C C U G Gc Gc Au Au Gc Gc Gacc

Fig. 1.1.3. The identical sequence partition CGGAAGGUCC (gray) of the GGAA- and the UCCG reference (center) allows the design of two competing secondary structures within a single sequence (below).

(predicted)

equilibria of short RNA sequences. The NH protons that are involved in Watson-Crick pairing (''imino protons'') are easily detectable for RNA in submillimolar concentrations. In principle, each Watson-Crick base pair is assigned to a single signal. Because of base-pair fraying at the double helix end, the signal of the terminal base pair is broadened or not detectable at room temperature. Usually, the dispersion of the NH signals is good.

Figure 1.1.4 shows the NH spectra of our sequence ensemble. The spectrum of the sequence construct rGACCGGAAGGUCCGCCUUCC has the characteristics of slow exchange and reflects both sets of NH signals as measured for the individual reference hairpins rGACCGGAAGGUC and rCGGAAGGUCCGCCUUCC. This is clear evidence that rGACCGGAAGGUCCGCCUUCC indeed occurs as a two-state equilibrium of defined secondary structures [30]. It must also be stressed that the GGAA conformer is 3-fold less populated than the UCCG conformer, even though the GGAA-hairpin reference is a more stable secondary structure motif than the UCCG-hairpin reference. A likely explanation is that the GGAA conformer has an overhang which is fully complementary to the loop region and can therefore provide a higher forward rate constant.

Comparative imino proton NMR spectroscopy as described above proved a useful

Comparative imino proton NMR spectroscopy - assignment of the conformational equilibrium

'imino protons'

numeric weighted addition of reference spectra

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