Chemical Diversity of Locked Nucleic Acid-Modified Antisense Oligonucleotides Allows Optimization of Pharmaceutical Properties

The identification of molecules that can modulate RNA or protein function and the subsequent chemical and structural optimization to refine such molecules into drugs is a key activity in drug discovery. Here, we explored the extent to which chemical and structural differences in antisense oligonucleotides, designed as gapmers and capable of recruiting RNase H for target RNA cleavage, can affect their functional properties. To facilitate structure-activity learning, we analyzed two sets of iso-sequential locked nucleic acid (LNA)-modified gapmers, where we systematically varied the number and positions of LNA modifications in the flanks. In total, we evaluated 768 different and architecturally diverse gapmers in HeLa cells for target knockdown activity and cytotoxic potential and found widespread differences in both of these properties. Binding affinity between gapmer and RNA target, as well as the presence of certain short sequence motifs in the gap region, can explain these differences, and we propose statistical and machine-learning models that can be used to predict region-specific, optimal LNA-modification architectures. Once accessible regions in the target of interest have been identified, our results show how to refine and optimize LNA gapmers with improved pharmacological profiles targeting such regions. [Display omitted]