A new paper published in Nature Biotechnology this week titled, “Template-independent enzymatic synthesis of RNA oligonucleotides,” provides details of a method for synthesizing single-stranded RNA that was developed by scientists at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School (HMS). The technology, which is being commercialized by EnPlusOne Biosciences, produces single-stranded RNA with efficiencies and purities that are comparable to traditional chemical synthesis using water and enzymes, and without using a template sequence.
Following their success against COVID-19 infections, interest in RNA-based therapeutics is growing rapidly. And as demand for RNA drugs continues to grow and “additional products come to market, we will exceed the current global supply of acetonitrile, the organic solvent used in chemical RNA synthesis methods,” said co-first author Jonathan Rittichier, PhD, a former postdoctoral fellow at the Wyss and HMS.
Besides reducing the creation of toxic synthesis byproducts, the technology that Rittichier and his collaborators, which includes George Church, PhD, Wyss core faculty member and HMS professor of genetics, have developed incorporates common molecular modifications found in RNA drugs today and can be used with novel RNA chemistries to develop new therapies. “Delivering RNA drugs to the world at these scales requires a paradigm shift to a renewable, aqueous synthesis, and we believe our proprietary enzymatic technology will enable that shift,” Rittichier said.
The starting point for the technology is an enzyme from Schizosaccharomyces pombe, a strain of yeast, that is used to form RNA strands. The scientists engineered a more efficient version of the enzyme that can also incorporate nonstandard nucleotides into RNA—a necessary improvement since every FDA-approved RNA drug includes modified nucleotides that increase their stability in the body or equip them with new functions. Their approach also uses milder methods to remove protecting groups which are added to nucleotides to protect them during the synthesis process.
Furthermore, the researchers modified the nucleotides by attaching a chemical group called a “blocker” that ensures that nucleotides are added to the RNA chain one at a time. Once the desired nucleotide has been added to the link, the blocker is removed to allow the next nucleotide in the sequence to bind. This two-step process is simpler and uses fewer reagents than the typical four-step chemical synthesis. According to the scientists, their process is also 95% efficient and can build RNA molecules as long as 23 nucleotides. However, they noted that “overall yields in bulk phase were lower than those in standard RNA oligonucleotide synthesis” likely due to “multiple rounds of purification after both extension and deblocking.”
“Enzymatic nucleotide synthesis technologies offer many advantages as an alternative to chemical-based methods,” Church said in a statement. “This platform can help unlock the immense potential of RNA therapeutics in a sustainable way, especially manufacturing high-quality guide RNA molecules for CRISPR/Cas gene editing.”
Rittichier, Church, and others launched EnPlusOne Biosciences in 2022 to commercialize the technology with funding from Northpond Ventures, Breakout Ventures, and Coatue. The company is currently using the technology to manufacture small interfering RNAs that could be used to treat various diseases.
