Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics...

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Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry

Transcript of Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics...

Page 1: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Structure determination: Nucleic acid

May 13, 2004

TIGP Chemical Biology and Molecular Biophysics

Lou-Sing Kan, Ph. D.

Institute of Chemistry

Page 2: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Structure and nomenclature of nucleic acid bases and sugars

Page 3: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Oligonucleotide chain (sugar-phosphate backbone)

Page 4: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

The Watson-Crick base pairs The non Watson-Crick base pairs

Page 5: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

The structures of DNA

Page 6: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

CruciformSupercoil Tetra-stranded

Other structures of nucleic acids

Triple-stranded

Hairpin loop

Bulge loop

Internal loop

Mismatch

Knots

Page 7: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Nucleic acids: RNA and DNA

Ribonucleic Acids (RNA) composed by adenosine, guanosine, cytidine, uridine

Page 8: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Spectra of nucleosides in DMSO-d6

Adenosine

Cytidine

Guanosine

Uridine

www.aist.go.jp/RIODB/SDBS/menu-e.html

Page 9: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Deoxyribonucleic Acids (DNA) composed by deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine

Page 10: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Comparison of nucleoside and deoxyribonucleoside in DMSO-d6

Comparison of nucleoside in DMSO-d6 and D2O

Adenosine

Deoxyadenosine

Cytidine in DMSO-d6

Cytidine in D2O

Page 11: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Structure and nomenclature of furanose and deoxyfuranose

Page 12: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Sugar-phosphate backbone conformation

Page 13: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

The conformation of glycosidic bond

Page 14: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Proton NMR study on two structures of 3'-O-(Acetylimino)3'-de(phosphinico)-thymidylyl-(3’,5')-deoxythymidine in aqueous solution

O

O3'

NCAC

H3CO

OH

HONH

N

O

O

NH

N

O

O

'

NC

''

OAC

Tetrahedron 59 (2003) 1981-1988

Page 15: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.
Page 16: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Table I. Temperature dependent chemical shifts of TNT

TN H6 C5-CH3 H1 H2 H2 H3 H4 H5, H5 35℃ 7.48 1.74 6.13 2.47 2.21 4.69 4.14 3.63

25℃ 7.50 1.74 6.14 2.47 2.21 4.70 4.15 3.63

20℃ 7.50 1.74 6.14 2.46 2.21 4.71 4.15 3.63

15℃ 7.51 1.74 6.11 2.46 2.21 4.71 4.15 3.64

10℃ 7.51 1.73 6.12 2.49/2.41* 2.27/2.17 4.71 4.16/4.14 3.64

0℃ 7.53 1.73 6.14 2.49/2.41 2.27/2.17 4.73/4.71 4.17/4.13 3.64

NT H6 C5-CH3 H1 H2 H2 H3 H4 H5, H5 35℃ 7.29 1.74 6.09 2.24 2.32 4.34 4.02 3.87, 3.95

25℃ 7.30 1.74 6.10 2.24 2.32 4.33 4.03 3.88, 3.96

20℃ 7.31 1.74 6.11 2.23 2.32 4.32/4.39 4.03 3.88, 3.95

15℃ 7.32/7.28 1.74 6.11/6.19 2.23 2.32 4.32/4.40 4.04 3.90, 3.95

10℃ 7.33/7.28 1.73 6.12/6.21 2.21/2.21 2.31/2.36 4.32/4.41 4.04/4.01 3.90, 3.95

0℃ 7.34/7.29 1.73 6.14/6.23 2.21/2.21 2.31/2.36 4.33/4.42 4.06/4.01 3.94, 3.94

-COCH3

35℃ 2.03

25℃ 2.06/1.98

20℃ 2.07/1.96

15℃ 2.07/1.96

10℃ 2.07/1.95

0℃ 2.08/1.95

Table II. Coupling constants of sugar proton resonances

JH1-H2 JH1-H2 JH3-H2 JH3-H2 JH3-H4 JH4-H5 JH4-H5

TN 6.14 8.19 6.14 2.30 2.3 4.35 4.35

NT 6.66 6.66 6.14 6.14 4.10 7.42 3.58

Page 17: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.
Page 18: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

major minor

N

3'T

O

O

CH3 5'T

N

3'T

O

H3C

5'TO

A) Conformation of major form of -5 C

B) Conformation of minor form of -5 C

1) 2)

2)1)

Page 19: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

The Application of Chemical Shift

Proton magnetic resonance studies on the nonexchangeable protons of ribosyl ApApGpCpUpU

Biochemistry, 14 (1975) 4847-4863 & 4864-4869.

Page 20: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.
Page 21: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.
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Biopolymers, 15 (1976) 2277-2286.

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Page 24: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.
Page 25: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

The Application of Coupling Constant

Page 26: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

The Application of NOE

Detection of a guanine/adenine base pair in a decadeoxyribonucleotide by proton magnetic resonance spectroscopy

Proc. Natl. Acad. Sci., 80 (1983) 4263-4265.

Science, 238 (1987) 498-504.

(CCAAGATTGG)2

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Page 28: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

The Application of T1

Internal loop vs. hairpin

Biochemistry, 27 (1988) 3024-3031.

Page 29: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

O

HO

N

N N

N

OH

NH2

H H

OH

Page 30: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

2D COSY or DQF-COSY

2D TOCSY

2D NOESY

2D E.COSY

2D PE.COSY

Methods

ppm

2.53.03.54.04.55.05.56.06.5 ppm

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

Page 31: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

Assignments based on structure

Page 32: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

K. C. Wuthrich, NMR of Proteins and Nucleic Acids, John Wiley & Sons (1986)

Page 33: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

The Study of Triplex Formation of TCTCTCCTCTCTAGAGAG

Page 34: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.
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The assignment

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Biophysical J., (2002) 82, 3170-3180.

Page 38: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.
Page 39: Structure determination: Nucleic acid May 13, 2004 TIGP Chemical Biology and Molecular Biophysics Lou-Sing Kan, Ph. D. Institute of Chemistry.

References

Edwin D. BeckerHigh Resolution NMR, Theory and Chemical Applications, 3rd EditionAcademic Press, 2000.Ray FreemanMagnetic Resonance in Chemistry And MedicineOxford, 2003Joseph P. HornakThe Basic of NMRhttp://www.cis.rit.edu/htbooks/nmr/bnmr.htm

General

T.C. FarrarAn Introduction To Pulse NMR SpectroscopyFarragut Press, Chicago, 1987.H. Gunther"Modern pulse methods in high-resolution NMR spectroscopy."Angew. Chem.. Int. Ed. Engl.22:350-380 (1983)

Basic Pulse NMR

Ad BaxTwo-Dimensional Nuclear Magnetic Resonance in LiquidDelft University Press, 1982Richard R. Ernst, Geoffrey Bodenhausen, Alexander WokaunPriciples of NMR in One and Two DimensionsOxford, 1987

2D NMR

Peter BiglerNMR Spectroscopy Processing StrategiesVCH, 1997

Data Process

H. Duddeck, W. DietrichStructure Elucidation by Modern NMR, A WorkbookSpringer-Verlag, 1989

Application: small molecules

Kurt WuthrichNMR of Proteins and Nucleic AcidsJohn Wiley & Sons, 1986

C. A. G. HaasnootNMR in Conformation Analysis of Bio-organic Molecules

Application: Peptides and Proteins

Application: Nucleic Acids

G. C. K. RobertsNMR of Macromolecules, A Practical ApproachIRL Press, 1995.

Application: Others

S. Braun, H.-O. Kalinowske, S. Berger100 and More Basic NMR ExperimentsVCH, 1996S. W. HomansA Dictionary of Concepts in NMROxford, 1992Handbook of High Resolution Multinuclear NMRJohn Wiley & Sons, 1981

Dictionary