Nucleic Acids Unit: Nucleic Acids Nucleus DNA structure DNA replication.
NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH...
Transcript of NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH...
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NUCLEIC ACIDS
Basic terms and notions
Presentation by
Eva Fadrná
adapted by Radovan Fiala
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RNA vs DNA
Single strand A-RNA B-DNA duplex
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Length of NA
Total length of DNA in a human cell 1 m (1000 km)�
DNA in typical human chromozome 1 cm (10 km)�
DNA from bacterial chromozome 1 mm
Diameter of typical human cell 0.01 mm
Diameter of folded DNA 0.1 µm (0.1 m)�
Diameter of DNA fiber 1 nm (1 mm)�
Diameter of atom 1 Å
(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
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Nukleotide chainH –
H –
H –
N
N
N9
N
O5’
O2’O3’
NH2
C1’
C3’
C4’
C5’O4’
C2’
– H
– H
– H
– H
H|
– H
H –
H –
H –
N
N
N9
N
O5’
O2’O3’
NH2
C1’
C3’
C4’
C5’O4’
C2’
– H
– H
– H
– H
H|
– H
P
O
O
H –
H –
H –
N
N
N9
N
O5’
O2’O3’
NH2
C1’
C3’
C4’
C5’O4’
C2’
– H
– H
– H
– H
H|
– H
P
O
O
H –
H –
H –
N
N
N9
N
O5’
O2’O3’
NH2
C1’
C3’
C4’
C5’O4’
C2’
– H
– H
– H
– H
H|
– H
P
O
O
nucl
eotid
e
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Groovesmajor vs. minor
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RNA vs DNA
uridinedeoxythymidine
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Nukleotide/nukleoside
base
sugar (ribose/deoxyribose)�
phosphate
+
nukleoside
+
nukleotide
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Bases
Adenin (Ade) �Guanin (Gua) �
Cytosin (Cyt)�Thymin (Thy) � Uracil (Ura) �
PU
RIN
ES
PY
RIM
IDIN
ES
sugar
DNA RNA
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Base numbering
PURINES PYRIMIDINES
N
N
NN
NN
H
3
2
1
4
57 6
9
83
6
5
1
4
2
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Base tautomerismenol ↔↔↔↔ keto
enamin ↔↔↔↔ imin
C = C – N CH – C = N – HH
H→→→→←←←←
C = C – O – H CH – C = O→→→→←←←←
fysiolog. conditions
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Base tautomerism enol ↔↔↔↔ keto enamin ↔↔↔↔ imin
C = C – N CH – C = N – H
H
H
C = C – O – H CH – C = O→→→→←←←← →→→→←←←←
N
HN
N
N
N
N
N
HN
H
O
OH
H
HO
OH
HOO
O O
→→→→←←←← →→→→←←←← →→→→←←←←
4 tautomers
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Sugar - pentoses
OHO
HO
OHOH
CH2 OHO
HO
OH
CH2
ß – D - ribose 2 – deoxy – ß – D - ribose
RNA DNA2’
1’
5’
4’
3’
semiacetal hydroxyl group
2’
1’
5’
4’
3’
semiacetal hydroxyl group + base N-glycosidic bond nukleoside
C1´ - N1
C1´ - N9
pyrimidinespurines
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Nukleosides
N
N
N9
N
OHO
OHOH
NH2
O N1
HN
OHO
OHHO
O
N-glykosidic bond
pyrimidinespurines
C1´ - N1C1´ - N9
C1’ C1’
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Nukleosides
Ribonukleosidesuridine = Ucytidine = Cadenosine = Aguanosine = G
Deoxyribonukleosidesdeoxythymidine = dTdeoxycytidine = dCdeoxyadenosine = dAdeoxyguanosine = dG
OHO
(OH) �HO
base
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Phosphate group
O = P – OH
orthophosphoric acid
H3PO4
N
N
N9
N
OHO
OHOH
NH2
C1’
OH
OH
N
N
N9
N
O
OHOH
NH2
C1’
O = P – O
O
O
-
-
acid alcohol+ ester
+
adenosine adenosine(mono)phosphate (AMP)�
adenosine
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Nukleotides
Ribonucleotidesuridyl acid = uridine – 5´monophosphate = UMP, pUcytidyl acid = cytidin -“- = CMP, pCadenyl acid = adenosin -“- = AMP, pAguanyl acid = guanosin -“- = GMP, pG
Deoxyribonucleotidesdeoxytymidyl acid = 2´deoxythymidine-5´-monophosphate = dTMP, pdTdeoxycytidyl acid = -“- cytidin -“- = dCMP, pdCdeoxyadenyl acid = -“- adenosin -“- = dAMP, pdAdeoxyguanyl acid = -“- guanosin -“- = dGMP, pdG
O
(OH) �HO
baseO = P – O
OH
OH
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Phosphate group
N
N
N9
N
OHO
OHOH
NH2
C1’
N
N
N9
N
O
OHOH
NH2
C1’
O = P – O
OH
O -
N
N
N9
N
OHO
OHO
NH2
C1’
N
N
N9
N
O
OHOH
NH2
C1’
O = P – O
O -
alcohol acid+ diester(ester)�
nukleoside-nukleotide
ApA
5’
3’
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Nucleotide chain
H –
H –
H –
N
N
N9
N
O5’
O2’O3’
NH2
C1’
C3’
C4’
C5’O4’
C2’
– H
– H
– H
– H
H|
– H
H –
H –
H –
N
N
N9
N
O5’
O2’O3’
NH2
C1’
C3’
C4’
C5’O4’
C2’
– H
– H
– H
– H
H|
– H
P
O
O
H –
H –
H –
N
N
N9
N
O5’
O2’O3’
NH2
C1’
C3’
C4’
C5’O4’
C2’
– H
– H
– H
– H
H|
– H
P
O
O
H –
H –
H –
N
N
N9
N
O5’
O2’O3’
NH2
C1’
C3’
C4’
C5’O4’
C2’
– H
– H
– H
– H
H|
– H
P
O
O
5’
3’
nucl
eotid
e
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Torsion angle
A
B C
D
�
�
⊕⊕⊕⊕
<0o, 360o>
<-180o, 180o>
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Torsion angle
synclinal (sc)�
anticlinal (ac)�
antiperiplanar (ap)�
synperiplanar (sp)�
+gauche (+g)�-gauche (-g)�
trans (t)�
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Torsion angles in NA
Sugar-phosphate backbone
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Torsion angles cont.
ααααββββγγγγδδδδεεεεζζζζχχχχ
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Torsion angle χSYN:
Pyrimidines: O2 above the sugar ringPurines: 6-member purine ring above the sugar ring
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Torsion angle χOrientation around the C1’ – N glycosidic bond
SYN
ANTI
O4’ – C1’ – N1 – C2 pyrimidinesO4’ – C1’ – N9 – C4 purines
<0o, 90o>
<270o, 360o><90o, 270o>
χ
χ
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Torion χ – border intervals
SYN
ANTI<0o, 90o>
<270o, 360o><90o, 270o>
χ
χ
high-syn (corresponds to +ac) … 90o + intrudes into anti
high-anti (corresponds -sc) … 270o + intrudes into syn
∈
∈
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Torsion angles in DNA
Angle B-DNA A-DNAα -40.7 -74.8β -135.6 -179.1γ -37.4 58.9δ 139.5 78.2ε -133.2 -155.0ζ -156.9 -67.1χ -101.9 -158.9
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Sugar conformation
O3’
C5’
δδδδ
δδδδ … C5’ – C4’ – C3’ – O3’
endocyclic
exocyclic
δ, ν3 relation
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„Puckering“ of the sugar ring
Envelope 4 atoms in a plane, the 5th above or below
Twist 3 atoms in a plane, the 4th
and the 5th on the oposite sides of the plane
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Definition of the puckering modes
The sugar ring is not planar
C1’ – O4’ – C4’ plane
Envelope C3’-endo 3E (prevalent in RNA)�
Envelope C2’-endo 2E (prevalent in DNA)�
symmetric Twist C2’-exo-C3’-endo 32T
Non-symmetric Twist C3’-endo-C2’-exo 3T2
With respect to C5’- endo- exo
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Pseudorotation cycleTheoretically – infinite number of conformations, can be characterized bymaximum torsion angle (degree of pucker) and pseudorotation phase angleTorsion angles are not independent (ring closed)�
Pseudorotation phase angle P
P = 0o :symmetric Twist C2’-exo-C3’-endo 32T
P = 180o :asymmetric Twist C2’-endo-C3’-exo 23T
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νmax amplitude
Maximum out-of-plane pucker
νmax = ν2 / cos(P) �
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P, νj relation
j = 0 .. 4
P value defines unambiguously all endocyclic torsion angles ν0 to ν4
Sum of all 5 ν = 0ν2 = νmax ⋅ cos(P + (j - 2) ⋅ 144°) �ν0 + ν1 + ν2 + ν3 + ν4 = 0
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P in nucleic acids
NORTH
SOUTH
0o ≤ P ≤ 36o north (prevalent in RNA)�
144o ≤ P ≤ 190o south (prevalent in DNA)�
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Helical parametersaxis-base, axis-base pairintra-base pairinter-base or inter-base pair
Distance/shift Angle
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Helical…D … displacement from helical axis
θθθθR … rollθθθθT … tilt
t …twist = 360o / n
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Helical parametersfor A and B DNA
Global B-DNA A-DNAX disp. 0.0 -5.28Y disp. 0.0 0.0Inclin 1.46 20.73Tip 0.0 0.0Shear 0.0 0.0Stretch 0.0 0.01Stagger -0.08 -0.04Buckle 0.0 0.0Propeller -13.3 -7.5Openning 0.0 -0.02Shift 0.0 0.0Slide 0.0 0.0Rise 3.38 2.56Tilt 0.0 0.0Roll 0.0 0.0Twist 36.00 32.70Bases per turn 360/36=10 360/32.7=11
Shifts in Å, angles in degrees
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Base pairing
Watson-Crick pairs
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Base pairing
Hoogsteen and reverse Hoogsteen
pairs
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A and B double helix
A-RNA
B-DNA
Ball and stick
models
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A and B helices
A-DNA B-DNA A-RNA with
bulge
![Page 41: NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH SOUTH 0o ≤P ≤36 o north (prevalent in RNA ) 144 o ≤P ≤190 o south (prevalent](https://reader036.fdocuments.us/reader036/viewer/2022071100/5fd8c609dbb46a548171f8aa/html5/thumbnails/41.jpg)
A and B helices
A-DNA B-DNA A-RNA with bulge
View tilted by 32° to show grooves
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Nuclear properties of selected isotopes
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Spin systems in ribose and deoxyribose
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Spin systems in nucleic acid bases
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1H chemical shift ranges in DNA and RNA
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1H chemical shift ranges in DNA and RNA
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1H NMR spectra of d(GCATGC)
Single strand
Duplex
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1H NMR spectra in D2O and H2O
d(GCATTAATGC)2
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1H COSY spectrum of DNA
d(CGCGAATTCGCG)2
a H2’-H2’’b H4’-H5’,5’’
H5’-H5’’c H3’-H4’d H2’,2’’-H3’e H1’-H2’,2’’f H5-H6 (Cyt)�g CH3-H6 (Thy)�
![Page 50: NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH SOUTH 0o ≤P ≤36 o north (prevalent in RNA ) 144 o ≤P ≤190 o south (prevalent](https://reader036.fdocuments.us/reader036/viewer/2022071100/5fd8c609dbb46a548171f8aa/html5/thumbnails/50.jpg)
1H NOESY spectrum of DNAd(
CG
CG
AA
TT
CG
CG
) 2
![Page 51: NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH SOUTH 0o ≤P ≤36 o north (prevalent in RNA ) 144 o ≤P ≤190 o south (prevalent](https://reader036.fdocuments.us/reader036/viewer/2022071100/5fd8c609dbb46a548171f8aa/html5/thumbnails/51.jpg)
Water Suppression
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WATERGATE
![Page 55: NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH SOUTH 0o ≤P ≤36 o north (prevalent in RNA ) 144 o ≤P ≤190 o south (prevalent](https://reader036.fdocuments.us/reader036/viewer/2022071100/5fd8c609dbb46a548171f8aa/html5/thumbnails/55.jpg)
Structure Determination Procedure
![Page 56: NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH SOUTH 0o ≤P ≤36 o north (prevalent in RNA ) 144 o ≤P ≤190 o south (prevalent](https://reader036.fdocuments.us/reader036/viewer/2022071100/5fd8c609dbb46a548171f8aa/html5/thumbnails/56.jpg)
Resonance Assignment
![Page 57: NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH SOUTH 0o ≤P ≤36 o north (prevalent in RNA ) 144 o ≤P ≤190 o south (prevalent](https://reader036.fdocuments.us/reader036/viewer/2022071100/5fd8c609dbb46a548171f8aa/html5/thumbnails/57.jpg)
Sequential connectivitieswith exchangeable protons
d(GGAATTGTGAGCGG) �d(CCGCTCACAATTCC) �
imino-imino imino-amino
![Page 58: NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH SOUTH 0o ≤P ≤36 o north (prevalent in RNA ) 144 o ≤P ≤190 o south (prevalent](https://reader036.fdocuments.us/reader036/viewer/2022071100/5fd8c609dbb46a548171f8aa/html5/thumbnails/58.jpg)
Sequential resonance assignments
d(GCATTAATGC) 2
![Page 59: NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH SOUTH 0o ≤P ≤36 o north (prevalent in RNA ) 144 o ≤P ≤190 o south (prevalent](https://reader036.fdocuments.us/reader036/viewer/2022071100/5fd8c609dbb46a548171f8aa/html5/thumbnails/59.jpg)
Connectivities between H1’ and H6/8d(CGCGAATTCGCG) 2
i intra-residues sequential
![Page 60: NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH SOUTH 0o ≤P ≤36 o north (prevalent in RNA ) 144 o ≤P ≤190 o south (prevalent](https://reader036.fdocuments.us/reader036/viewer/2022071100/5fd8c609dbb46a548171f8aa/html5/thumbnails/60.jpg)
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Assignment of Sugar-Phosphate Backbone
HP-COSY HP-TOCSY
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J-couplings from COSY spectra
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P determination from J-couplings
1’2’
2’3’
1’2’’
3’4’
2’’3’
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Equilibrium of N and S conformations
3’-endo
2’-endo
![Page 72: NUCLEIC ACIDS - NCBRfiala/Graphics/NuclAcidsNMR_1.pdf · 2010. 4. 13. · P in nucleic acids NORTH SOUTH 0o ≤P ≤36 o north (prevalent in RNA ) 144 o ≤P ≤190 o south (prevalent](https://reader036.fdocuments.us/reader036/viewer/2022071100/5fd8c609dbb46a548171f8aa/html5/thumbnails/72.jpg)
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