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. Sei. Books, 2000. Wuthrich, K., NMR of Proteins and Nucleic Acids, Wiley, 1986. Review articles 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. Single strand A-RNA B-DNA duplex 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 cm 1 mm 0.01 mm 0.1 |um 1 nm 1 A =^> 1 chromozome would be 10km long with fiber diameter of 1 mm and it would fold into 10 cm diameter =^> extraordinary DNA RNA vs DNA deoxythymidine uridine Nukleotide/nukleoside base + sugar (ribose/deoxyribose) nukleoside + phosphate nukleotide Base numbering Base tautomerism fysiolog. conditions C -C = 0 C = C-N H C -C=N-H Base tautomerism enamin|<-> imin C C-O- c C-N Sugar - pentoses HO CH, semiacetal hydroxyl group RNA OH OH (&y D - ribose semiacetal hydroxyl group + base DNA 2 - deoxy - R> - D - ribose nukleoside C1'-N1 C1' - N9 pyrimidines purines Nukleosides Nukleosides Deoxyribonukleosides deoxythymidine = dT deoxycytidine = dC deoxyadenosine = dA deoxyguanosine = dG HO (OH) Phosphate group + adenosine OH OH orthophosphonc , adenosine acid h3po4 adenosine(mono)phosphate (AMP) Nukleotides 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 deoxycytidyl acid = -"- cytidin -"- = dCMP, pdC deoxyadenyl acid = -"- adenosin -"- = dAMP, pdA deoxyguanyl acid = -"- guanosin -"- = dGMP, pdG = dTMP, pdT Phosphate OH OH acid (ester) OH OH nukleoside-nukleotide diester ApA Nucleotide chain Torsion angle Torsion angle synperiplanar synclinal anticlinal (ac) antiperiplanar Torsion angles in NA Torsion angles cont. l o M 03'(j-1) «VT* 05'@ C5'® C3'® 03'® P~(iVl)~ " 05'(i+l) ci (0 = 03 (i-1) - P (J) - 051 (i) - C51 (J) ß (j) = P (i) - 051 (i) - C51 (i) - C41 (i) V® = 05l(i)-C5l(i)-C+lCi)-C3lCi) 5(i) = C5\0-C4X:0-C3l(j)-Ü3l(i)' e(i) = C4-1 (j) - C31 (i) - 031 (j) - P (i+1) C(i) = C31 © - 03' ffi - P (j+1) - 05' (j+1) chain direction mic lern tide unit i P(j+1) 05'(i+l) Torsion angle % SYN: Torsion angle % Orientation around the C1' - N glycosidic bond <270°, 360°> Torion % - border intervals Torsion angles in DNA Angle B-DNA A-DNA a -40.7 -74.8 ß -135.6 -179.1 Y -37.4 58.9 5 139.5 78.2 8 -133.2 -155.0 c -156.9 -67.1 X -101.9 -158.9 Sugar conformation „Puckering" of the sugar ring 4 atoms in a plane, the 5th above or below 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 With respect to C5' - endo Envelope C3'-endo 3E (prevalent in RNA) Envelope C2'-endo 2E (prevalent in DNA) symmetric Twist C2'-exo-C3'-endo 3 T 21 Non-symmetric Twist C3'-endo-C2'-exo 3T2 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) vmax amplitude Maximum out-of-plane pucker max = VolCOS(P P, Vj relation P in nucleic acids SOUTH C2'-endo c5' s 1 o3' C2 -erdo 0° < P < 36° north (prevalent in ) 144°< P< 190° south (prevalent in DNA) Helical parameters Helical... Helical parameters Base pairing Watson-Crick pairs Base pairing H A H2N. 2hJ H" " - -O CH- NN 1hJHN H 0 Hoogsteen and reverse Hoogsteen pairs H H / H-N N 2hJ NN N- .N H — N C H o A and B double helix A and B helices A-RNA with bulge A and B helices Nuclear properties of selected isotopes Isotope Y x 10 V at 11.74T Natural Sensitivity (I=V2) (rad T s ) (MHz) Abundance (%) Bal.a Abs.fc 1 H 26.75 500.0 99.98 1.00 1.00 13c 6.73 125.7 1.11 1.6xl0~2 1.8xl0"4 15 N -2.71 50.7 0.37 l.OxlO"3 3.8xl0~6 31 P 10.83 202.4 100 6.6xl0~2 6.6xl0~2 1 Relative sensitivity at constant field for equal number of nuclei. Product of relative sensitivity and natural abundance. Spin systems in ribose and deoxyribose 5' O-CH Base 0- 0-CH2 Base 0 H h^-tfH B-D-Rjbose XWTPMA 'C—c 0^ H 2" 2'-Deoxy-f3-D-Ribos XAMWTNP Spin systems in nucleic acid bases INH* ^C2 6C ° XNX H Cytosine.C AX H; N 0 II CH: Thymine, T A3X o Uracil, U AX N :NH?: H- * 4 7 \ N Adenine, A A+ A o ii iH=!Nrc%N/c-N/ Guanine, G A C-H H chemical shift ranges in DNA and RNA H chemical shift ranges in DNA and RNA Code 8 (ppm) Commpnts 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 spectrum of d(CGCGAATTCGCG) 1H NMR spectra in H20 1H COSY spectrum of DNA d(CGCGAATTCGCG)2 - s b ■ i■■■■i■-■■i■■■■i■■■■i■ ■ ■ -1 ■ ..i......... i.... i.... i.... i.... i.. 3.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm -1.5 2.0 -25 a H2'-H2" -3.0 b H4,-H5,,5" -3.5 HS'-HS" -4.0 H3-H4' c 4.5 d H2T,2"-H3' -5.0 e H1'-H2',2" -5.5 6.0 f H5-H6 (Cyt) -6.5 g CH3-H6 (Thy) 7.0 -7.5 8.0 ppm 1H TOCSY spectrum of DNA 1H NOESY spectrum of DNA in D20 1H NOESY spectrum of DNA in H20 b c ll ; ll I i 1 CytHS 1 f i CytH4 ! AdeH2 CytH4(HB) * ■ > * I If -ilCi * *i t r 14 13 12 11 10 9 7 d(CGCGAATTCGCG)2 a H imino - H imino b H imino - H amino H imino - AdeH2,CytH5 c H amino - H amino H amino - AdeH2,CytH5,H6 d H imino - TCH3 4 3 2 1 ppm Water Suppression The presence of an intense solvent resonance necessitates an impractical high dynamic range. 110 M vs How do we set distance information? o o Nuclear Overhauser effect (< 6A) a and t pose problems Determinants of 31P chem shift, e and t correlate. rc = -317-1.23 e p 8 'JP5'-H5'(H5") JH4'-H5'(H5") JP3-H3' JHl'-C6 (UJP5'-C4' JC3'-H5'(H5") JP3-C2' JH1'-C2 (UATJ 3 t 3 ~JP3'-C4' JH1'-C8 (A,G) 3jHl'^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-Bond constraints What do we want? • Low energy structures Methods • Distance Geometry • Simulated annealing, rMD •Torsion angle dynamics (DYANA) • Mardigr as/IRMA/Morass optimize conditions pH, I, T. 1D NMR Assignments spin system sequential long range Distance constraints Torsion constraints Distance Geometry/ simulated annealing Initial structure(s) 1 Reffine structure(s) NOESY. TOCSY, COSY NOESY, COSY Use contraints to calculate structure Identify additional constraints (side chains, additional long range contacts etc) rMD calculations Structures \ y Additional Experiments f Dynamics y Mutants Interaction with target/drug