Systematic Evaluation of Position-Specific Tolerability of Seven Backbone and Ribose Modifications in Fully Chemically Stabilized siRNAs

Chemically modified short interfering RNAs (siRNAs) unequivocally represent a groundbreaking class of drugs. The deliberate chemical modification of the natural structure has been pivotal to their resounding success. Specific modifications at certain positions bolster their potency, safety, stability, and specificity. In clinical research, 2'-<i>O</i>-methyl and 2'-fluoro are the most used modifications. The effects of a wide range of chemical changes in fully modified siRNAs have not been thoroughly evaluated for tolerability. In this study, we utilized two sequences in a fully modified siRNA to systematically assess the tolerability of single nucleotide backbone and sugar modifications, including deoxyribonucleic acid, 2'-<i>O</i>-(2-methoxyethyl), locked nucleic acid, unlocked nucleic acid, mismatches, butane diol substitution, and butane diol insertion. We synthesized 522 siRNA variants and evaluated their efficacy <i>in vitro</i>. Our findings demonstrate that individual tolerability is significantly influenced by the modification's sequence, pattern, and position, with limited universal principles identifiable from this dataset. The efficacy results are probably driven by the thermodynamic balance defined by a combination of parameters. The framework presented here will serve as a reference dataset to facilitate the expansion of chemical diversity in therapeutic siRNAs.