Heteronuclear NMR of Nucleic Acids In most cases, requires samples isotopicaly enriched by 13C and 15N (except for HSQC, HMQC) Assignment uses NOE or through-bond experiments Traditional constraints (NOEs, J-couplings) Novel constraints (RDCs, residual CSA) Studies of intramolecular dynamics Sample preparation J. Flinders, T. Dieckmann / Progress in Nuclear Magnetic Resonance Spectroscopy 48 (2006) 137–159 Labeled NTPs Available commercially Can be grown in cells E. Coli 13C-glucose, 15N-ammonium M. Methylotropus 13C-methanol, 15N-ammonia I. (H2O) Correlation of exchangeable protons with 15 N 2D 1 H-15 N HSQC NH imino optimized (Gua and Ura) 2D 1 H-15 N HSQC NH2 amino optimized (Cyt, Gua, Ade) Sequential assignment of exchangeable protons 3D NOESY-HSQC (1 H-1 H-15 N, imino 15 N edited NOESY) imino-imino and imino-amino interactions 3D NOESY-HSQC (1 H-1 H-15 N, amino 15 N edited NOESY) amino-imino interactions II. (H2O) Assignment of non-exchangeable protons with NOE connectivities to imino and amino protons 3D NOESY-HSQC (1 H-1 H-15 N, imino/amino 15 N edited NOESY) interactions of aromatic protons with imino and amino groups Assignment procedure for labeled NA NOE based (I) Assignment procedure for labeled NA NOE based (II) III. (2 H2O) Identification of hydrogen and carbon atoms in sugars 2D 1 H-13 C CT-HSQC identification of H-C pairs 3D HCCH-COSY identification of neighboring C-H groups 3D HCCH-RELAY H1′′′′-C2′′′′/C3′′′′ correlation 3D HCCH-TOCSY H1′′′′-C2′′′′/C3′′′′/C4′′′′/C5′′′′ correlation Identification of hydrogen and carbon atoms in bases 2D 1 H-13 C CT-HSQC identification of H-C pairs 2D/3D HCCH-COSY H5-H6 and C5-C6 correlations in pyrimidines Sequential assignment 3D NOESY-HSQC (1 H-1 H-13 C), H6/8-H1′′′′, H6/8-H2′′′′ correlations IV. (2 H2O) Assignment of 31 P resonances 1 H-31 P HETCOR/heteroTOCSY Assignment procedure for labeled NA Through bond correlations (I) I. (H2O) Correlation of exchangeable protons with 15 N 2D 1 H-15 N HSQC NH imino optimized (Gua and Ura) 2D 1 H-15 N HSQC NH2 amino optimized (Cyt, Gua, Ade) II. (H2O) Correlation of imino and amino protons with nonexchangeable base protons HCCNH-TOCSY / HNCCH-TOCSY Assignment procedure for labeled NA Through bond correlations (II) III. (2 H2O) Correlation of non-exchangeable protons with 13 C 2D 1 H-13 C CT-HSQC identification of H-C pairs 3D HCCH-COSY identification of neighboring C-H groups 3D HCCH-TOCSY H1′′′′-C2′′′′/C3′′′′/C4′′′′/C5′′′′ correlation Identification of hydrogen and carbon atoms in bases 2D 1 H-13 C CT-HSQC identification of H-C pairs 2D/3D HCCH-COSY H5-H6 and C5-C6 correlations in pyrimidines HCCH-TOCSY / 1 H-13 C HMBC H2-H8 correlations in Ade Sugar-base correlations HsCsN and HbCbN HsCsNCbHb / HsCsNHb IV. (2 H2O) Sequential assignment of 31 P resonances across the sugar-phosphate backbone HCP / PCH / PCCH-TOCSY / HPHCH Correlation of exchangeable protons with 15N Gradient sensitivity enhanced HSQC Kay, Keifer, Saarinen, JACS 1992. A U C G G C G C C G U U C G 1 5 10 Correlation of exchangeable and nonexchangeable protons HCCNH-TOCSY, Fiala et al. JACS 1996, Sklenar et al. J. Biomol. NMR 1996. Correlation across the hydrogen bond HNN-COSY experiment Dingley and Grzesiek, JACS 1998 Identification of hydrogen and carbon atoms in bases and sugars Constant-time 1H-13C HSQC experiment base sugar Assignment of non-exchangeable protons: HCCH-type experiments Sugar to base correlation – the HCN experiment Sklenar et al., J. Biomol. NMR 1993, 1994, Fiala et al., J. Biomol. NMR 1998, 2000. Sugar to phosphate correlation – the HCP experiment Dipolar couplings •Dipolar couplings add to J couplings •They show up as a field or alignment media dependence of the coupling •If the overall orientation of the molecule is known the orientation of the vectors can be determined