Template-independent enzymatic synthesis of RNA oligonucleotides

Abstract

RNA oligonucleotides have emerged as a powerful therapeutic modality to treat disease, yet current manufacturing methods may not be able to deliver on anticipated future demand. Here, we report the development and optimization of an aqueous-based, template-independent enzymatic RNA oligonucleotide synthesis platform as an alternative to traditional chemical methods. The enzymatic synthesis of RNA oligonucleotides is made possible by controlled incorporation of reversible terminator nucleotides with a common 3'-O-allyl ether blocking group using new CID1 poly(U) polymerase mutant variants. We achieved an average coupling efficiency of 95% and demonstrated ten full cycles of liquid phase synthesis to produce natural and therapeutically relevant modified sequences. We then qualitatively assessed the platform on a solid phase, performing enzymatic synthesis of several N + 5 oligonucleotides on a controlled-pore glass support. Adoption of an aqueous-based process will offer key advantages including the reduction of solvent use and sustainable therapeutic oligonucleotide manufacturing.

Fig. 1. General overview of the controlled, template-independent enzymatic RNA oligonucleotide synthesis process.

a, Three primary components are required for carrying out an enzymatic extension: 3′-blocked reversible terminator nucleotides, enzymes capable of their robust and indiscriminate incorporation, and an initiator oligonucleotide. The reversible terminator group stops uncontrolled polymerization by the enzyme and limits extension to a single incorporation event. The initiator oligonucleotide may vary in terms of sequence and length. It can also be bound to a solid support or feature other modifications such as a 5′-fluorophore or functional handle. b, A typical cycle of enzymatic synthesis begins with (1) extension of the initiator oligonucleotide in the presence of an RT-NTP and enzyme. A deblocking step (2) then occurs to remove the reversible terminator group from the extended oligonucleotide, allowing the next cycle of synthesis to commence. When the desired length and composition have been reached, the final oligonucleotide product is isolated. Source data

Fig. 2. Preparation of 3′-O-allyl ether ATP and UTP.

a, Preparation of 3′-O-allyl ether ATP. b, Preparation of 3′-O-allyl ether UTP.

Fig. 3. Preparation of 3′-O-allyl ether GTP and CTP.

a, Preparation of 3′-O-allyl ether GTP. b, Preparation of 3′-O-allyl ether CTP.

Fig. 4. Initial evaluation of 3′-O-allyl ether RT-NTPs as building blocks for controlled, enzymatic RNA oligonucleotide synthesis.

a, A complete set (A, U, G, C) of 3′-O-allyl ether NTPs were tested for enzymatic incorporation and deblocking using a liquid bulk phase reaction scheme, where N is the length of the initiator, N + 1* is the extension intermediate with the 3′-O-allyl ether group as represented by the asterisk, and N + 1 is the deblocked product for each base. b, MALDI-TOF mass spectrometry was used to verify NTP extension to N + 1* by the poly(U) mutant variant and subsequent deblocking of the allyl ether group to N + 1; the masses of all resultant oligonucleotides are given and compared with that of the 19-nt initiator. c, Kinetic profile for each 3′-O-allyl ether NTP, obtained and analyzed with denaturing gel electrophoresis; reaction samples were taken at 1, 5, 10, 20 and 30 min. Control reactions (N) included all reaction components except NTP. This direct comparison was performed once but is a compilation of several independent experimental repeats with similar results. d, MALDI-TOF was used to assess the efficiency of two controlled, enzymatic synthesis cycles in which all N + 2* combinations of base extensions were produced; the masses of all resultant oligonucleotides are given and compared with that of the 19-nt initiator. The observed and calculated m/z values for all oligonucleotide synthesis products generated by MALDI-TOF analysis, as well as their respective theoretical molecular weights, are summarized in Supplementary Table 1. Phos., triphenylphosphine; intens., intensity. Source data

Fig. 5. Results of multicycle enzymatic synthesis to produce natural RNA oligonucleotides using the 3′-O-allyl ether NTP set.

a, An N + 5 RNA oligonucleotide with the sequence N + U-U-U-C-G* was produced in the liquid bulk phase, where the asterisk represents a 3′-O-allyl ether group. b, MALDI-TOF mass spectrometry was used to track the outcome of the extension and deblocking steps during each cycle of enzymatic synthesis. c, The isolated purity of the growing oligonucleotide and final product was determined after each cycle using LC/MS at 260 nm and 649 nm; the results are summarized in the table. d, An N + 10 RNA oligonucleotide with the sequence N + A-C-A-C-C-U-U-A-A-C* was also produced in the liquid bulk phase. e, High-resolution gel electrophoresis was used to analyze the success of each cycle after the sequence had been enzymatically synthesized with an imager set to collect the Cy5 signal. This analysis was conducted once. f, The final N + 10* oligonucleotide product was also assessed with MALDI-TOF and summarized along with any major impurities detected. Further data are given in Supplementary Table 2 regarding the observed and calculated m/z values for all oligonucleotide synthesis products and impurities generated by MALDI-TOF analysis, as well as their respective theoretical molecular weights. Obs., observed.

Fig. 6. Compatibility summary of modified 3′-O-allyl ether and PS 3′-O-azido methyl ether RT-NTP sets and results of multicycle synthesis to produce a fully modified RNA oligonucleotide.

a, Modified 3′-O-allyl ether and PS 3′-O-azido methyl ether RT-NTPs were evaluated by performing an initial N + 1* extension, a deblocking reaction and, if possible, an N + 2* extension. A green checkmark indicates a successful reaction, and a red cross-out indicates an unsuccessful reaction. Reactions that were not attempted are indicated by a yellow bar. Each individual cycle step was evaluated using MALDI-TOF mass spectrometry (Supplementary Figs. 11–13). b, MALDI-TOF assessment of enzymatic extension reactions using a set of 3′-O-propargyl ether NTPs (A, U, G, C) to install a functional handle onto oligonucleotides. c, A fully modified N + 10 oligonucleotide with the sequence N + A_f-A_f-C_m-C_m-U_f-U_f-C_m-U_f-A_p was synthesized using modified RT-NTPs, where f is 2′-fluoro, m is 2′-methoxy and p is 3′-O-propargyl. d, MALDI-TOF mass spectrometry was used to verify extension using the modified RT-NTPs during each cycle of enzymatic synthesis. e, The expected oligonucleotide sequences and their calculated and observed m/z values from MALDI-TOF analysis are summarized in the table. Further data are provided in Supplementary Table 2 regarding the observed and calculated m/z values for all oligonucleotide synthesis products and impurities generated by MALDI-TOF analysis, as well as their respective theoretical molecular weights. Calc., calculated.

Fig. 7. Overview and demonstration of a solid support system for controlled enzymatic RNA oligonucleotide synthesis.

a, Outlined of a general scheme: an initiator oligonucleotide (black) is bound to LCAA-CPG with a Bis(NHS)PEG5 linker (pink) using NHS conjugation chemistry. The initiator harbors a deoxy- or riboinosine base (red) for recognition by E. coli endonuclease V, which cleaves the desired oligonucleotide product (blue) immediately downstream of the inosine base. The oligonucleotide product can then be isolated from the CPG solid support. b, To demonstrate the viability of the CPG solid support, 5× N + 5 oligonucleotides were enzymatically synthesized in a stir reactor format. Their sequences comprised natural and modified bases, with one partially modified with 2′-fluoro groups and another fully modified with both 2′-fluoro and 2′-methoxy groups. c, MALDI-TOF mass spectrometry was used to evaluate the oligonucleotide material cleaved from the solid support. d, Summary of enzymatic synthesis, including a high-level description of the major and minor products found. Further data are provided in Supplementary Table 3 regarding the observed and calculated m/z values for all oligonucleotide synthesis products and impurities generated by MALDI-TOF analysis, as well as their respective theoretical molecular weights. Seq., sequence; oligo., oligonucleotide.