Synthesis and Properties of Nucleobase-Sugar Dual Modified Nucleic Acids: 2<b>′</b>‑OMe-RNA and scpBNA Bearing a 5‑Hydroxycytosine 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

Development and multicenter validation of an LC–MS-based bioanalytical method for antisense therapeutics: supplementary figures

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC–MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC–MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study.Mipomersen was measured by ion-pairing LC– MS (IP-LC–MS) as a model ASO using different LC–MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC–MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC–MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.</p