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. 2022 May 31;7(23):20420-20427.
doi: 10.1021/acsomega.2c02742. eCollection 2022 Jun 14.

Optimization of Automated Synthesis of Amide-Linked RNA

Julien A Viel  1 , Chandan Pal  1 , Eriks Rozners  1
Affiliations

Optimization of Automated Synthesis of Amide-Linked RNA

Julien A Viel et al. ACS Omega. .

Abstract

The recent FDA approval of several antisense and siRNA drugs illustrates the utility of nucleic acid chemical modifications, but numerous challenges remain for generalized nucleic acid therapeutics, urging the exploration of new modification strategies. Replacing backbone phosphates with amides has shown promise for enhancing siRNA activity, specificity, and nuclease resistance; however, amide-linked RNA has not been fully explored due to lengthy and low yielding manual amide coupling procedures. We have addressed this by automating the assembly of amide-linked RNA using an Expedite 8909 nucleic acid synthesizer and optimizing solid-phase synthesis conditions to achieve 91-95% yields in just 5 min of coupling time. The optimized protocol allowed synthesis of a 21-nucleotide-long siRNA guide strand having six consecutive amide linkages at the 3'-end with an overall yield of ∼1%. Our results show that the steric hindrance caused by bulky 2'-O protecting groups and steric hindrance of the solid support are the key optimization variables for improving the amide couplings.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Structures of RNA, amide-linked RNA (AM1), and monomers for the preparation of AM1.
Scheme 1
Scheme 1. Structures and Synthesis of RNA Monomers for Functionalization of Solid Support (3) and Optimization of Amide Couplings (2b and 2d)
Figure 2
Figure 2
Sequence (AM1 amide linkages are highlighted in red) and RP-HPLC chromatograms of crude synthesis and purified (inset) siRNA G6. The major peak that contained G6 was isolated as shown by the blue lines. Conditions for all traces: Agilent Bio PLRP-S column (100 Å, 8 μm, 4.6 × 150 mm) at 65 °C, a gradient of acetonitrile in 50 mM triethylammonium acetate buffer, pH 7.2 (buffer A), at 1.0 mL/min. Buffer B was a 40:60 mixture of acetonitrile and buffer A. Gradient method: 0 min–10% B; 5 min–22% B; 25 min–32% B; 30 min–37% B. The slight difference in retention times of crude and purified G6 was likely caused by the presence of residual triethylammonium trihydrofluoride and other salts in the crude synthesis mixture.
See this image and copyright information in PMC

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