Abstract
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 13C,15N labeled RNA oligonucleotide was performed using new Exclusive COSY (E.COSY) type multidimensional heteronuclear NMR experiments designed to measure 3J(H4‘,H5‘) homonuclear and 2J(C4‘,H5‘) heteronuclear coupling constants. The experiments were demonstrated on a uniformly 13C,15N labeled 19-mer RNA hairpin (5‘-rGCACCGUUGGUAGCGGUGC-3‘) derived from the RNA I transcript involved in Col E1 replication control. From the small 3J(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 2J(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.
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