NUCLEIC ACIDS. Basic terms and notions. Presentation by Eva Fadrná adapted by Radovan Fiala

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1 UCLEIC ACIDS Basic terms and notions Presentation by Eva Fadrná adapted by Radovan Fiala

2 RA vs DA Single strand A-RA B-DA duplex

3 Length of A Total length of DA in a human cell 1 m (1000 km) DA in typical human chromozome 1 cm (10 km) DA from bacterial chromozome 1 mm Diameter of typical human cell 0.01 mm Diameter of folded DA 0.1 µm (0.1 m) Diameter of DA fiber 1 nm (1 mm) Diameter of atom 1 Å (multiplied by 10 6 ) 1 chromozome would be 10km long with fiber diameter of 1 mm and it would fold into 10 cm diameter extraordinary DA flexibility

4 ukleotide H C3 O3 H H O5 C5 H O4 H C4 O O P 9 C1 H C2 H O2 H H 2 H C3 O3 H H O5 C5 H O4 H C4 O O P H 9 C1 H C2 H O2 H H 2 H C3 O3 chain H H O5 C5 H O4 H C4 O O P H 9 C1 H C2 H O2 H H 2 H C3 H H O5 C5 H O4 H C4 H 9 C1 H C2 H nucleotide H 2 H O3 O2 H

5 Grooves major vs. minor

6 RA vs DA deoxythymidine uridine

7 ukleotide/nukleoside base + sugar (ribose/deoxyribose) nukleoside + phosphate nukleotide

8 DA Bases RA Guanin (Gua) Adenin (Ade) PURIES PYRIMIDIES Thymin (Thy) sugar Cytosin (Cyt) Uracil (Ura)

9 Base numbering PURIES PYRIMIDIES H

10 Base tautomerism enol keto fysiolog. conditions C = C O H CH C = O enamin imin H C = C H CH C = H

11 Base tautomerism enol keto enamin imin H C = C O H CH C = O C = C CH C = H H 4 tautomers O OH OH O H H O H O H HO HO

12 Sugar - pentoses HO RA CH 2 O OH 1 2 semiacetal hydroxyl group HO CH 2 O OH 2 1 DA OH OH OH ß D - ribose 2 deoxy ß D - ribose semiacetal hydroxyl group + base -glycosidic bond nukleoside C1-1 C1-9 pyrimidines purines

13 ukleosides purines pyrimidines C1-9 H 2 C1-1 O H HO O 9 C1 -glykosidic bond HO O O 1 C1 OH OH HO OH

14 ukleosides Ribonukleosides uridine cytidine adenosine guanosine = U = C = A = G HO O base Deoxyribonukleosides deoxythymidine deoxycytidine deoxyadenosine deoxyguanosine = dt = dc = da = dg HO (OH)

15 Phosphate group H 2 H 2 OH O = P OH OH HO O 9 C1 O O = P O O - - O 9 C1 + adenosine OH OH OH OH acid orthophosphoric acid H 3 PO 4 + alcohol ester adenosine adenosine(mono)phosphate (AMP)

16 ukleotides OH O = P O OH O base Ribonucleotides uridyl acid = uridine 5 monophosphate = UMP, pu cytidyl acid = cytidin - - = CMP, pc adenyl acid = adenosin - - = AMP, pa guanyl acid = guanosin - - = GMP, pg HO (OH) Deoxyribonucleotides deoxytymidyl acid = 2 deoxythymidine-5 -monophosphate = dtmp, pdt deoxycytidyl acid = - - cytidin - - = dcmp, pdc deoxyadenyl acid = - - adenosin - - = damp, pda deoxyguanyl acid = - - guanosin - - = dgmp, pdg

17 H 2 Phosphate H 2 group HO O OH OH O = P O O - alcohol OH 9 C1 O 9 C1 H 2 OH OH + acid diester (ester) HO 5 O O O = P O O - OH 9 C1 3 OH O OH 9 C1 H 2 nukleoside-nukleotide ApA

18 H 2 H C3 O3 H H O5 C5 H O4 H C4 O 5 nucleotide O P 9 C1 H C2 H O2 H H C3 O3 H H O5 C5 H O4 H C4 O O P H 9 C1 H C2 H O2 H H 2 H C3 O3 H H O5 C5 H O4 H C4 O O 3 P H 9 C1 H C2 H O2 H H 2 H C3 O3 H H O5 C5 H O4 H C4 H 9 C1 H C2 H O2 H H 2 H ucleotide chain

19 Torsion angle A D B C <0 o, 360 o > <-180 o, 180 o >

20 Torsion angle synperiplanar (sp) -gauche (-g) +gauche (+g) synclinal (sc) anticlinal (ac) antiperiplanar (ap) trans (t)

21 Torsion angles in A Sugar-phosphate backbone

22 Torsion angles cont. α β γ δ ε ζ χ

23 Torsion angle χ SY: Pyrimidines: O2 above the sugar ring Purines: 6-member purine ring above the sugar ring

24 Torsion angle χ Orientation around the C1 glycosidic bond O4 C1 1 C2 pyrimidines O4 C1 9 C4 purines SY χ <0 o, 90 o > <270 o, 360 o > ATI χ <90 o, 270 o >

25 Torion χ border intervals high-syn (corresponds to +ac) 90 o + high-anti (corresponds -sc) 270 o + intrudes into anti intrudes into syn SY χ <0 o, 90 o > <270 o, 360 o > ATI χ <90 o, 270 o >

26 Torsion angles in DA Angle B-DA A-DA α β γ δ ε ζ χ

27 Sugar conformation C5 δ endocyclic O3 δ C5 C4 C3 O3 δ, ν3 relation exocyclic

28 Puckering of the sugar ring Envelope 4 atoms in a plane, the 5 th above or below Twist oposite plane 3 atoms in a plane, the 4 th and the 5 th on the sides of the

29 Definition of the puckering modes The sugar ring is not planar C1 O4 C4 plane Envelope C3 -endo With respect to C5 - endo - exo 3 E (prevalent in RA) Envelope C2 -endo 2 E (prevalent in DA) symmetric Twist C2 -exo-c3 -endo 3 2 T on-symmetric Twist C3 -endo-c2 -exo 3 T 2

30 Pseudorotation cycle Theoretically infinite number of conformations, can be characterized by maximum torsion angle (degree of pucker) and pseudorotation phase angle Torsion angles are not independent (ring closed) Pseudorotation phase angle P P = 0 o : symmetric Twist C2 -exo-c3 -endo 3 2 T P = 180 o : asymmetric Twist C2 -endo-c3 -exo 2 3 T

31 ν max amplitude Maximum out-of-plane pucker ν max = ν 2 / cos(p)

32 P, ν j relation P value defines unambiguously all endocyclic torsion angles ν 0 to ν 4 ν 2 = ν max cos(p + (j - 2) 144 ) ν 0 + ν 1 + ν 2 + ν 3 + ν 4 = 0 j = Sum of all 5 ν = 0

33 P in nucleic acids ORTH SOUTH 0 o P 36 o north (prevalent in RA) 144 o P 190 o south (prevalent in DA)

34 axis-base, axis-base pair intra-base pair inter-base or inter-base pair Helical parameters Distance/shift Angle

35 Helical D displacement from helical axis t twist = 360 o / n θ R roll θ T tilt

Helical parameters for A and B DA Global B-DA A-DA Shifts in Å, angles in degrees X disp. 0.0-5.28 Y disp. 0.0 0.0 Inclin 1.46 20.73 Tip 0.0 0.0 Shear 0.0 0.0 Stretch 0.0 0.01 Stagger -0.

36 Helical parameters for A and B DA Global B-DA A-DA Shifts in Å, angles in degrees X disp Y disp Inclin Tip Shear Stretch Stagger Buckle Propeller Openning Shift Slide Rise Tilt Roll Twist Bases per turn 360/36=10 360/32.7=11

37 Base pairing Watson-Crick pairs

38 Base pairing Hoogsteen and reverse Hoogsteen pairs

39 A and B double helix A-RA Ball and stick models B-DA

40 A and B helices A-DA B-DA A-RA with bulge

41 A and B helices A-DA B-DA A-RA with bulge View tilted by 32 to show grooves

42 uclear properties of selected isotopes

43 Spin systems in ribose and deoxyribose

44 Spin systems in nucleic acid bases

45 1 H chemical shift ranges in DA and RA

46 1 H chemical shift ranges in DA and RA

47 1 H MR spectra of d(gcatgc) Single strand Duplex

48 1 H MR spectra in D 2 O and H 2 O d(gcattaatgc) 2

49 1 H COSY spectrum of DA a b c d e f g H2 -H2 H4 -H5,5 H5 -H5 H3 -H4 H2,2 -H3 H1 -H2,2 H5-H6 (Cyt) CH 3 -H6 (Thy) d(cgcgaattcgcg) 2

50 1 H OESY spectrum of DA d(cgcgaattcgcg)2

51 Water Suppression

54 WATERGATE

55 Structure Determination Procedure

56 Resonance Assignment

57 Sequential connectivities with exchangeable protons d(ggaattgtgagcgg) d(ccgctcacaattcc) imino-imino imino-amino

58 Sequential resonance assignments d(gcattaatgc) 2

59 Connectivities between H1 and H6/8 d(cgcgaattcgcg) 2 i intra-residue s sequential

65 Assignment of Sugar-Phosphate Backbone HP-COSY HP-TOCSY

69 J-couplings from COSY spectra

70 P determination from J-couplings

71 Equilibrium of and S conformations 3 -endo 2 -endo

NMR of Nucleic Acids. K.V.R. Chary Workshop on NMR and it s applications in Biological Systems November 26, 2009

NMR of Nucleic Acids. K.V.R. Chary Workshop on NMR and it s applications in Biological Systems November 26, 2009 MR of ucleic Acids K.V.R. Chary chary@tifr.res.in Workshop on MR and it s applications in Biological Systems TIFR ovember 26, 2009 ucleic Acids are Polymers of ucleotides Each nucleotide consists of a