Molecular Recognition in Purine-Rich Internal Loops:  Thermodynamic, Structural, and Dynamic Consequences of Purine for Adenine Substitutions in 5‘(rGGCAAGCCU)₂^†,‡

The contribution of amino groups to the thermodynamics, structure, and dynamics of tandem A·A mismatches is investigated by substitution of purine (P) for adenine (A) within the RNA duplex, 5‘(rGGCAAGCCU)₂, to give 5‘(rGGCPAGCCU)₂, 5‘(rGGCAPGCCU)₂, and 5‘(rGGCPPGCCU)₂. The 5‘(rGGCAAGCCU)₂ duplex has sheared A_anti·A_anti (A·A trans Hoogsteen/Sugar-edge) pairs in which the A5 amino group is involved in hydrogen bonds but the A4 amino group is not [Znosko, B. M., Burkard, M. E., Schroeder, S. J., Krugh, T. R., and Turner, D. H. (2002) Biochemistry 41, 14969−14977]. In comparison to 5‘(rGGCAAGCCU)₂, replacing the amino group of A4 with a hydrogen stabilizes the duplex by 1.3 kcal/mol, replacement of the A5 amino group destabilizes the duplex by 0.6 kcal/mol, and replacement of both A4 and A5 amino groups destabilizes the duplex by 0.8 kcal/mol. In NMR structures, the P·A noncanonical pairs of the 5‘(rGGCPAGCCU)₂ duplex have a sheared anti−anti structure (P·A trans Hoogsteen/Sugar-edge) with P4·A5 interstrand hydrogen bonding and A5 bases that interstrand stack, similar to the structure of 5‘(rGGCAAGCCU)₂. In contrast, the A·P pairs of the 5‘(rGGCAPGCCU)₂ duplex have a face-to-face conformation (A·P trans Watson−Crick/Watson−Crick) with intrastrand stacking resembling typical A-form geometry. Although the P5 bases in 5‘(rGGCPPGCCU)₂ are involved in an interstrand stack, the loop region is largely undefined. The results illustrate that both hydrogen-bonded and non-hydrogen-bonded amino groups play important roles in determining the thermodynamic, structural, and dynamic characteristics of purine rich internal loops.