Synthetic biology researchers at Northwestern University say they have developed a system that can rapidly create cell-free ribosomes in a test tube, then select the ribosome that can perform a certain function. The system, called ribosome synthesis and evolution (RISE), is an important step toward using ribosomes beyond their natural capabilities, according to the scientists, who add that the key feature of RISE is the ability to evolve ribosomes without cell viability constraints. The result, they note, could be new ways to synthesize materials, like nylon, or therapies like new antibiotics that could address rising antibiotic resistance. The research study, “In vitro ribosome synthesis and evolution through ribosome display,” appears in Nature Communications.

“Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 107 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 103 rRNA variants,” write the investigators.

“We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.”

“Ribosomes have an extraordinary capability as the protein synthesis machinery of the cell,” said Michael Jewett, PhD, Walter P. Murphy Professor of Chemical and Biological Engineering and director of the Center for Synthetic Biology at Northwestern’s McCormick School of Engineering, who led the research. “But to synthesize proteins beyond those found in nature, we have to design and modify the ribosome to work with non-natural substrates. Developing ribosomes in vitro is an important part of that system, and we are very excited to have this new capability.”

Jewett and his group developed the new RISE system to overcome those cell viability constraints and ultimately repurpose the ribosome in ways that have never been possible before. By building DNA that encodes for ribosome mutants, the system can make hundreds of thousands of mutant ribosomes within hours. Using magnetic beads, researchers can then select ribosomes with functions that they want. This platform sets the stage to understand the fundamental constraints of the ribosome’s active site and create new biopolymers that could transform society. Additionally, the method could potentially be used to manufacture new materials to improve soldier and police protection.

“We validated the RISE method by selecting highly active ribosomes that are resistant to the antibiotic clindamycin from a library of variants,” Jewett said. “Our hope is that others will be able to use this platform to select for ribosomes that can carry out a new function.”

With the ability to evolve ribosomes at hand, Jewett’s team has separately been trying to understand which parts of the ribosome are amenable to change. Jewett and his team have also mapped out the nucleotides of the active site of the ribosome to find out which nucleotides could be changed without breaking the ribosome. By building and testing every possible single nucleotide mutation in the active site, 180 in total, the researchers were surprised to find that 85 percent of these nucleotides possessed some flexibility and could be altered.

“It proves to us that you can change almost every nucleotide in the active site and still get a functional ribosome. This is so exciting for synthetic biology,” Jewett said.

Last year, the researchers also published a paper in which they developed a set of design rules that guide how ribosomes can incorporate new kinds of monomers not found in nature. Together, the group’s collection of papers provides a comprehensive platform for transforming the ribosome into a machine that can create new kinds of therapeutics and materials, explained Jewett.

“Right now, the ribosome is a chef that can only make certain meals,” he said. “We want to create many chefs that can make many different cuisines. This is a huge step forward toward that vision.”

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