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Over a hundred non-canonical nucleotides have been found in DNA and RNA. Many of them are sensitive toward nucleophiles. Because known oligonucleotide synthesis technologies require nucleophilic conditions for deprotection, currently there is no suitable technology for their synthesis. The recently disclosed method regarding the use of 1,3-dithian-2-yl-methyl (Dim) for phosphate protection and 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) for amino protection can solve the problem. With Dim–Dmoc protection, oligodeoxynucleotide (ODN) deprotection can be achieved with NaIO 4 followed by aniline. Some sensitive groups have been determined to be stable under these conditions. Besides serving as a base, aniline also serves as a nucleophilic scavenger, which prevents deprotection side products from reacting with ODN. For this reason, excess aniline is needed. Here, we report the use of alkyl Dim (aDim) and alkyl Dmoc (aDmoc) for ODN synthesis. With aDim–aDmoc protection, deprotection is achieved with NaIO 4 followed by K 2 CO 3 . No nucleophilic scavenger such as aniline is needed. Over 10 ODNs including one containing the highly sensitive N 4 -acetylcytidine were synthesized. Work on extending the method for sensitive RNA synthesis is in progress.
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This content will become publicly available on November 22, 2025
Sensitive RNA Synthesis Using Fluoride-Cleavable Groups for Linking and Amino Protection
A chemical method suitable for the synthesis of RNAs containing modifications such as N4-acetylcytidine (ac4C) that are unstable under the basic and nucleophilic conditions used by standard RNA synthesis methods is described. The method uses the 4-((t-butyldimethylsilyl)oxy)-2-methoxybutanoyl (SoM) group for the protection of exo-amino groups of nucleobases and the 4-((t-butyldimethylsilyl)oxy)-2-((aminophosphaneyl)oxy)butanoyl (SoA) group as the linker for solid phase synthesis. RNA cleavage and amino deprotection are achieved using fluoride under the same conditions used for the removal of the 2′-OH silyl protecting groups. Using the method, a wide range of electrophilic and base-sensitive groups including those that play structural and regulatory roles in biological systems and those that are artificially designed for various purposes are expected to be able to be incorporated into any position of any RNA sequences. As a proof of concept, a 26-mer RNA containing the highly sensitive ac4C epitranscriptomic modification was successfully synthesized and purified with RP HPLC. MALDI MS analysis indicated that the ac4C modification is completely stable under the fluoride deprotection conditions. The sensitive RNA synthesis method is expected to be able to overcome the long lasting obstacle of accessing various modified sensitive RNAs to projects in areas such as epitranscriptomics, molecular biology and the development of nucleic acid therapeutics.
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- PAR ID:
- 10559357
- Publisher / Repository:
- RSC and ACS
- Date Published:
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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