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

Oligonucleotide chain (sugar-phosphate backbone)

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

The structures of DNA

CruciformSupercoil Tetra-stranded

Other structures of nucleic acids

Triple-stranded

Hairpin loop

Bulge loop

Internal loop

Mismatch

Knots

Nucleic acids: RNA and DNA

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

Spectra of nucleosides in DMSO-d6

Adenosine

Cytidine

Guanosine

Uridine

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

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

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

Structure and nomenclature of furanose and deoxyfuranose

Sugar-phosphate backbone conformation

The conformation of glycosidic bond

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

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

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)

The Application of Chemical Shift

Proton magnetic resonance studies on the nonexchangeable protons of ribosyl ApApGpCpUpU

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

Biopolymers, 15 (1976) 2277-2286.

The Application of Coupling Constant

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

The Application of T1

Internal loop vs. hairpin

Biochemistry, 27 (1988) 3024-3031.

O

HO

N

N N

N

OH

NH2

H H

OH

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

Assignments based on structure

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

The Study of Triplex Formation of TCTCTCCTCTCTAGAGAG

The assignment

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

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