A novel PH-cT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. application to HIV-2...

A quantitative analysis of JPH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal 3JPH3', 3JPHS' and 3JPHS" scalar couplings can be obtained by monitoring the intensity decay of the P1-H3'(i-1) peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two 3JPH5' and 3JPH5" scalar couplings even in the presence of overlapped H5'/H5" resonances, since the quantitative information is extracted from the intensity decay of the P-H3' peak. Moreover, the relaxation of the H3' proton is considerably slower than that of the H5'/H5" geminal protons and the commonly populated conformations of the phosphate backbone are associated with large 3JPH3' couplings and relatively small 3JPH5'/H5". These two facts lead to optimal signal-to-noise ratio for the P-H3' correlation compared to the P-H5'/H5" correlation. The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10-15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (DPH) as well. We will present qualitative results for the measurement of P-Hbase and P-H2' dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the DPHS for the 30mer RNA.