The growing demand for nucleic acid drugs and vaccines highlights the urgent need for improved technologies to produce long DNA and RNA sequences with Chemical Modifications. Unlike DNA, surprisingly little effort has been devoted to making therapeutic RNA Oligonucleotides longer than the short sequences (20-80 nt) used for small interfering RNA (siRNA) and microRNA (miRNA).
In view of this, George M. Church, Erkin Kuru et al. from Harvard Medical School report an alternative, reliable method for the Enzymatic Synthesis of template-independent RNA oligonucleotides, both natural and with therapeutically relevant modifications. Using mutant polymerases, the researchers demonstrated controlled enzymatic synthesis of chemically modified RNA oligonucleotides up to 10 nt. This strategy has the potential to surpass existing methods and enable sustainable production of long (>200 nt) RNA sequences with a wide range of chemical modifications at discrete, user-defined locations.
RNA holds great promise in biomedicine as a means of delivering Vaccines and therapeutic proteins, as well as regulating gene expression and splicing. Many of these applications require chemical modifications. Without such modifications, RNA oligonucleotides cannot readily penetrate cells, are susceptible to rapid nuclease-mediated degradation and renal filtration, and have low target affinity and delivery specificity. More than 170 naturally occurring chemical modifications have been identified in the four RNA forms, providing a layer of transcriptional regulation and structural complexity. Despite steady progress in identifying and synthesizing these modified RNA Building Blocks, determining their exact chemical nature and functional roles, both for known structures and, more importantly, for unknown structures, remains challenging. The field of modified RNA would benefit greatly from new synthetic methods that allow the production of longer (>200 nt) sequences and the introduction of multiple chemical modifications at specific positions.
| Cat# | Product Name | Purity | Inquiry |
| X22-09-ZQ071 | Adenosine 5'-triphosphate (ATP) sodium salt solution, 100 mM | >98% | Inquiry |
| X22-09-ZQ072 | Guanosine 5′-triphosphate (GTP) sodium salt solution, 100 mM | >98% | Inquiry |
| X22-09-ZQ073 | Cytidine-5'-triphosphate (CTP) sodium salt solution, 100 mM | >98% | Inquiry |
| X22-09-ZQ074 | Uridine 5'-triphosphate (UTP) sodium salt solution, 100 mM | >98% | Inquiry |
| X22-09-ZQ075 | Adenosine 5'-triphosphate (ATP) tris solution, 100 mM | >98% | Inquiry |
| X22-09-ZQ076 | Guanosine 5′-triphosphate (GTP) tris solution, 100 mM | >98% | Inquiry |
| X22-09-ZQ077 | Cytidine-5'-triphosphate (CTP) tris solution, 100 mM | >98% | Inquiry |
| X22-09-ZQ078 | Uridine 5'-triphosphate (UTP) tris solution, 100 mM | >98% | Inquiry |
| X22-09-ZQ079 | 5-Me-CTP solution, 100 mM | >98% | Inquiry |
| X22-09-ZQ080 | 5-O-Me-UTP solution, 100 mM | >98% | Inquiry |
| View Other Nucleotide Products | |||
There are two main methods for producing RNA oligonucleotides: chemical synthesis and in vitro transcription using RNA polymerase. Chemical synthesis relies on the sequential assembly of appropriately protected phosphoramidite building blocks on a solid support. Even at an industrial scale, it can easily generate short RNA sequences up to about 100 nucleotides long containing a variety of chemical modifications. However, longer sequences cannot be obtained because the coupling yield decreases rapidly with increasing length, and the rather harsh synthesis conditions coupled with Acetylation errors easily lead to single-base deletions or indels. Chemical synthesis also produces a large amount of hazardous waste, making large-scale production unsustainable.
Alternatively, in vitro transcription uses RNA polymerases (e.g., T7, SP6, and T3) to catalyze the polymerization of nucleotides based on a DNA template. Chemically modified nucleotides can be incorporated as long as they are tolerated by the polymerase. The efficacy of this approach has been fully demonstrated in the industrial-scale production of mRNA vaccines, which are long (>4,000 nt) oligonucleotides containing one modified nucleotide, primarily N1-methylpseudouridine. Despite its attractive properties, in vitro transcription offers limited flexibility with respect to chemical modification, as not all RNA polymerases accept all nucleotide analogs as substrates, and there is little control over the positioning of the modified residues.
Controlled enzymatic synthesis of oligonucleotides is an alternative de novo approach that merges Solid-phase Chemical Synthesis and polymerase-based incorporation of nucleoside triphosphates. In this strategy, temporarily protected nucleotides are incorporated into primers, primarily by template-independent polymerases. After removal of excess reagents, the protected moieties are unblocked and additional cycles of synthesis can be initiated. Enzymatic synthesis is particularly well developed for DNA. In fact, benchtop synthesizers based on this technology have been commercialized and have recently broken through the kilobase limit. In contrast, controlled enzymatic synthesis of chemically modified RNAs has completely eluded researchers, and the first examples are only beginning to be reported. This discrepancy may stem from the easier synthesis and enhanced stability of DNA compared to RNA building blocks, coupled with intensive research activity on the design of more efficient DNA polymerase variants.
The study describes a significant advance in the template-independent enzymatic synthesis of natural and modified RNA oligonucleotides. To this end, the authors engineered the poly(U) polymerase (PUP) from yeast to tolerate and efficiently incorporate nucleotides blocked at the 3' position with an allylic functional group, without the use of a template. This modification has previously been found on DNA nucleotides in the context of DNA sequencing synthesis. The mutant PUP readily incorporated all four blocked nucleotides within minutes with coupling efficiencies of more than 95%. After primer extension, unblocking was achieved within 10-15 minutes by applying a mild palladium-based deallylation reaction. Impressively, up to 10 consecutive cycles of controlled enzymatic RNA synthesis could be performed in solution. The method is also compatible with solid-phase synthesis, where the extension products can be easily removed by endonuclease cleavage of the inosine moiety included in the primer sequence. Furthermore, the process does not involve any organic solvents and reagents, thus reducing the environmental impact.
Interestingly, nucleotides with therapeutically relevant modifications are also excellent substrates for PUPs. These include oligonucleotides with 2' modifications such as 2'-fluoro and 2'-methoxy, as well as nucleotides that introduce terminal permanent blocking moieties such as 3'-O-propargyl modifications, functional handles that may facilitate further RNA modification via Click Chemistry. However, due to the sensitivity of α-phosphorothioate modifications to palladium-mediated deprotection steps, blocked nucleotides must be replaced with analogs equipped with a 3'-O-azidomethyl moiety.
Fig. 1 Comparison of natural and modified RNA oligonucleotide synthesis methods. Left, phosphoramidite-based chemical synthesis can generate RNA sequences up to ~100 nucleotides with a variety of modifications. Middle, in vitro transcription using RNA polymerase can obtain long (>4,000-nt) RNA sequences with limited modifications. Right, controlled enzymatic RNA synthesis of up to 10 cycles can generate natural and therapeutically relevant RNA oligonucleotides with the potential to be extended to long sequences. (Hollenstein, 2024)
Although this study has clearly demonstrated that controlled enzymatic synthesis is a very promising approach for the de novo production of RNA oligonucleotides, several issues need to be addressed before this approach can be widely adopted. These drawbacks include lower coupling yields than solid-phase synthesis, relatively long deblocking times, the need for alternative blocking groups in the presence of phosphorothioate modifications, and the potential for the appearance of side products such as 2′,5′-branched RNAs. These limitations could undoubtedly be overcome by evaluating alternative blocking groups for the nucleotides, adopting multiplexed synthesis, or replacing the solid support system with Microarrays or microfluidic devices. Further engineering of PUPs could also yield more efficient and processive Enzyme variants, thereby increasing coupling efficiency to the level of controlled enzymatic DNA synthesis or solid-phase synthesis.
This study represents an important milestone in the development of alternative biocatalytic strategies for the production of RNA oligonucleotides and modified nucleic acids, and has important implications for many practical applications. With this and related emerging methods, sustainable production of RNA oligonucleotides, including longer sequences modified at specific positions, is becoming a reality.
Reference
