NMR studies of base-pairing in 15N-labeled ribotetranucleotide GGCUp

95% N-15 enriched ribotetranucleotide GGCUp was prepared. Its aqueous solution gives three imino-proton resonances at 10.6, 12.0 and 13.6 ppm. The peak at 10.6 ppm split by N-15 into a doublet with coupling constant 90 Hz. On the preirradiation at this peak, the intensity of the 12.0 ppm peak decreased and the peak at 13.6 ppm remained unchanged. Therefore the 10.6 ppm peak was unambiguously assigned to the G imino proton and the 12.0 ppm peak to the U imino proton in the G:U base pair. By raising temperature the former two peaks coalesced at around 25 degrees C, while the signal of the G imino proton in the G:C pair was observed up to 35 degrees C. The G:U base pair seems to be disrupted first, then the break of the G:C pair is followed. Temperature dependences of the CH proton, N-15 and P-31 nuclei resonances were also measured.