STABILITIES OF CONSECUTIVE A.C, C.C, G.G, U.C, AND U.U MISMATCHES IN RNA INTERNAL LOOPS - EVIDENCE FOR STABLE HYDROGEN-BONDED U.U AND C.C+ PAIRS

The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA almost-equal-to UU > GG greater-than-or-equal-to CA greater-than-or-equal-to AA = CU = UC greater-than-or-equal-to CC greater-than-or-equal-to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a T(M) of 44.8-degrees-C at 1 X 10(-4) M and a DELTA-G-degrees-37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a T(M) of 33.2-degrees-C and DELTA-G-degrees-37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the T(M) increases by 12-degrees-C, and DELTA-G-degrees-37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with T(M) increases of 5 and 3-degrees-C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with T(M) increases of 0.8 and 1.4-degrees-C, respectively, upon lowering the pH to 5.5. CD spectra of sequences with CC, CU, UC, and UU internal loops are typical of A-form conformation. CD spectra of AC, CA, and GG have a positive band at 280 nm, similar to that observed for GA and AA internal loops (SantaLucia et al., 1990). CD spectra of all sequences studied, except rCGCCCGGCG, are independent of pH. For rCGCCCGCG, a weak negative band at 300 nm is observed at pH 7, but at pH 5.5 a weak positive band is observed. Current algorithms for the prediction of RNA secondary structure assume that the stability of internal loops is not sequence-dependent or depends only on stacking. This study indicates these approximations are wrong. The measured internal loop free energy increments range from -0.6 to +2.3 kcal/mol, and do not correlate with known stacking parameters.