field of synthetic biology," Geary added.

The researchers are currently working on expanding the ROAD software to use RNA nanotechnology within cells. Although the scientists are unsure of how cells will react to synthetic RNA structures, the team hopes that special RNA origami will be able to act as biosensors in cells and as scaffolds to regulate gene expression as part of the rapidly emerging field of metabolic engineering.

"We want to be able to switch enzymes around at will, rearranging their order, under genetic control," Rothemund said. "If we were awesome protein engineers, we could rearrange the enzymes as desired. But we (humans) aren't that awesome at protein engineering (yet), and so, making RNA origami scaffolds, augmented with various RNA widgets that attract the protein enzymes … is the fastest way to engineer the sorts of molecular factories we want."

The study, "RNA origami design tools enable cotranscriptional folding of kilobase-sized nanoscaffolds," published Monday in Nature Chemistry, was authored by Cody Geary, Aarhus University and California Institute of Technology; Guido Grossi, Ewan K. S. McRae and Ebbe S. Andersen, Aarhus University; and Paul W. K. Rothemund, California Institute of Technology.

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