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Proton nuclear magnetic resonance has been used to examine the effect of both noncomplementary and G.U oppositions in the duplexes formed by the synthetic pentaribonucleotides CpApApUpG, CpApUpUpG, CpApGpUpG, and CpApCpUpG. The lack of any sigmoidal behavior in the chemical shift vs. temperature plots of the base protons in the individual pentaribonucleotides indicates that duplexes with noncomplementary base oppositions of the type: formula: (see text), (where X = A, U, G, or C) do not form. Variable temperature spectra of the mixture of CpApGpUpG and CpApUpUpG were recorded over the range of 70--10 degrees C. The chemical shift vs. temperature plot of the purine aromatic protons displayed sigmoidal curves. This demonstrated both duplex formation and the presence of a G.U. base pair. The average Tm of the duplex was found to be 23.4 +/- 2.0 degrees C. This is similar to that of the duplex formed by CpApUpG (24.0 +/- 1.0 degrees C) but less than the Tm of the following duplexes: CpApApUpG:CpApUpUpG (Tm = 28.5 +/- 2.1 degrees C), CpApGpUpG:CpApCpUpG (Tm = 38.4 +/- 0.6 degrees C) and CpApUpApUpG (Tm = 41.5 +/- 1.1 degrees C). The G.U base pair has a Tm (20.0 degrees C) significantly lower than the rest of the duplex (24 +/- 1 degree C) and is a region of local instability within the double helix. This 1H NMR study is the first to investigate both the formation and relative stability of an internal G.U. base pair neighboring regular Watson--Crick base pairs.
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