> NUCLEIC ACIDS Basic terms and notions Presentation by Eva Fadrná adapted by 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 Bowater, R. P., Waller, Z. AE., In: eLS. John Wiley & Sons, Chichester, 2014. Wijmenga, S. S., van Buuren, B. N. M., Progr. NMR Spect. 32, (1998), 287-387. Furtig, B. et al., ChemBioChem 4 (2003), 936-962. RNA vs DNA > > Single strand A-RNA B-DNA duplex > > Length of NA Total length of DNA in a human cell 1 m (1000 km)‏ DNA in typical human chromozome 1 cm (10 km)‏ DNA from bacterial chromozome 1 mm Diameter of typical human cell 0.01 mm Diameter of folded DNA 0.1 m (0.1 m)‏ Diameter of DNA fiber 1 nm (1 mm)‏ Diameter of atom 1 Å (multiplied by 106)‏  1 chromozome would be 10km long with fiber diameter of 1 mm and it would fold into 10 cm diameter  extraordinary DNA flexibility Nukleotide chain H – H – H – N N N9 N O5’ O2’ O3’ NH2 C1’ C3’ C4’ C5’ O4’ C2’ – H – H – H – H H | – H H – H – H – N N N9 N O5’ O2’ O3’ NH2 C1’ C3’ C4’ C5’ O4’ C2’ – H – H – H – H H | – H P O O H – H – H – N N N9 N O5’ O2’ O3’ NH2 C1’ C3’ C4’ C5’ O4’ C2’ – H – H – H – H H | – H P O O H – H – H – N N N9 N O5’ O2’ O3’ NH2 C1’ C3’ C4’ C5’ O4’ C2’ – H – H – H – H H | – H P O O Grooves > major vs. minor minor groove: O2 v pyrimidinech, N3 v purinech RNA vs DNA > > uridine deoxythymidine Nukleotide/nukleoside > > > > > > > base sugar (ribose/deoxyribose)‏ phosphate + nukleoside + nukleotide Bases > > > > > Adenin (Ade)‏ Guanin (Gua)‏ Cytosin (Cyt)‏ Thymin (Thy)‏ Uracil (Ura)‏ > sugar > > DNA RNA 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 > > > > > > Base tautomerism enol  keto enamin  imin C = C – N CH – C = N – H H H > >   > > C = C – O – H CH – C = O > >   > fysiolog. conditions lisi se polohou H a dvojne vazby Base tautomerism enol  keto enamin  imin C = C – N CH – C = N – H H H C = C – O – H CH – C = O     N HN N N N N N HN H O OH H HO OH HO O O O       4 tautomers lisi se polohou H a dvojne vazby Sugar - pentoses OH O HO OH OH 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 Nukleosides N N N9 N O HO OH OH NH2 O N1 HN O HO OH HO O N-glykosidic bond pyrimidines purines C1´ - N1 C1´ - N9 C1’ C1’ > > > > > > Nukleosides Ribonukleosides uridine = U cytidine = C adenosine = A guanosine = G Deoxyribonukleosides deoxythymidine = dT deoxycytidine = dC deoxyadenosine = dA deoxyguanosine = dG O HO (OH)‏ HO > base Phosphate group O = P – OH orthophosphoric acid H3PO4 N N N9 N O HO OH OH NH2 C1’ OH OH N N N9 N O OH OH NH2 C1’ O = P – O O O - - acid alcohol + ester + adenosine adenosine(mono)phosphate (AMP)‏ adenosine Zmatek v názvosloví: fosfát – ester i sůl > > > > > Nukleotides Ribonucleotides uridyl acid = uridine – 5´monophosphate = UMP, pU cytidyl acid = cytidin -“- = CMP, pC adenyl acid = adenosin -“- = AMP, pA guanyl acid = guanosin -“- = GMP, pG Deoxyribonucleotides deoxytymidyl acid = 2´deoxythymidine-5´-monophosphate = dTMP, pdT deoxycytidyl acid = -“- cytidin -“- = dCMP, pdC deoxyadenyl acid = -“- adenosin -“- = dAMP, pdA deoxyguanyl acid = -“- guanosin -“- = dGMP, pdG O (OH)‏ HO > > base O = P – O > > OH OH Phosphate group N N N9 N O HO OH OH NH2 C1’ N N N9 N O OH OH NH2 C1’ O = P – O OH O - N N N9 N O HO OH O NH2 C1’ N N N9 N O OH OH NH2 C1’ O = P – O O - alcohol acid + diester (ester)‏ nukleoside-nukleotide ApA 5’ 3’ Zmatek v názvosloví: fosfát – ester i sůl Nucleotide chain H – H – H – N N N9 N O5’ O2’ O3’ NH2 C1’ C3’ C4’ C5’ O4’ C2’ – H – H – H – H H | – H H – H – H – N N N9 N O5’ O2’ O3’ NH2 C1’ C3’ C4’ C5’ O4’ C2’ – H – H – H – H H | – H P O O H – H – H – N N N9 N O5’ O2’ O3’ NH2 C1’ C3’ C4’ C5’ O4’ C2’ – H – H – H – H H | – H P O O H – H – H – N N N9 N O5’ O2’ O3’ NH2 C1’ C3’ C4’ C5’ O4’ C2’ – H – H – H – H H | – H P O O 5’ 3’ Torsion angle > > > > A B C D > > > > >    0o, 360o -180o, 180o Torsion angle > > > > > > > synclinal (sc)‏ anticlinal (ac)‏ antiperiplanar (ap)‏ synperiplanar (sp)‏ +gauche (+g)‏ -gauche (-g)‏ trans (t)‏ Torsion angles in NA > Sugar-phosphate backbone Torsion angles cont. >        > Torsion angle  SYN: Pyrimidines: O2 above the sugar ring Purines: 6-member purine ring above the sugar ring Torsion angle  > Orientation around the C1’ – N glycosidic bond > > > > > > SYN ANTI O4’ – C1’ – N1 – C2 pyrimidines O4’ – C1’ – N9 – C4 purines 0o, 90o 270o, 360o 90o, 270o > >   Obrazek je obracene oproti kruhu s popisem uhlu!!! Torion –border intervals > > > > > SYN ANTI 0o, 90o 270o, 360o 90o, 270o > > >   high-syn (corresponds to +ac) … 90o + intrudes into anti > high-anti (corresponds -sc) … 270o + intrudes into syn > >   > Torsion angles in DNA Angle B-DNA A-DNA  -40.7 -74.8  -135.6 -179.1 g -37.4 58.9  139.5 78.2  -133.2 -155.0  -156.9 -67.1  -101.9 -158.9 Sugar conformation > > O3’ C5’ > >   … C5’ – C4’ – C3’ – O3’ > > > endocyclic exocyclic , 3 relation „Puckering“ of the sugar ring > > Envelope 4 atoms in a plane, the 5th above or below Twist 3 atoms in a plane, the 4th and the 5th on the oposite sides of the plane Definition of the puckering modes > > > > > > > The sugar ring is not planar C1’ – O4’ – C4’ plane Envelope C3’-endo 3E (prevalent in RNA)‏ Envelope C2’-endo 2E (prevalent in DNA)‏ symmetric Twist C2’-exo-C3’-endo 32T Non-symmetric Twist C3’-endo-C2’-exo 3T2 With respect to C5’ - endo - exo 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 = 0o : symmetric Twist C2’-exo-C3’-endo 32T P = 180o : asymmetric Twist C2’-endo-C3’-exo 23T max amplitude > > Maximum out-of-plane pucker max = 2 / cos(P)‏ > > P, j relation j = 0 .. 4 P value defines unambiguously all endocyclic torsion angles 0 to 4 > > > Sum of all 5  = 0 2 = max  cos(P + (j - 2)  144)‏ 0 + 1 + 2 + 3 + 4 = 0 P in nucleic acids > > NORTH SOUTH > > 0o  P  36o north (prevalent in RNA)‏ 144o  P  190o south (prevalent in DNA)‏ Helical parameters > > > > > axis-base, axis-base pair intra-base pair inter-base or inter-base pair Distance/shift Angle 3 translations, 3 rotations, + X and Y displacement, axis inclination, axis tip. Všechny tyto parametry – tzv. globální – uvažují existenci helikální osy. Ale! pro vellmi deformované struktury to nefunguje – musí existovat lokální axis systém, založený na vztahu k předchozímu páru bazí. Helical… > > D … displacement from helical axis R … roll T … tilt > t …twist = 360o / n Pár bazí není centrován kolem osy helixu, ale posunut poněkud vedle. Posunutí … displacement D. Není kolmý k helikální ose … Tilt, Roll. Báze nejsou koplanární … propeller twist. Twist t = 360/n … rotace mezi nukleotidem a jeho nejblizsim sousedem > > Helical parameters for A and B DNA Global B-DNA A-DNA 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 -0.08 -0.04 Buckle 0.0 0.0 Propeller -13.3 -7.5 Openning 0.0 -0.02 Shift 0.0 0.0 Slide 0.0 0.0 Rise 3.38 2.56 Tilt 0.0 0.0 Roll 0.0 0.0 Twist 36.00 32.70 Bases per turn 360/36=10 360/32.7=11 > Shifts in Å, angles in degrees Base pairing Watson-Crick pairs Base pairing Hoogsteen and reverse Hoogsteen pairs A and B double helix A-RNA > > B-DNA Ball and stick models > > A and B helices A-DNA B-DNA A-RNA with bulge > > > A and B helices A-DNA B-DNA A-RNA with bulge View tilted by 32 to show grooves 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 spectra of d(GCATGC) > > Single strand Duplex > 1H NMR spectra in D2O and H2O d(GCATTAATGC)2 > > 1H COSY spectrum of DNA d(CGCGAATTCGCG)2 a H2’-H2’’ b H4’-H5’,5’’ H5’-H5’’ c H3’-H4’ d H2’,2’’-H3’ e H1’-H2’,2’’ f H5-H6 (Cyt)‏ g CH3-H6 (Thy)‏ 1H NOESY spectrum of DNA > Water Suppression > WATERGATE Structure Determination Procedure > Resonance Assignment > Sequential connectivities with exchangeable protons > > > d(GGAATTGTGAGCGG)‏ d(CCTTAACACTCGCC)‏ imino-imino imino-amino > Sequential resonance assignments > d(GCATTAATGC)2 > Connectivities between H1’ and H6/8 > > d(CGCGAATTCGCG)2 i intra-residue s sequential > > Assignment of Sugar-Phosphate Backbone HP-COSY HP-TOCSY > 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