Enol form of uracil in the GU"wobble"pair. NMR evidence

The idea of the enol form of nucleotides being responsible for the formation of some noncanonical pairs was put forward by Watson and Crick in their first paper on DNA structure [1]. In the context of this hypothesis, the GU pair may contain either the enol form of uracil and the keto form of guanine or the enol form of guanine and the keto form of uracil. Later, in 1966, Francis Crick proposed the “wobble base pair” structure for the GU pair [2]. There are some structural data suggesting that the structure of the RNA/DNA double helix is accurately described by the earlier hypothesis. Based on X-ray structure data, Weixlbaumer et al. [3] concluded that modified uridine and guanine could pair up if uridine exists in enol form. The authors, however, interpreted this fact as an unexpected violation of the “standard GU wobble geometry”. Xray methods cannot directly identify the enol form of uracil; thus, that study [3] could only provide indirect evidence. A direct proof of uracil existence in enol form can be obtained by using NMR spectroscopy. For the GU pair, the proton spectrum demonstrates two protons attached to heteroatoms in the 10-15 ppm region. For the “wobble base pair” structure (A), both protons taking part in the formation of hydrogen bonds between G and U must have almost equal values of chemical shifts, but this is not the case [4]. Signals from amino group protons are outside the range given above (they are in a stronger field). For the structure based on the enol form of uracil, (C-4 atom is enolized, structure B), G and U could be linked by three hydrogen bonds, and the three protons involved in this should be noticeably nonequivalent, as they are positioned between different pairs of heteroatoms: oxygen-oxygen, nitrogen-nitrogen, and nitrogen-oxygen. Importantly, structure B is very close imitates the classic Watson-Crick base pair GC (structure C).