NUCLEIC ACIDS Basic terms and notions Presentation by Eva Fadrná adapted by Radovan Fiala RNA vs DNA Length of NA Total length of DNA in a human cell DNA in typical human chromozome DNA from bacterial chromozome Diameter of typical human cell Diameter of folded DNA Diameter of DNA fiber Diameter of atom 1 m (1000 km} 1 cm (10 km} 1 mm 0.01 mm 0.1 |Lim (0.1 m} 1 nm (1 mm} o 1 A (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/nukleoside Base numbering I / H-N=0-HO \ H N-0 = 0 / \ H UILUI UILUBU9 I / 0 = 0-HO \ _ I / H-0-0 = 0 \ 019>| <-» |0U9 WSU9W0}rH2} 9SBg Sugar - pentoses semiacetal hydroxyl group + base N-glycosidic bond C1'-N1 C1'-N9 nukleoside pyrimidines purines Nukleosides Nukleosides Ribonukleosides uridine = U cytidine = C adenosine = A guanosine = G Deoxyribonukleosides deoxythymidine = dT deoxycytidine = dC deoxyadenosine = dA deoxyguanosine = dG base HO HO (OHJ Phosphate group N OH I J-1 = P-OH HO- I 1-1 OH OH OH acid + alcohol orthophosphoric . K adenosine acid H3P04 N I = P-0 I N OH OH + adenosine ester adenosine(mono)phosphate (AMP| Nukleotides OH I P-0 I OH Ribonucleotides uridyl acid = uridine - 5'monophosphate = UMP, pU cytidyl acid = cytidin -"- = CMP, pC adenyl acid = adenosin -"- = AMP, pA guanyl acid = guanosin -"- = GMP, pG base HO (OHJ Deoxyribonucleotides deoxytymidyl acid = 2'deoxythymidine-5'-monophosphate = dTMP, pdT deoxycytidyl acid = -"- cytidin -"- = dCMP, pdC deoxyadenyl acid = -"- adenosin -"- = dAMP, pdA deoxyguanyl acid = -"- guanosin -"- = dGMP, pdG 05'—C5'-H I ^04' H"C4' XC1'-H \ / H-C3'—C2'-H A3' OV-H ■05'—C5'-H I ^04' H "C4" XC1'- H \ / H -C3'--C2'- H oV oV-H ■05'—C5'-H / ^04' H_C4' ^C1'-H \ / H -C3'--C2'- H OV ^2'-H ■05'—C5'-H I ^04' H"C4' XC1'-H \ / H -C3'--C2'- H 0"3' A?'"" Nucleotide chain Torsion angle <0°, 360°> <-180°, 180°> Torsion angle synperiplanar (sp} -gauche (-gj +gauche (+gj synclinal (scj anticlinal (acj HHBjB| antiperiplanar (apj trans (tj Torsion angles in NA Sugar-phosphate backbone Torsion angles cont. o M 03'(j-1) " " ~P®""" era ■ C+'CQ C3'@ ~P(iVl)~ " 05'(i+l) öl© = 03(i-l)-P(i)-05'(J)-C5'(i) ß (i) = P (J) - OS1 (J) - C51 (i) - C4-' (J) y(0 = O5'(i)-C5,(0-C4-,(J)-C3,(j) Ö(J) = G5li)^-G'+^—JC3l(ii-Q3l"Ö e® = C4-' (i) - C3' (i) - 03' (i) - P (i+1) t(i) = C3' (i) - 03' (i) - P (i+1) - 05' (i+1) Torsion angle % SYN: Pyrimidines: 02 above the sugar ring Purines: 6-member purine ring above the sugar ring Torsion angle % Orientation around the C1' - N glycosidic bond 04'-C1'-N1-C2 pyrimidines 04'-C1'-N9-C4 purines SYN X <0°, 90°> + <270°, 360°> ANTI X <90°, 270°> Torion % - border intervals high-syn (corresponds to +ac) ... 90° + intrudes into anti high-anti (corresponds -sc) ... 270° + intrudes into syn Torsion angles in DNA Angle B-DNA A-DNA a -40.7 -74.8 ß -135.6 -179.1 y "37.4 58.9 I-> 5 139.5 78.2 e -133.2 -155.0 C -156.9 -67.1 l=>X -101.9 -158.9 „Puckering" of the sugar ring Envelope 4 atoms in a plane, the 5th above or below HI 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'-04'-C4' plane With respect to C5' - endo - exo Envelope C3'-endo 3E (prevalent in I NA} Envelope C2'-endo 2E (prevalent in I \} symmetric Twist C2'-exo-C3'-endo 3 T Non-symmetric Twist C3'-endo-C2'-exo 3T, 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! I Pseudorotation phase angle P & w &» -endo tan P = (V4 + Vi)-(V3 +%) 2 .V,. (sin 36°+ sin 72° 1 P = 0° : symmetric Twist C2'-exo-C3'-endo 32T P = 180° : asymmetric Twist C2'-endo-C3'-exo 23T vmax amplitude P in nucleic acids 0° < P < 36° north (prevalent in RNAJ 144°< P< 190° south (prevalent in DNA} Helical parameters axis-base, axis-base pair intra-base pair inter-base or inter-base pair Helical... D ... displacement from helical axis t ...twist = 360°/ n 0R ... roll 6V ... tilt Helical parameters for A and B DNA Global X disp. Y disp. Inclin Tip_ Shear Stretch Stagger Buckle Propeller Openninq Shift Slide Rise Tilt Roll Twist Bases per turn B-DNA 0.0 0.0 1.46 0.0 0.0 0.0 -0.08 0.0 -13.3 0.0 0.0 0.0 3.38 0.0 0.0 36.00 360/36=10 A-DNA -5.28 0.0 20.73 0.0 0.0 0.01 -0.04 0.0 -7.5 -0.02 0.0 0.0 2.56 0.0 0.0 32.70 360/32. Shifts in A, angles in degrees 7=11 Base pairing Watson-Crick pairs Base pairing H f A 1 H"--\ /CH* \\ / \1hj™ ) \ H 0 N h c / i H H-N H 0 Hoogsteen and reverse Hoogsteen pairs N H \ /"-H—N U A> Ml K H 0 A and B double helix hÜ HSU KiÜfl A-RNA Ball and ■l«-aSüta^^' "fl — ill stick m I models Er*■•^■■Saa11 B-DNA A and B helices View tilted by 32° to show grooves Nuclear properties of selected isotopes -7 Isotope Y x 10 -H 2 4 9 / 1 " / Adenine, A Guanine,G A* A A 1H chemical shift ranges in DNA and RNA H chemical shift ranges in DNA and RNA Code rS (ppm) Comments 2' 1.8-3.0 2'H, 2"H in DNA 4' ,5» 3.7-4.5 4'H, 5'H, 5"H in DNA 3« 4.4-5.2 3'H in DNA 3.7-5.2 2'H, 3'H, 4'H, 5*H, 5"H in RNA 1« 5.3-6.3 l'H CH3 1.2-1.6 CH3 of T 5 5.3-6.0 5H of C and U 6 7.1-7.6 6H of C, T and U 2,8 7.3-8.4 8H of A and G, 2H of A -NH2* 6.6-9.0 NH2 of A, C and G ^ NH* 10 - 15 Ring NH of G, T and U 1H NMR spectra ot d(GCATGC) 1H NMR spectra in D20 and H20 A D20 2H.8I jjjjli1' HDO H.6H ITH 5H 3-H In H.5'H,5'H CH3 I 2'H2"H f 8 B H20 1 I * ! 7 6 5 d(GC A 3 2 ô(ppm) ;ATTAATGC)2 -NH2,2H.8H,6H i i i i 1 i 1 % 13 12 ö(ppm) 8 7 1H NOESY spectrum of DNA Water Suppression The presence of an intense solvent resonance necessitates an impractical high dynamic range. 110 M vs ). In general: RNA (A type double helix) C3f endo. DNA (B type double helix) C2T endo. Vi = d>m cos (P + 144 (j-2)) N (Northern) (Southern) Deoxyribose; ^'Jhi-hz * 10 Hz J-couplings from COSY spectra P determination from J-couplings Equilibrium of N and S conformations 3'-endo J(HV-H2') Distance information determines the glycosidic torsion angle > How do we get distance information? o Nuclear Overfiauser effect (< 6A) 031 041 a and t pose problems Determinants of31P chem shift. 8 and t correlate, £ = -317-1.23 e V2 OS' JpS-HS'fHS") ,JP5'-C4' Y X 5jH4'-H5'(H5") JP3-H3' JH1'-C6 (UAT) JC3'-H5'(H5") JP3-C2' "JH1'<:2 (uicJ) JP3'-C4' '^Hl'-CS (A,G) 3%r<:4 (A,G) Structure Determination: I) Assignment II) Local Analysis •glycosidic torsion angle, sugar puckering,backbone conformation base pairing III) Global Analysis •sequential, inter strand/cross strand, dipolar coupling Nucleic Acids have few protons..... •NOE accuracy > account for spin diffusion •Backbone may be difficult to fully characterize > especially a and t •Dipolar couplings What do we know? • Distance, Torsion, H-Boncl constraints What do we want? • Low energy structures Methods • Distance Geometry • Simulated annealing, rMD •Torsion angle dynamics (DYANA) • Mardigras/IRMA/Morass optimize conditions pH, J, T. 1D NMR Assignments spin system sequential Jong range NOESY, TOCSY, COSY Distance constraints Torsion constraints NOESY, COSY Distance Geometry/ simulated annealing Use contraints to calculate structure Initial structure(s) Identify additional constraints (side chains, additional long range contacts etc) Reffine structure(s) rMD calculations I Structures T Additional Experiments ^ Dynamics Mutants Interaction with target/drug