Although glycosidic bonds in purines typically involve the N9 position, the chemical synthesis of adenosine produces N7-ribofuranosyladenine (7A) as a kinetically favorable ribosylation product. Similarly, in the synthesis of LNA-adenosine (AL), a minor product, N7-LNA-adenosine (7AL), is observed. While extensive research has focused on investigating the properties of N9-regioisomers of adenosine, 7A has been largely overlooked and considered as a side-product. In this study, we conducted comprehensive experimental and computational investigations to elucidate the structural and thermodynamic properties of 7A and 7AL. Our results reveal that 7A and 7AL primarily enhance the thermodynamic stability of 1 × 1 mismatches when paired with purines but decrease stability when paired with pyrimidines. Utilizing nuclear magnetic resonance and computational techniques, we discovered that 1 × 1 7A:A and 7AL:A prefer anti-anti conformations, while 1 × 1 7A:G and 7AL:G prefer syn-anti orientations, both forming two hydrogen bond states, resulting in enhanced duplex stabilities. Altogether, these findings underscore the unique properties of 7A and 7AL when incorporated in RNA, which could advance structure-based RNA studies and potentially be utilized to modulate binding affinity, selectivity and biostability of RNA molecules.
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.