Nucleic Acid NMR

Part II

Resonance Assignment DNA/RNA (Homonuclear)

Resonance Assignmnent DNA/RNA (Homonuclear)

7.4

αC8

A5

ppm

5.45.65.86.06.2 ppm

7.6

8.0

T6

C10T7C2

G1G9G3

A48.2

7.8

7.2

NOESY Connectivity (e.g. α C Decamer)NOESY Connectivity (e.g. α C Decamer)

G1-H1’

G1-H8

7.4

αC8

A5

ppm

5.45.65.86.06.2 ppm

7.6

8.0

T6

C10T7C2

G1G9G3

A48.2

7.8

7.2

7.4

αC8

A5

ppm

5.45.65.86.06.2 ppm

7.6

8.0

T6

C10T7C2

G1G9G3

A48.2

7.8

7.2

7.4

αC8

A5

ppm

5.45.65.86.06.2 ppm

7.6

8.0

T6

C10T7C2

G1G9G3

A48.2

7.8

7.2

2'2''

2'2''

2'2''

G

αC

T3'-3'

5'-5'

H

H

H

5’- CATGCATG GTACGTAC – 5’

DNA Miniduplex

31P NMR

Two- and Three-dimensional 31P-driven NMR Procedures for completeassignment of backbone resonances in oligodeoxyribonucleotides. G.W. Kellogand B.I. Schweitzer J. Biomol. NMR 3, 577-595 (1993).

31P NMR

ppm

4.04.24.44.64.85.05.2 ppm

�-2.0

�-1.5

�-1.0

�-0.5

1.0

0.5

0.0

P7

P8

P3

P2

P6P4

P1

P5

P9

ppm

4.04.24.44.64.85.05.2 ppm

�-2.0

-�1.5

�-1.0

-�0.5

1.0

0.5

0.0

P6

P4

P8P2P9P1P7P5

P3

ppm

4.04.24.44.64.85.05.2 ppm

�-1.5

�-1.0

�-0.5

1.0

0.5

0.0

P2

P3

P5P6 P7

P1P4

P9P8

AlphaC

3’ 4’ 5’,5’’

;****************************************;mwgcorrpt, AMX version;X-H correlation. H-detected;Sklenar et al., 1986, FEBS, 208, 94-98;****************************************d12=20up2=p1*2

1 ze d11 dhi2 d113 d12 p2 ph0 d2 lo to 3 times l1 d3 (p3 ph2):d d0 (p1 ph1) (p3 ph1):d go=2 ph31 d11 wr #0 if #0 id0 ip2 zd lo to 3 times td1 do exit

ph0=0ph1=0ph2=0 0 2 2ph31=2 2 0 0

;>>>>>>>>>>>>>>>DELAYS;d0 = 3us;d2 = 50ms;d3 = 3us;d11= 30 msec

;>>>>>>>>>>>>>>>PULSES;p1 = 90 deg proton pulse hl1 = 1;p2 = 180 deg proton pulse hl1 = 1;p3 = 90 deg X pulse;>>>>>>>>>>>>>>>LOOP-COUNTER;l1 = loop counter for presaturaton;l1*d2 = relaxation delay (l1=40, d2=50ms >>2s);>>>>>>>>>>>>>>>COMMENTS;rd=pw = 0, nd0 = 2, in0 = 1/(2*SW);ns = 4*n, ds = 4, MC2= TPPI;-----------------------END of PROGRAM---------------

Backbone Experiments:

Harald Schwalbe, Wendelin Samstag, Joachim W. Engels, Wolfgang Bermel, and Christian Griesinger,"Determination of 3J(C,P) and 3J(H,P) Coupling Constants in Nucleotide Oligomers", J. Biomol. NMR 3, 479-486

Z. Wu, N. Tjandra, and A. Bax, Measurement of H3’-31P dipolar couplings in a DNA oligonucleotide byconstant-time NOESY difference spectroscopy, J. Biomol. NMR 19, 367–370 (2001).

G. M. Clore, E. C. Murphy, A. M. Gronenborn, and A. Bax, Determination of three-bond H3’-31P couplings innucleic acids and protein-nucleic acid complexes by quantitative J correlation spectroscopy, J. Mag. Reson.134, 164–167 (1998).

BioNMR in Drug Research 2003Editor(s): Oliver ZerbeMethods for the Measurement of Angle Restraints from Scalar, Dipolar Couplings and from Cross-CorrelatedRelaxation: Application to BiomacromoleculesChapter Author: Christian Griesinger:J-Resolved Constant Time Experiment for the Determination of the Phosphodiester Backbone Angles α and ζ.

RNA DNA

Heteronuclear Methods

Structure Determination:

I) Assignment

II) Local Analysis•glycosidic torsion angle, sugar puckering,backbone conformationbase 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 •Dipolar couplings

What do we know?What do we know?••Distance, Torsion, H-Bond constraintsDistance, Torsion, H-Bond constraintsWhat do we want?What do we want?••Low energy structuresLow energy structures

MethodsMethods••Distance GeometryDistance Geometry••Simulated annealing,Simulated annealing, rMD rMD••Torsion angle dynamics (DYANA)Torsion angle dynamics (DYANA)••MardigrasMardigras/IRMA/Morass/IRMA/Morass

1D NMR

optimize conditionspH, I, T.

Assignments spin system sequential long range

NOESY, TOCSY, COSY

Distance constraintsTorsion constraints

Distance Geometry/simulated annealing

NOESY, COSY

Initial structure(s)

Use contraints to calculate structure

Identify additional constraints(side chains, additional long range contacts etc)

Reffine structure(s) rMD calculations

Structures

Additional ExperimentsDynamics

MutantsInteraction with target/drug

Dipolar Couplings… is the kink real ?

• Dipolar couplings add to J coupling• 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

N

NN

N

NH2

H

HO

HO

HHHH

OH

H

Aligned with pf1

αA

NOE RDC + NOE

RMSD (all atoms) 0.66 C3' DG5 1 -- H3' C4' DT 2 -- H4' C6 DT 2 -- H6 C1' DC 4 -- H1' C1' ADA 5 -- H1' C4' ADA 5 -- H4' C2 ADA 5 -- H2 C4' DC 6 -- H4' C8 DA 8 -- H8 C1' DC 9 -- H1' C3' DC 9 -- H3' C6 DC 9 -- H6 C1' DG3 10 -- H1' C4' DG3 10 -- H4' C1' DC5 11 -- H1' C4' DC5 11 -- H4' C1' DT 13 -- H1' C6 DT 13 -- H6 C4' DC 14 -- H4' C6 DC 14 -- H6 C8 DG 15 -- H8 C1' DT 16 -- H1' C1' DG 17 -- H1' C3' DC3 20 -- H3' C4' DC3 20 -- H4'

General references, NMR techniques, sample preparation, analysis

BioNMR in Drug Research. Edited by Oliver Zerbe, 2002 Wiley VerlagWijmenga, S. S., Mooren, M. M. W. and Hilbers, C. W. (1993) in Roberts, G. C. K. (ed.) NMR of

Macromolecules; A Practical Approach. Oxford University Press, NY.Zidek L., Stefl R and Sklenar V. (2001) "NMR methodology for the study of nucleic acids"Curr.

Opin. Struct. Biol., 11, 275-28

NMR structure determination: DNA DNA/RNA, pseudorotation analysis, dynamics.See also referenced quoted in the listed papers

Altona, C., Francke, R., de Haan, R., Ippel, J. H., Daalmans, G. J., Westra Hoekzema, A. J. A.and van Wijk, J. (1994) Magn. Reson. Chem., 32, 670-678.

Aramini, J. M., Cleaver, S. H., Pon, R. T., Cunningham, R. P. & Germann, M.W: SolutionStructure of a DNA Duplex Containing an a-Anomeric Adenosine: Insights into SubstrateRecognition by Endonuclease IV. J. Mol. Biol. (2004), 338, 77-91.

Aramini, J. M., Mujeeb, A., Ulyanov, N. B. & Germann, M. W.: Conformational Dynamics in Mixeda/b- Oligonucleotides Containing Polarity Reversals: A Molecular Dynamics Study usingTime-averaged Restraints. J. Biomol. NMR, (2000), 18, 287-303.

Aramini, J. M. & Germann, M. W. NMR solution structure of a DNA/RNA hybrid containing analpha anomeric thymidine and polarity reversals. Biochemistry, (1999), 38, 15448-15458.

Donders, L. A., de Leeuw, F. A. A. M. and Altona, C. (1989) Magn. Reson. Chem., 27, 556-563.van Wijk, J., Huckriede, B. D., Ippel, J. H. & Altona, C. (1992) Methods Enzymol., 211, 286-306.Bax, A., Lerner, L.. "MEASUREMENT OF H-1-H-1 COUPLING-CONSTANTS IN DNA

FRAGMENTS BY 2D NMR." . J Magn Reson. 79 429 - 438, 1988..Szyperski, T., Fernández, C., Ono, A., Kainosho, M. and Wüthrich, K. (1998) Measurement of

Deoxyribose 3 JHH Scalar Couplings Reveals Protein-Binding Induced Changes in theSugar Puckers of the DNA. J. Am. Chem. Soc. 120, 821- 822

Iwahara J, Wojciak JM, Clubb RT. (2001), An efficient NMR experiment for analyzing sugar-puckering in unlabeled DNA: application to the 26-kDa dead ringer-DNA complex. J MagnReson. 2001, 153, 262

Multinuclear experiments, DNA/RNA

Pardi, A. and Nikonowicz, E.P. (1992) Simple procedure for resonance assignment of the sugarprotons in 13C labeled RNA J. Am. Chem. Soc., 114, 9202–9203

Sklénar, V., Miyashiro, H., Zon, G., Miles, H.T., Bax, A. (1986) Assignment of the 31P and 1Hresonances in oligonucleotides by two-dimensional NMR spectroscopy FEBS Lett., 208,94–9

Varani, G., Aboul-ela, F., Allain, F., Gubser, C.C. (1995) Novel three-dimensional 1H–13C–31Ptriple resonance experiments for sequential backbone correlations in nucleic acids J.Biomol. NMR, 5, 315–3

Legault, P., Farmer, B.T. II , Mueller, L. and Pardi, A. (1994) Through-bond correlation ofadenine protons in a 13C-labeled ribozyme. J. Am. Chem. Soc., 116, 2203-2204

Marino, J.P, Prestegard, J.H. & Crothers, D.M. (1994) Correlation of adenine H2/H8 resonancesin uniformly 13C labeled RNAs by 2d HCCH-TOCSY: a new tool for 1H assignment. J. Am.Chem. Soc., 116, 2205-2206

Sklenár, V., Peterson, R.D., Rejante, M.R., Wang, E. & Feigon, J. (1993) Two-dimensional triple-resonance HCNCH experiment for direct correlation of ribose H1 and Base H8, H6 protonsin 13C, 15N-labeled RNA oligonucleotides. J. Am. Chem. Soc., 115, 12181-12182

Sklenár, V. Peterson, R.D., Rejante, M.R. & Feigon, J. (1994) Correlation of nucleotide base andsugar protons in 15N labeled HIV RNA oligonucleotide by 1H-15N HSQC experiments. J.Biomol. NMR, 4, 117-122

P Schmieder, J H Ippel, H van den Elst, G A van der Marel, J H van Boom, C Altona, and HKessler (1992) Heteronuclear NMR of DNA with the heteronucleus in natural abundance:facilitated assignment and extraction of coupling constants. Nucleic Acids Res. 25;4747–4751.

H. Schwalbe, J. P. Marino, G. C. King, R. Wechselberger, W. Bermel, and C. Griesinger (1994)"Determination of a complete set of coupling constants in 13C-labeled oligonucleotides", J.Biomol. NMR 4, 631-644

Trantirek L., Stefl R., Masse J.E., Feigon J. and Sklenar V. (2002)"Determination of the glycosidictorsion angles in uniformly 13C-labeled nucleic acids from vicinal coupling constants3J(C2)/4-H1' and 3J(C6)/8-H1'" J. Biomol. NMR., 23(1):1-12

Szyperski, T., Ono, A., Fernández, C., Iwai, H., Tate, S., Wüthrich, K. and Kainosho, M. (1997)Measurement of 3JC2'P Scalar Couplings in a 17 kDa Protein Complex with 13 C,15N-Labeled DNA Distinguishes the B I and BII Phosphate Conformations of the DNA. J. Am.Chem. Soc. 119, 9901 -990

Szyperski, T., Fernandez, C., Ono, A., Wüthrich, K. and Kainosho, M. (1999) The { 31P}-Spin-echo-difference Constant-time [ 13C,1 H]-HMQC Experiment for SimultaneousDetermination of 3 JH3'P and 3JC4'P in Nucleic Acids and their Protein Complexes. J.Magn. Reson. 140, 491 -494.

H. Schwalbe, W. Samstag, J. W. Engels, W. Bermel, and C. Griesinger, (1993) "Determination of3J(C,P) and 3J(H,P) Coupling Constants in Nucleotide Oligomers", J. Biomol. NMR 3, 479-486

C. Richter, B. Reif, K. Wörner, S. Quant, J. W. Engels, C. Griesinger, and H. Schwalbe (1998)"New Experiment for the Measurement of 3J(C,P) Coupling Constants including 3J(C4'i,Pi)and 3J(C4'i,P i+1) coupling constants in Oligonucleotides" J. Biomol. NMR 12, 223-23