Unusual nucleotide conformations in GNRA and UNCG type tetraloop hairpins:evidence from Raman markers assignments

High resolution NMR data on UNCG and GNRA tetraloops (where N is any of the four nucleotides and R is a purine) have shown that they contain ribonucle osides with unusual 2'-endo/anti and 3'-endo/syn conformations, in addition to the 3'-endo/anti ones which are regularly encountered in RNA chains. in the current study, Raman spectroscopy has been used to probe these nucleos ide conformations and follow the order (hairpin) to disorder (random chain) structural transitions in aqueous phase in the 5-80 degrees C temperature range. Spectral evolution of GCAA and GAAA tetraloops, as formed in very sh ort hairpins with only three G . C base pairs in their stems (T-m > 60 degr ees C), are reported and compared with those previously published on UUCG a nd UACG tetraloops, for which the syn orientation of the terminal guanine a s well as the 2'-endo/anti conformation of the third rC residue have been c onfirmed by means of vibrational marker bands, Raman data obtained as a fun ction of temperature show that the first uracil in the UUCG tetraloop is st acked and the two middle residues (rU and rC) are in the 2'-endo/anti confo rmation, in agreement with the previously published NMR results, As far as the new data concerning the GNRA type tetraloops are concerned, they lead u s to conclude that: (i) in both cases (GCAA and GAAA tetraloops) the adenin e bases are stacked; (ii) the second rC residue in the GCAA tetraloop has a 3'-endo/anti conformation; (iii) the sugar pucker associated with the thir d rA residue in both tetraloops possibly undergoes a 3'-endo/2'-endo interc onversion as predicted by NMR results; (iv) the stem adopts a regular A-for m structure; (v) all other nucleosides of these two GNRA tetraloops possess the usual 3'-endo/anti conformation.