Surprising Sequence Effects on GU Closure of Symmetric 2 × 2 Nucleotide RNA Internal Loops

Abstract

GU base pairs are important RNA structural motifs and often close loops. Accurate prediction of RNA structures relies upon understanding the interactions determining structure. The thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs are not well understood. Here, several self-complementary oligonucleotide sequences expected to form duplexes with 2 × 2 nucleotide internal loops closed by GU pairs were investigated. Surprisingly, nuclear magnetic resonance revealed that many of the sequences exist in equilibrium between hairpin and duplex conformations. This equilibrium is not observed with loops closed by Watson-Crick pairs. To measure the thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs, non-self-complementary sequences that preclude formation of hairpins were designed. The measured thermodynamics indicate that some internal loops closed by GU pairs are unusually unstable. This instability accounts for the observed equilibria between duplex and hairpin conformations. Moreover, it suggests that future three-dimensional structures of loops closed by GU pairs may reveal interactions that unexpectedly destabilize folding.

Figure 1

Plot of 1/T_M vs log C_T for 5′CGGGCAUCCG at 1 M Na⁺. Error bars are ±1 °C. The linear fit (shown with the line) to oligonucleotide concentrations between 12.5 and 140 μM gave values for ΔH°, ΔS°, and ΔG°₃₇ of –38.0 kcal/mol, –100.9 eu, and –6.74 kcal/mol, respectively. Values are similar to those reported previously: –39.5 kcal/mol, –106.3 eu, and –6.49 kcal/mol, respectively.

Figure 2

Imino proton NMR spectra of 1 mM and 0.02 mM 5′CGGGCAUCCG in 0.1 M Na⁺ at 5 °C. The additional peaks in the low concentration spectrum are likely due to a small amount of hairpin.

Figure 3

Plots of 1/T_M vs log C_T at 1 M Na⁺ for 5′CGGGCUUCCG (blue), 5′GGCGAAUGCC (green), 5′GCGUGCUUUGCG/3′CGCAUUCGACGC (orange), and 5′GCUGAAUACG/3′CGAUAAGUGC (black). Error bars are ±1 °C.

Figure 4

Imino proton NMR spectra in 0.1 M Na⁺ for: (a) 5′CGGGCUUCCG; a 2D spectrum contains an exchange peak for G4 at 11.04/13.49 ppm, revealing two conformations in slow exchange, (b) 5′GGCGAAUGCC, (c) 5′CCUGUCUAGG; two resonances are overlapped at 13.3 ppm, (d) 5′GUCGCCUGAC. A 2D spectrum for sequence d reveals that the peak around 12.3 ppm contains two resonances for G8. In general, red and blue resonance assignments refer to hairpins and duplexes, respectively, as determined from 2D spectra.

Figure 5

Imino proton NMR spectra for 0.1 mM RNA in 1 M NaCl at 5 °C for duplexes with AU flanked 2×2 nt loops. (a) (5′CGGACAUCCG)₂, (b) (5′GAGACUUCUC)₂, and (c) (5′GCAUCUGC)₂.

Figure 6

Imino proton NMR spectra at 0.1 M Na⁺ and ~ 1 mM duplex for: (a) 5′GCGUGCUUUGCG/3′CGCAUUCGACGC, (b) 5′GCGUGUCUUGCG/3′CGCAUCUGACGC, (c) 5′GCGUGAAUAGCG/3′CGCAUAAGUCGC, (d) 5′GCGUGACUAGCG/3′CGCAUCAGUCGC, (e) 5′GCGUUUCGUGCG/3′CGCAGCUUACGC, and f) 5′GCGUGCCUUGCG/3′CGCAUCCGACGC.