Determination of γ and stereospecific assignment of H5′ protons by measurement of²J and³J coupling 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 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, ¹³N 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-s) 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.