Molecular Recognition in Purine-Rich Internal Loops: Thermodynamic, Structural, and Dynamic Consequences of Purine for Adenine Substitutions in 5'(rGGCAAGCCU)_2

摘要

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 teh 5'(rGGCPAGCCU)_2 duplex have a sheared anti-anti structure (P·A trans Hoogsteen/Sugar-edge) with P4·P5 interstand 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 interstand 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.