From U to mnm⁵Se²U: tuning base pairing preferences through 2-chalcogen and 5-methylaminomethyl modifications

5-Substituted uridines, 2-thiouridines and 2-selenouridines represent the most common wobble-positioned bacterial tRNA modifications, with the 5-methylaminomethyl (mnm5) substituent being particularly widespread. Their biological role in the precise recognition of synonymous purine-ending codons is still under investigation. Modified uridines are also known to enhance the stability and base-pairing specificity of therapeutic nucleic acids. However, a full understanding of the O2/S2/Se2 chalcogen effect, particularly in combination with the naturally occurring mnm⁵ substituent, remains limited. To address this, a systematic comparative study was conducted on the thermodynamic and structural contributions of mnm⁵ and 2-chalcogen modifications to RNA duplex properties. We found that chalcogens modulate the stability of duplexes containing opposing adenosine in the following order: uridines < Se2-uridines < S2-uridines, with the mnm⁵ substituent exerting a significantly destabilizing effect. In duplexes with opposing guanosine, the influence of chalcogens is less pronounced, whether alone or in combination with mnm5, however, Se2-uridines promote duplex formation more effectively than their 2-thio and 2-oxo counterparts. This effect is likely associated with their high ionization propensity, as we demonstrated by pH-dependent melting studies. Overall, the base-pairing specificity for adenosine over guanosine was found to follow the order: uridines < Se2-uridines < S2-uridines, with the mnm⁵ group significantly reducing this specificity. All studied RNA duplexes exhibited circular dichroism (CD) spectra characteristic of A-RNA double stranded helices. To afford the above data, the first chemical synthesis of an mnm⁵Se²U-modified RNA oligomer was developed.