Evaluation of 3',4'-cyclopropyl monomers for oligonucleotide therapeutic applications

Modern oligonucleotide based therapeutics rely on chemical modification to achieve the desired potency, stability and safety for therapeutic applications. In this study, we investigated the biophysical characteristics and potency of the 3',4'-cyclopropyl (Cp) uridine monomer and its 5'-vinyl phosphonate (VP) and ethyl phosphonate (EP) analogues. We incorporated these modifications into 9-mer strands, antisense oligonucleotides (ASOs), and small interfering RNAs (siRNAs) to identify position-dependent trends. Incorporation of these specific chemical modifications into 9-mer oligonucleotides resulted in a narrow range of thermal effects, from marginal stabilization to mild destabilization per modification. These modifications, when incorporated into ASOs, resulted in significant destabilizing effects, particularly when placed in the wings of the gapmer and as the number of modifications increased. However, these modifications caused relatively less destabilization in siRNAs compared to ASOs. Parallel in vitro assessments revealed that Cp modifications slightly improved the potency of ASO constructs but yielded no increase for siRNA molecules. In vitro snake venom phosphodiesterase (SVPD) assays demonstrated that Cp-modified ASOs exhibit significantly enhanced nuclease resistance over the unmodified parent sequence, sometimes providing near-complete protection. This differential effect on stability and potency across the different oligonucleotide lengths and modalities warrants further investigation to fully delineate the mechanism of action of these modifications and to further optimize their application in therapeutic oligonucleotide design. Here, based on these results, we propose Cp monomers as a novel class of molecules for oligonucleotide therapeutic applications.