Synthesis and Properties of Nucleobase-Sugar Dual Modified Nucleic Acids: 2′-OMe-RNA and scpBNA Bearing a Nucleobase

Naturally occurring 5-hydroxycytosine (5-OHCyt), which is associated with DNA damage, was recently found to reduce the hepatotoxicity of antisense oligonucleotides (ASOs) without compromising its antisense activity when used as a replacement for cytosine (Cyt). Additionally, sugar modified nucleic acids, such as 2 '-O-methylribonucleic acid (2 '-OMe-RNA) and 2 '-O,4 '-C-spirocyclopropylene-bridged nucleic acid (scpBNA), have emerged as useful antisense materials. Herein, we aimed to combine these two advantages by designing dual modified nucleic acids 2 '-OMe-RNA-5-OHCyt and scpBNA-5-OHCyt bearing the 5-OHCyt nucleobase to develop efficient and safe ASOs. We describe the synthesis of 2 '-OMe-RNA-5-OHCyt and scpBNA-5-OHCyt phosphoramidites and their incorporation into oligonucleotides (ONs). The duplex-forming ability and base discrimination properties of 2 '-OMe-RNA-5-OHCyt-and scpBNA-5-OHCyt-modified ONs were similar to those of 2 '-OMe-RNA-Cyt-and scpBNA-mCyt-modified ONs, respectively. We also synthesized two 2 '-OMe-RNA-5-OHCyt-modified ASOs, and one of the two was found to exhibit reduced hepatotoxicity while retaining target mRNA knockdown activity in in vivo experiments.

Automated summary

Naturally occurring 5-hydroxycytosine (5-OHCyt) has been found to reduce the hepatotoxicity of antisense oligonucleotides (ASOs) while maintaining its antisense activity. Sugar modified nucleic acids, such as 2'-O-methylribonucleic acid (2'-OMe-RNA) and 2'-O,4'-C-spirocyclopropylene-bridged nucleic acid (scpBNA), are useful antisense materials. In this study, dual modified nucleic acids 2'-OMe-RNA-5-OHCyt and scpBNA-5-OHCyt were designed and synthesized, and they showed similar duplex-forming ability and base discrimination properties as 2'-OMe-RNA-Cyt and scpBNA-mCyt modified oligonucleotides.