Determination of γ and Stereospecific Assignment of H5‘ Protons by Measurement of2Jand3JCoupling Constants in Uniformly13C Labeled RNA

The conformational analysis of the backbone angle γ (O5‘−C5‘−C4‘−C3‘) and the stereospecific assignment of the H5‘ (pro-S), H5‘ (pro-R) diastereotopic protons in a uniformly 13 C, 15 N labeled RNA oligonucleotide was performed using new Exclusive COSY (E.COSY) type multidimensional heteronuclear NMR experiments designed to measure 3 J (H4‘,H5‘) homonuclear and 2 J (C4‘,H5‘) heteronuclear coupling constants. The experiments were demonstrated on a uniformly 13 C, 15 N labeled 19-mer RNA hairpin (5‘-rGCACCGUUGGUAGCGGUGC-3‘) derived from the RNA I transcript involved in Col E1 replication control. From the small 3 J (H4‘,H5‘) couplings constants observed for the RNA hairpin, it was concluded that all γ angles assume a gauche + rotamer conformation (γ = 60°). From the signs of the 2 J (C4‘,H5‘) coupling constants, the H5‘ protons were stereospecifically assigned. In the helical stem region of the hairpin, the H5‘ (pro-S) protons were found to resonate downfield (∼0.4 ppm) of the H5‘ (pro-R) protons. In the loop region of the hairpin, the chemical shift differences between the H5‘ (pro-S), H5‘ (pro-R) resonances were found to be smaller, and in most cases, the H5‘ (pro-S) protons were found to resonate upfield of the H5‘ (pro- R) protons. The different chemical shift patterns observed for the H5‘ (pro-S) and H5‘ (pro-R) protons in the two secondary structure elements are discussed.