Versatile and inexpensive enzymatic RNA oligonucleotide synthesis approach with the potential to greatly facilitate the development of novel RNA therapeutics
Synthetic RNA oligonucleotides designed as specific successions of the four nucleobases A, U, G, and C that mimic naturally occurring RNA species are the key components of diverse RNA-based therapies. These include RNA therapeutics that can partially or completely turn off the expression of disease-causing genes (antisense and interfering RNAs), help replace or supplement dysfunctional or insufficiently produced proteins (mRNAs), are developed as vaccines for cancer and infectious diseases, or either deliver sensitive therapeutic payloads or function as chemical drugs themselves by directly binding and inhibiting a target molecule (RNA aptamers). In addition, RNA oligonucleotides are vastly deployed in genome editing technologies.
Despite the rapidly growing market and increasing demand for RNA oligonucleotides, their production still primarily relies on antiquated chemical synthesis methods. Current methods often yield suboptimal amounts and purities of the final product, and are limited by sequence length and the ability to efficiently incorporate non-standard nucleobases common to many RNA therapeutics. In addition, current methods use large quantities of environmentally harmful solvents as well as lengthy purification steps that drastically increase the overall cost of RNA oligonucleotide synthesis, especially at the scales needed for wide distribution of high-quality therapeutics to patients. Furthermore, these limitations also prevent researchers from creating novel RNA-based therapeutics in the first place.
A research team in the Wyss Institute’s Synthetic Biology platform lead by George Church, Ph.D., is developing a new enzymatic-based, template-independent RNA oligonucleotide synthesis technology to address the current limitations of traditional chemical synthesis. Their scalable, flexible, and cost-efficient synthesis method uses a set of proprietary engineered enzymes and novel nucleotide building blocks to produce accurate, high quality RNA oligonucleotide sequences comprised of natural and non-natural bases at efficiencies that diminish the need for post-reaction purification. In addition to providing a substantially “greener” approach to oligonucleotide synthesis via aqueous reaction conditions, this technology could potentially enable the synthesis of highly customized and significantly longer RNA oligonucleotides. Current chemical synthesis techniques can synthesize RNA oligonucleotides up to 120 nucleotides, but for extreme costs and at small scales.
With a newly developed blocking strategy that allows the synthesis system to tightly control nucleotide additions at each step of the growing RNA oligonucleotide chain, the overall accuracy of synthesis is greatly increased – conventional methods are prone to the accumulation of premature truncation products, nucleobase depurination, and insertions/deletions. Importantly, this level of control also enables the introduction of modified oligonucleotides – many of which thus far have been inaccessible as building blocks for RNA therapeutics – at defined positions. In contrast, existing some RNA synthesis methods introduce a modified nucleotide, like for example a modified A nucleotide at all A-specific positions of the growing RNA oligonucleotide sequence. Modified nucleotide building blocks can stabilize RNA oligonucleotide products, uniquely endow RNA oligonucleotides with functional properties, and facilitate delivery to their molecular targets within the body’s tissues.