USE OF ULTRA-STABLE UNCG TETRALOOP HAIRPINS TO FOLD RNA STRUCTURES - THERMODYNAMIC AND SPECTROSCOPIC APPLICATIONS

RNA molecules of >20 nucleotides have been the focus of numerous recen t NMR structural studies, Several investigators have used the UNCG fam ily of hairpins to ensure proper folding. We show that the UUCG hairpi n has a minimum requirement of a two base-pair stem, Hairpins with a C G loop closing base pair and an initial 5'CG or 5'GC base pair have a melting temperature similar to 55 degrees C in 10 mM sodium phosphate, The high stability of even such small hairpins suggests that the hair pin can serve as a nucleation site for folding, For high resolution NM R work, the UNCG loop family (UACG in particular) provides excellent s pectroscopic markers in one-dimensional exchangeable spectra, in two-d imensional COSY spectra and in NOESY spectra that clearly define it as forming a hairpin, This allows straightforward initiation of chemical shift assignments.