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 the presence of the 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. The duplexes were designed to contain seventeen base pairs with the sequence 5'- GUUGACUU*UUAAUCAAC-3'/3'-CAACUGA(A/G)AAUUAGUUG-5', where U* denotes U, S²U, Se²U or their mnm⁵-substituted analogs. We found that chalcogens modulate the stability of duplexes with 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. The base pairing specificity for A over G was found to follow the order: uridines < Se²-uridines < S²-uridines. The degenerate behaviour of mnm⁵-uridines toward A and G was observed manifested by their tendency to reduce the base pairing discrimination between purine nucleosides. All studied RNA duplexes exhibited circular dichroism (CD) spectra characteristic of A-RNA double stranded helices. In addition, the first chemical synthesis of an mnm⁵Se²U-modified RNA oligomer is reported.