NMR OF NUCLEIC ACIDS Radovan Fiala Literature Books Saenger, W., Principles of Nucleic Acid Structure, Springer 1984. Bloomfield, V. A., Crothers, D. M., Tinoco, I., Nucleic Acids, Structures, Properties, and Functions, Univ. Sci. Books, 2000. Wuthrich, K., NMR of Proteins and Nucleic Acids, Wiley, 1986. Review articles Can be downloaded from https://web.ncbr.muni.cz/~fiala/ Bowater, R. P., Waller, Z. AE., DNA Structure, In: eLS. John Wiley & Sons, Chichester, 2014. Wijmenga, S. S., van Buuren, B. N. M., The use of NMR methods for conformational studies of nucleic acids, Progr. NMR Spect. 32, (1998), 287-387. Furtig, B. et al., NMR of RNA, ChemBioChem 4 (2003), 936-962. Base numbering PURINES PYRIMIDINES N N N N N N H 3 2 1 4 5 7 6 9 8 3 6 5 1 4 2 Sugar - pentoses OH O HO OHOH CH2 OH O HO OH CH2 ß – D - ribose 2 – deoxy – ß – D - ribose RNA DNA 2’ 1’ 5’ 4’ 3’ semiacetal hydroxyl group 2’ 1’ 5’ 4’ 3’ semiacetal hydroxyl group + base N-glycosidic bond nukleoside C1´ - N1 C1´ - N9 pyrimidines purines Other aldopentoses: arabinose, xylose, lyxose Base pairing Watson-Crick pairs Base pairing Hoogsteen and reverse Hoogsteen pairs Nuclear properties of selected isotopes Spin systems in ribose and deoxyribose Spin systems in nucleic acid bases 1H chemical shift ranges in DNA and RNA 1H chemical shift ranges in DNA and RNA 1H NMR spectrum of d(CGCGAATTCGCG) 1H NMR spectra in H2O Watson-Crick d(CGCGAATTCGCG)2 B-helix Hoogsteen d(AAT GGG T GGG TTT GGG T GGG TAA) G-quadruplex 1H COSY spectrum of DNA d(CGCGAATTCGCG)2 1H TOCSY spectrum of DNA d(CGCGAATTCGCG)2 1H NOESY spectrum of DNA in D2O d(CGCGAATTCGCG)2 1H NOESY spectrum of DNA in H2O d(CGCGAATTCGCG)2 [H2O]=55,000 mM [Protein]< 5 mM [H2O]/[Protein]>11,000 Water suppression Sample : 1 mM TEP-I in 90% H2O/10%D2O, pH 6.0, 290 K. Water suppression WATERGATE Structure Determination Procedure Resonance Assignment Sequential connectivities with exchangeable protons Dickerson’s dodecamer d(CGCGAATTCGCG)2 imino-imino imino-amino Sequential resonance assignments d(CGCGAATTCGCG)2 H6/8-H2’,2’’/Me H6/8-H1’ Assignment based on H6/8-H1’ connectivities d(CGCGAATTCGCG)2 Assignment based on H6/8-H1’ connectivities d(CGCGAATTCGCG)2 Assignment based on H6/8-H1’ connectivities d(CGCGAATTCGCG)2 Assignment based on H6/8-H1’ connectivities d(CGCGAATTCGCG)2 Assignment based on H6/8-H1’ connectivities d(CGCGAATTCGCG)2 Assignment based on H6/8-H1’ connectivities d(CGCGAATTCGCG)2 Assignment based on H6/8-H1’ connectivities d(CGCGAATTCGCG)2 Assignment based on H6/8-H2’2’’ connectivities d(CGCGAATTCGCG)2 31P spectrum of DNA d(CGCGAATTCGCG)2 Phosphate buffer Assignment of Sugar-Phosphate Backbone HP-COSY HP-TOCSY 1H - 31P correlation spectrum 31P 1H 3’ 4’, 5’, 5’’ d(CGCGAATTCGCG)2 J-couplings from COSY spectra P determination from J-couplings 1’2’ 2’3’ 1’2’’ 3’4’ 2’’3’ Equilibrium of N and S conformations 3’-endo 2’-endo 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 RNA preparation 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 Assignment of imino protons in loops Correlation of exchangeable and non-exchangeable protons Labeled samples – HCCNH-TOCSY Fiala et al. JACS 1996, Sklenar et al. J. Biomol. NMR 1996. Assignment of imino protons in loops Correlation of exchangeable and non-exchangeable protons Unabeled samples – JRHMBC Phan, Journal of Biomolecular NMR, 16: 175–178, 2000. “Green Monkey” 5'-CGGGCGGGCGCGAGGGAGGGC-3' (21 nt) Correlation across the hydrogen bond HNN-COSY experiment Dingley and Grzesiek, JACS 1998 A U C G G C G C C G U U C G 1 5 10 N1(G) N3(U) N3(C) 15N 2hJNN ~ 5.5 Hz 1hJHN ~ 1.0 Hz 1H G10 G2 G1 G12 G9 U11 A4(N1) folded U6 C5 C14 C3C13 Identification of hydrogen and carbon atoms in bases and sugars Constant-time 1H-13C HSQC experiment base sugar 13C 1H 1H 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 Sugar to phosphate correlation – the HCP experiment real spectra – nt-14 RNA with UUCG loop 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