Nucleic Acid NMR
Part II
O3’
α and ζ pose problems!
à determinants of 31P chem shift!
!
ε and ζ correlate. ζ = -317-1.23 ε !
nucleotide unit
α
β
γ
ν4
O4’
ν0
ν3
δ
ε
χ
ν1
ν2
ζ
O5’
Ranges !
χ
α
β
γ
δ
ε
ζ
B-DNA
Bf-DNA
Af-DNA
!-119
!-102
!-154
!-61
!-41
!-90
!180
!136
!-149
!57
!38
!47
!122
!139
! 83
!-187
!-133
!-175
! -91!
!-157!
! -45!
!
!
!
Sanger, Principles of nucleic acid Structures!
Springer 1984
!
Σ Backbone Experiments
• Z. Wu, N. Tjandra, and A. Bax, Measurement of H3 -31P dipolar couplings in a DNA oligonucleotide by
constant-time NOESY difference spectroscopy, J. Biomol. NMR 19, 367-370 (2001).
• A. Bax, N. Tjandra, W. Zhengrong. Measurements of 1H-31P dipolar couplings in a DNA oligonucleotide
by constant time NOESY difference spectroscopy, J. Mol. Biol., 19, 367-270, 91 ( 2001). • G. M. Clore, E. C. Murphy, A. M. Gronenborn, and A. Bax, Determination of three-bond H3 -31P couplings
in nucleic acids and protein-nucleic acid complexes by quantitative J correlation spectroscopy, J. Mag.
Reson. 134, 164-167 (1998).
• H. Schwalbe, W. Samstag, J. W. Engels, W. Bermel, & C. Griesinger, "Determination of 3J(C,P) and
3J(H,P) Coupling Constants in Nucleotide Oligomers", J. Biomol. NMR 3, 479-486 (1993).
• BioNMR in Drug Research 2003 Edito: O. Zerbe
Methods for the Measurement of Angle Restraints from Scalar, Dipolar Couplings and from CrossCorrelated Relaxation: Application to Biomacromolecules Chapter Author: Christian Griesinger:
J-Resolved Constant Time Experiment for the Determination of the Phosphodiester Backbone Angles α
and ζ.
591
Imino protons and pH!
Nucleic Acids
Acids Research,
Research,1994,
1996,Vol.
Vol.22,
24,No.
No.14
Nucleic
591
à  pH may change structure!
!pH changes may hide or show weak base pairs!
!
!
à  Buffer changes spectral properties!
!e.g. phosphate vs Tris buffer!
!
à Some DNA structures are incredibly stable!
Figure 3 – A) schematic structure of the c-m
the imino protons of the G tetrads are colo
green: bottom face of the quadruplex.
(600MHz spectrum of 0.5mM DNA, 308K 2
absence (bottom) and presence of co
iminoprotons corresponds to panel A. C
presence and absence of compound 10.
pH 7.0!
586–595
Nucleic Acids Research, 1996, Vol. 24, No. 4
 1996 Oxford University Press
Acid-induced exchange of the imino proton in
G·C pairs
Sylvie Nonin1,2, Jean-Louis Leroy1 and Maurice Guéron1,*
e 3. Imino proton spectra of d(ATATAGATCTATAT). The neutral pH
um
(top) is assigned
to the Watson–Crick
TheNo.
weak4spectrum
Nucleic
Acids Research,
1996, duplex.
Vol. 24,
Groupe
Biophysique
et due
de l’URA
1254protons
du CNRS, 91128 Palaiseau, France and
d 111
p.p.m.dewhich
appearsdeatl’Ecole
lower Polytechnique
pH is probably
to imino
2
CEA-Service
de
Biologie
et
Génétique
Moléculaire,
DBCM/DSV,
CEN
Saclay, 91191 Gif-sur-Yvette, France
paired or Hoogsteen-paired nucleosides from partially dissociated
es or from single strands. This spectrum (and also the peak of the
9, 1995;
and Accepted January
8, 1996
alReceived
T1 of November
the duplex)
is Revised
exchange-broadened
upon
addition of a proton
or (formiate, 0.17 M), whereas relaxation of the G6 imino proton in the
pH 11.0!
 1996 Oxford University Press
induced exchange of the imino proton in
Base Pair Lifetime!
H!
H!
O!
H!
H!
+ Catalyst!
AxC base pair life times
0.25
GC!
τex (s)
0.20
0.15
0.10
AT!
0.05
0.00
τex
=
τop
+
1
αKdKtr[Catalyst]
0
50
100
150
200
250
1
[Catalyst]
Mazurek et al. PNAS 2009
31
P backbone perturbations 
Base pair lifetimes
 < 5ms,
 6 ms - 30 ms,
 31 ms - 65 ms.
Resonance Assignment DNA/RNA (Homonuclear)
A)  Non Exchangeable Protons!
!
!•Aromatic Spin Systems
!!
!
!•Sugar Spin Systems !
!
!
!•Sequential Assignment
!
!
B)  Exchangeable Protons
!
!NOESY, DQFCOSY, TOCSY
!
!DQFCOSY, TOCSY!
!
!NOESY, 31P-1H HETCOR!
!
!1D, NOESY (11, WG, etc)!
C)  Correlation of Exchangeable !
!and Non Exchangeable Protons
! NOESY (excitation sculpting)!
!
!!
Assignment of Non Exchangeable Protons
Base and Sugar
COSY/TOCSY
TOCSY
C:
U:
T:
A:
H5-H6
H5-H6
CH3-H6
H8-H2 (H2 are generally difficult to assign)
COSY/TOCSY
H1 -H2 (H2 ) etc
NH2
O
U!
H
H2N
H
H
N
NH
C!
O
H
N
N
N
H
O
N
A!
N
N
H
J Zhang, A Spring, M W Germann J. Am. Chem. Soc. 131 5380. (2009
Sequential Assignment
NOESY Connectivity (e.g. α C Decamer)
ppm!
T6!
7.2!
7.4!
C2!
T7!
C10!
7.6!
α
C8!
7.8!
G3!
G9!
G1
C2
G1!
G1-H8!
8.0!
A5!
8.2!
A4!
6.2!
6.0!
5.8!
G1-H1 !
5.6!
5.4!
ppm!
G3
ppm!
T6!
7.2!
7.4!
C2!
T7!
C10!
7.6!
α
C8!
7.8!
G3!
G9!
G1
C2
G1!
8.0!
A5!
8.2!
A4!
6.2!
6.0!
5.8!
5.6!
5.4!
ppm!
G3
ppm!
T6!
7.2!
7.4!
C2!
T7!
C10!
7.6!
α
C8!
7.8!
G3!
G9!
G1
C2
G1!
8.0!
A5!
8.2!
A4!
6.2!
6.0!
5.8!
5.6!
5.4!
ppm!
G3
alphaC!
5 -G C G A A T T α!C! G C!
C G α!C! T T A A G C G-5 !
ppm!
T6!
7.2!
H!
C2!
T7!
C10!
7.4!
7.6!
α
C8!
7.8!
G3!
G9!
G1!
8.0!
T!
2'2''!
3'-3'!
α
C!
G!
H! 2'2''!
H!
A5!
8.2!
A4!
6.2!
6.0!
5.8!
5.6!
5.4!
ppm!
2'2''!
5'-5'!
DNA Miniduplex
5’- CATGCATG
GTACGTAC – 5’
Excercise
31P
NMR
5 ,5 !
4!
3!
ppm
-2.0
P3
-1.5
-1.0
ppm
P6
-2.0
-0.5
P7
AlphaC
P8
0.0
-1.5
0.5
P4
1.0
-1.0
5.2
P6
5.0
4.8
4.6
4.4
4.2
4.0
ppm
P5
P4
P1
-0.5
ppm
P2
P9
P2
-1.5
0.0
P3
-1.0
P
-0.5
0.5
P
P
0.0
P8
1.0
0.5
5.2
5.0
4.8
4.6
4.4
4.2
4.0
ppm
1.0
P3
5.2
5.0
4.8
4.6
4.4
4.2
4.0
ppm
RREIIBTr − ZF29R, 1:1 (excess), 298K
B) Exchangeable Protons
1D Imino Proton Spectrum
Free RREIIBTr, 298K
U66 G53 U43 G64 G76 G42 G67 G46 G77 U45 G55 G41 14.0
13.5
13.0
12.5
12.0
11.5
11.0
10.5
ppm
B) Exchangeable Protons
NOESY Imino Proton Region
G77!
U43!
G46!
G76!
G64!
G53!
U45!
G42!
U66!
C) Correlation between exchangeable and
non-exchangeable protons
H
O
H
N
N
N
A!
N
N
N
H
U
N
RNA!
O
H
H1'
H
N
H
O
H
N
N
G
N
N
N
H
H1'
H
H
C
N
O
N
DNA!
Heteronuclear Methods!
Resonance Assignment of RNA/DNA by Heteronuclear NMR!
13C and 15N correlations!
!
A)  Exchangeable Protons
!
!15N-1H HSQC !
!
!
!
!
!15N edited NOESY HSQC (3D)!
!
B)  Non Exchangeable Protons
!
!• Base/Sugar!
!
!
! !
!
!
!• Base-Sugar!
!
!
!
!• Sequential !
!
!
! !
!
!
!
C)  Correlation of Exchangeable !
!and Non Exchangeable Protons
!
D) !Base Pairing !
!
!
!!
!!
!13C-1H
HSQC !
!HCCH -TOCSY HCCH-COSY
!HCN, H(CNC)H, H(CN)H !
!2/3D!
!2/3D!
!13C
Edited NOESY-HSQC !
!PH, P(C)H, HCP !
!
!3/4D !
!2/3D!
!A, C, G, U, T- specific
!
13
! C Edited NOESY-HSQC!
!2D!
!3/4D!
!NN COSY!
A) Exchangeable Protons
15N-1H
G s U s HSQC
G55 (Low T)!
ppm
G77 146
G46 G67 G41 148
O!
N!
150
N!
N!
N!
152
154
156
N!
H!
Red = complexed Black = free RREIIBTr O!
158
H!
160
162
U66 14.0
U43 U45 13.5
13.0
12.5
12.0
N!
O!
11.5
ppm
N!
H!
H!
B) Non-exchangeable protons: CT-HSQC/HMQC
Spectrum: hsqc_base
Spectrum: hsqc_sugar
User: Alex Date: Mon Mar 8 21:50:36User:
2010 Alex Date: Mon Mar 8 21:51:00 2010
contours: low 1.80e+06 levels 40 factor 1.10
Positive contours: low 2.00e+06 levels 40Positive
factor 1.10
Negative contours: low -5.15e+05 levels 32 factor 1.10
Use Constant time experiments (CC couplings in F1 !)
8
6.2 6.0 5.8 5.6 5.4 5.2
NH2
7
135
135
140
140
145
145
150
150
N
90
N
90
O
N
NH2
CH even #C!
C8,C2,C5(pyr)!
2 ,3 ,4 !
95
95
100
! 1 - 13C (ppm)
N
NH
! 1 - 13C
N
(ppm)
O
100
CH odd #C!
C6,C1 ,C5 !
155
6.2 6.0 5.8 5.6 5.4 5.2
! 2 - 1H
(ppm)
155
7
8
! 2 - 1H
(ppm)
B) Non-exchangeable protons: HCCH-Type Experiments
F1 x F2: correlate a specific sugar 1H to its own sugar 1H’s and their respecSve 13C’s. HCCH COSY
HCCH TOCSY
6.0
62
64 H
66
3
68
- 13C (ppm)
INEPT
2
COSY
RELAY
TOCSY
g
4.0
C5’/H5’ C5’’/H5’’ O
74
N
CH
H
76
H
78
2
C
C
C
C
O
OH
70
C2’/H2’ C3’/H3’ N
O
72
74
H
76
H
78
80
80
C4’/H4’ 82
88
64
68
N
N
62
66
84
H
86
82
84
86
88
C1’/H1’ 90
90
92
92
94
94
6.0
5.5
5.0
1
6.0
- 13C (ppm)
INEPT
gg
1H
2
g
13C
4.5
- 1H : 6.047 (ppm)
F1
F1
72
13C
5.0
RREIIB-­‐Tr, ~300 uM, 298 K NH
70
1H
5.5
88
1
5.5
4.5
4.0
4.5
4.0
- 1H (ppm)
5.0
88
1
- H : 6.050 (ppm)
90
90
92
92
94
6.0
5.5
5.0
3
4.5
- 1H (ppm)
F3 x F2: Correlate each of its own sugar 1H’s to the 13C of a specific 1H. (HCCH TOCSY)
94
4.0
B) Non-exchangeable protons: HCN
1H
g13C g15N(F1)g 13C g 1H(F2)
U!
C!
A, G!
PL
B) Non-exchangeable protons: H(CNC)H & H(CN)H
H(CNC)H
H(CN)H
C) Correlation of Non-exchangeable and exchangeable 1H
G-specific H(NC)-TOCSY(C)H
PL
C) Correlation of Non-exchangeable and exchangeable 1H
A-specific (H)N(C)-TOCSY(C)H
PL
C) Correlation of Non-exchangeable and exchangeable 1H
U-specific H(NCCC)H
PL
C) Correlation of Non-exchangeable and exchangeable 1H
C-specific H(NCCC)H
PL
D) Direct Observation of Hydrogen Bonding by
2JNN
Couplings
O
D) Scalar Coupling Across H Bonds:
HNN-COSY
N
H
8294 J. Am. Chem. Soc., Vol. 120, No. 33, 1998
H
N
H
H
H N
H
Dingley and Grzesiek
N
N
15N carrier at 215 ppm, and the 13C carrier at 154 ppm.
the
N
H
H H
15
NN andH13C H
O was
O
Simultaneous
decoupling
applied during data acquisi2
!
H
N
JNN
tion.
5
H
N
1H (t )-HMQC-13C(t )-NOEA 3DH NOESY
was
recorded
as
a
3 UC 11
2
7 5
N
H
1H(t ) experiment
1N
9 with
Aoptimized
detection
N of imino-proton resonances
3
N
163WATERGATE,15 and radiation damping18 techby water flip-back,
C
H
N
O
H
H
C data matrix consisted
niques. The
of 46*(t11!) × 48*(t2) × 1024*(t3)
! acquisition times of 7 (t1), 12 (t2), and 68 ms (t3), and
data points 1
with
H
an NOE mixing time of 80 ms. The total experimental time was 60 h.
H
H H O resonance, the 13C carrier at
O positioned
H N on the
The 1HNcarrier was
2
2J
!
NN
7
15
15
110 ppm,
and
the
N
carrier
at
153
ppm.
N decoupling was applied
5
9
5
N data acquisition.
during
H
G 1N H
N 3C
H
3 were
1Hthe program nmrPipe,19 and peak
C Data sets
processed using
N
N
20
1 ! H determined
positions
with
PIPP.
Amplitudes of the time
H
O theHprogram
N
N
H
C
N Hin the quantitative
O
domain oscillations
JNN HNN-COSY data set were
1!
determined
nlinLS contained
N byH using the time domain fitting routine
H
N
H
N
19
in the H
NMRPipe package.
N
N
H
N H
O
≈Results
7 Hz; 1and
J ≈Discussion
90 Hz
!
Figure 1. Pulse sequence of the quantitative JNN HNN-COSY
experiment. Narrow and wide pulses correspond to flip angles of 90°
and 180°, respectively. RF power levels for high-power pulses are 29
A-U 1H pulses are
G-C!
applied at a field
(1H) and 5.8 kHz (15N). Low-power
15N of imino donor
1
1
! ppm (15N),
strength of 200 Hz. ! Carrier positions are! H2O ( H), 185
G s 140 – 15013ppm!
andU153
ppm ( C). Garp! decoupling (γB
!!2 ) 2.5 kHz) was applied
s 155 – 170 ppm
13
during the t1 period on the C channel. Delays: δ ) 2.25 ms; T ) 15
ms; ζa ) 2.5 ms; ζb ) 0.25 ms; ζc ) 2.25 ms; ζd ) 0.5 ms. Unless
indicated, all pulses are applied along the x axis. Phase cycling: φ1 )
x, y, -x, -y; φ2 ) R2, -R2 with R2 ) (y, -x, -y, x); φ3 ) R3, R3, -R3,
NH
-R3 with R3 ) (-y, x, y, -x); Acq. ) x, -y, -x, y. Quadrature • JNN
H
Homonuclear JNN couplings involving the imino 15N nuclei
detection in the t1 dimension was achieved by simultaneously increassignment
of GN1
– CN3byand
UN3
AN1!
in RNA were
observed and
quantified
using
theto
quantitative
15N of φ
menting
φ2 in !the States-TPPI manner. Gradients are sine-bell • Unambiguous
1 and
b.p.
acceptor
!
C s 190
– 205
shaped,
with
an ppm!
absolute amplitude of 25 G/cm at their center and
JNN correlation experiment2 depicted in Figure 1. The experi•
Q
uantitative
determination of JNN!
A
s
215
230
ppm
!
durations (polarities) of G1,2,3,4,5,6,7 ) 2.5 (+), 2.1 (-), 1.35 (+), 2.35
ment
is conceptually similar
to the quantitative 3JHNHA COSY
2
1/2]/(πT)!
|
J
|
=
atan[(-I
/I
)
NN
Na Nd
21 The
(+), 0.2 (+), 0.4 (+), and 0.101 ms (+).
experiment.
following product operator description will
be given for the uridine-adenosine (U-A) base pair (Figure 2A)
Here we report the direct observation of hydrogen bonding
where N3 of U is the donor nitrogen, H3 of U the hydrogen
1H (10 – 15 ppm)!
Imino
in Watson-Crick base pairs by a cross hydrogen bond scalar
bond proton, and N1 of A the acceptor nitrogen. The analogous
15
Dingley,
Grzesiek, S.,
Am.guanosine-cytidine
Chem. Soc., 1998,(G-C)
120 (33),
coupling between the imino N atom of the donor base
withA.J. & description
forJ.the
base 8293
pair –is7.!
15
the hydrogen bond acceptor N atom on the complementary
obtained by interchanging the U-A nuclei H3, N3, and N1 with
base. These 2JNN couplings yield valuable through-bond interthe G-C nuclei H1, N1, and N3 (Figure 2B). Magnetization is
HNN-COSY of Free RREIIB-Tr (300 µM)!
ppm
G77!
G41! G53!
G76! G46!G67!
G42! G64!
G-N1!
150
155
U-N3! 160
H
N
U45!
U43!
U66!
N
H
165
H
H
170
H
N7
5
9
N
175
H
A 1N
N
H
O
NN H H
H
N
H
N
N
H
O
H
O
N
H
U1
N3
N
3
C1
H !
C1 !
H
O
H
H
5
180
H
185
H
O
N
7
190
C-N3!
N9
HC1 !
H
N
195
C54!C44!
C79!C78!
C65!
200
C74!C51!
5
G 1N
3
N
O
N
A-N1! 220
N3
H
H
H
H
H
N H
N
C
1
H
N
H
HC1 !
O
H
N
N
N
N H
210
A-N3! 215
H
N
5
N
H
205
H
O
H
H
A52! A75!
225
14.0
13.5
13.0
12.5
12.0
11.5
ppm
Spring et al. unpublished!
Structure Determination:
I)
Assignment
II)
Local Analysis
•glycosidic torsion angle, sugar puckering,backbone conformation
base pairing
Global Analysis
•sequential, inter strand/cross strand, dipolar coupling
III)
Nucleic Acids have few protons…..
•NOE accuracy
> account for spin diffusion
•Backbone may be difficult to fully characterize
•Dipolar couplings
What do we know?
•Distance, Torsion, H-Bond constraints, Orientation
What do we want?
•Low energy structures in agreement with NMR
Optimize conditions!
pH, I, T.!
Assignments!
spin system!
sequential!
long range!
Constraints:!
Distance + Torsion!
!
Initial Structure!
!
Cyana. rMD. DG!
Mardigras/Corma!
rMD!
!
Evaluate/Refine!
Relaxation Matrix method:
use of longer mixing times
(need initial structure, dynamics!)
MD-Tar!
Dynamics!
Add experiments!
RDC!
etc!
Dipolar couplings!
• Dipolar couplings add to J coupling
• They show up as a field or alignment media dependence
• If the overall orientation of the molecule is known the orientation of
the vectors can be determined
!
B0
θ
S
I
IS!
IS!
!max!
D! =! D
IS!
!max!
D
1!
(!3!cos!2!θ! -! 1)! !
2!
µ!0γ! !I!γ!Sh
! !
=!-!
4!π!2!rIS!3!!
Sp borano modified DNA / RNA hybrid residual dipolar splittings!
---------------------------------------------------------------------!
First atom
Last atom
Calc.
Exp.
Deviation penalty !
---------------------------------------------------------------------!
C1' DA5
1 -- H1' DA5
1:
-0.308
-0.700
0.392
0.154 !
C1' DT
2 -- H1' DT
2:
7.435
7.400
0.035
0.001 !
C1' DG
3 -- H1' DG
3:
-0.788
-0.900
0.112
0.012 !
C1' DG
4 -- H1' DG
4:
-5.398
-5.500
0.102
0.010 !
!
SI_3
CαAG
20
Experimental (Hz)
R² = 0.98357
15
10
5
0
-4
-2
0
2
4
6
8
10
12
14
16
Calculated (Hz)
-5
SI_5
2.50
-10
Well Width (Å)
2.00
1.50
1.00
0.50
0.00
1.50
rMD with RDC!
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Average Restraint Distance (Å)
5.50
6.00
6.50
R.M.S.D. 0.63!
CαAG
Force Constant (k)a
246
154
92
1
0.70 (stdev 0.46)
30
30
30
30
exchangeable (total)
average well width (Å)
27
3.0
30
Endocyclic Torsion Angle Restraints
deoxyribose (pseudo rotation analysis)
average well width | r2- r3| / N
95
30
50
25
25
25
10
68
60, 80, 60, 65
18
varries 20 -50 depending on
# of data points available
50
Parameter
Quantitative Distance Restraints (RANDMARDI)
non exchangeable (total)
intra residue
inter residue (sequential)
inter residue (cross strand)
average well width (Å)
Watson Crick Restraints
distance
flat angle
Backbone Torsion Angle Restraints
DNA / RNA hybrid broad rsts
well width α β γ ζ (deg)
ε (CT NOESY) (deg)
average well width
50
Residual Dipolar Coupling
total RDC restraints
46
base (C6, C8, C2, C5)
24
1.0 (dwt)
sugar (C1')
sugar (C3')
12
10
1.0 (dwt)
1.0 (dwt)
Total Restraints
total restraints / residue
550
27.5
CORMA Rx Values
RX (number of unique cross-peaks)
Intra
Inter
Total
4.73 (93)
6.55 (44)
5.25 (134)
4.13 (143)
5.61 (77)
4.62 (220)
3.81 (136)
5.19 (83)
4.29 (291)
TM (ms)
75
125
250
Final Amber Parameters
Total Distance Penalty (kcal/mol)
Total Angle Penalty (kcal)/(mol)
Total Torsion Angle Penalty (kcal)/(mol)
Residual Dipolar Coupling (RDC) Allignment Constraint
55.4
0.24
4.6
4.9
Bundle of 10 Final Structures
Heavy Atom R.M.S.D.
0.63
a
kcal/(mol x unit of violation)
Johnson et al: DNA
sequence context conceals
α anomeric lesions. J. Mol
Biol. (2012) 416, 425-437.
Structural Basis of the RNase H1 Activity on Stereo Regular Borano
Phosphonate DNA / RNA Hybrids.!
Johnson et al, (2011) Biochemistry, 50, 3903-3912!
*
*
A
11B
brid
B
C
11B {1H}
-40
-45
-40
11B
11B}
R1HP {Hybrid
SP Hybrid
1H
B
C
-45
11B {1-40
H}
0.51H
0.5
0.5
0.1
0.1 ppm
-45
-45 -40
-40
-45
-45 -40
-40
*
0.5
0.5
T5 H4’
T5 H4’
G6 H4’
WATER
G6 H3’
T5 H6
G6 H8
-45
-40
0.1
0.1ppm
ppm
*
*
-40
0.5
0.5
0.1
0.1
*
*
-45
0.5
1H {11B}0.1 ppm
0.1
*
SP Hybrid
brid
D
**
*
*
-45
A
Sp!
-45 -40
D
G6 H5’1 / H5’2
Rp!
0.5
0.1
0.5
0.1 ppm
-45
-40
-45
-40
0.5
0.1
0.5
0.1 ppm
BH3
BH3
Molecular Details!
T8!
A15!
A7!
T16!
Michael Rettig, et al, 2013, ChemBioChem!
-1.0
-2.0
5! 6!
5 6 7
step!
0
Twist!
294°
B)50
Twist °
317°
40
30
20
DNA-NETROPSIN!
C)
0
10
Twist
Change(Complex-Control)
(Complex-Control)
Twist
Change
5! 6!
5 6 7
step!
1! 2! A! C! 5
1 2 3 4 5
basepair ste
1! 2! A! C! 5
1 2 3 4 5
basepair ste
8
6
Free DNA!
4
2
0
-2
2-3
T3-4 A5-6T 6-7A 7-8
G1-2G A
T C8-9 C
Base Step
Michael Rettig, et al, 2013, ChemBioChem!
7
5
Roll (Å)
Minor Groove Width (Å)
9
30
20
10
3
2
0
5
6
7
8
9
Base Pair Level
-20
0
-30
-10
0.5
8-9
7-8
6-7
5-6
4-5
3-4
2-3
1
9-10
Roll (°)
4
1
10
-20
3
-10
20
1-2
0
Bend à change in twist and roll!
collapse of minor groove!
2
0
0
3
10
4
20
5
6
7
8
9
10
30
base
pair step
Bend Angle (°)
Base Pair Level
50 Bent into MAJOR groove!
Control
Complex Bent into MINOR groove!
45
Michael Rettig, et al, 2013, ChemBioChem!
NOESY (Exchange) Peaks!
Methyl proton !
Methyl proton !
C
G
G
C
A
T 18
4! T
A
6! T
A
NOESY !
ROESY !
A
T 16
A
T 14
8! T
A
C
G
G
C
Methyl proton region of the a) 250 ms NOESY b) 150 ms ROESY spectrum of the
netropsin-CG/CG complex at 283 K at 600 MHz!
Michael Rettig, et al, 2012, J. Phys. Chem.!
2D Exchange Spectroscopy (EXSY)
Quantifying Exchange Processes
Figure 4. Temperature dependence of the methyl resonances of the a) 1:1 CG/CG-netropsin complex at 600 MHz a
complex (BPES buffer containing 20 mM NaCl) at 500 MHz. At 278 K T6 is not visible in a) due to signal overlap b
298 K as indicated by the asterisk. The Journal of Physical Chemistry B
A !
B
with equal integral can be observed
exclusively. We doAB
not observe any exchange
processes between
BB
increasing the temperature the
complexed and free DNA as expected for a very slow exchange
broaden and merge into one peak
rate between these states, and this is consistent with the SPR
at around 300 K. As temperatur
data. In addition to these 1D spectra, 2D NOESY experiments
AA
sharpening of the merged peaks c
were recorded for
assigning the resonances
of the ligand-DNA
BA
shift changes and signal broadenin
complexes. Surprisingly, strong crosspeaks between symmetry
T6/T16 and T8/T18 pairs of methy
related protons like e.g. T4 H6/T14 H6 or A7 H8/A17 H8 are
overlap the coalescence temperatur
observed for both DB921- and netropsin-DNA complexes.
for these resonances.
Similarly for the methyl groups, equivalent crosspeaks between
An estimate of the rate of exchang
the T6/T16 and T8/T18 (netropsin-DNA complex, Figure 3a)
binding sites was obtained by usin
or T4/T14 and T8/T18 (DB921-DNA complex, data not
for two-site exchange with equal po
shown) methyl protons are seen. The distance between these
protons >9−16 Å (standard B-DNA) is far too large to give rise
to possible NOE crosspeaks as NOEs
usually be detected
kcoal =resonances
(π /21/2)Δ
Figurecan
4. Temperature
dependence of the methyl
ofνthe a) 1:1 CG/
34
(BPES
between protons that are less thancomplex
6 Å apart
frombuffer
eachcontaining
other. 20 mM NaCl) at 500 MHz. At 278 K T6 is no
298
Kand
as
indicated
by theChem.
asterisk.
In addition, the evaluationCharles
of a L.related
netropsin-ATAT
DNA
Δν = 7 Hz is the chemical s
Perrin
Tammy
J. Dwyer,
Rev. 1990.where
90, 935-967
35
structure gives carbon−carbon distances of equivalent methyl
exchanging spins T4/T14. Thus, an
wi
exclusively.
We cannot
do not be
observe
groups >7.5−14 Å. Therefore, these
crosspeaks
due any exchange
constant processes
of 16 s−1 between
and an apparent
Microscopic Rearrangement of Bound Minor
Groove Binders Detected by NMR
à NMR sees exchange rates in the order of 20-60 ms at 300K!
à BUT It takes >2 s for the drug to dissociate off the DNA (SPR)!
C
G
C
G
G
C
G
C
A
T
A
T
T
A
T
A
A
T
A
T
T
A
T
A
A
T
A
T
T
A
T
A
C
G
C
G
G
C
G
C
Microstates!
Michael Rettig, et al, 2012, J. Phys. Chem.!
Microscopic Rearrangement of Bound Minor
Groove Binders Detected by NMR
+!
+!
+!
à  very common!
+!
+!
+!
+!
+!
à  difficult to see !
depending on k!
!
à  contributes to binding!
+!
+!
+!
+!
Bulk!
+!
+!
+!
+!
CC skin!
General references, NMR techniques, sample preparation,
analysis
BioNMR in Drug Research. Edited by Oliver Zerbe, 2002 Wiley Verlag
Wijmenga, 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: Solution
Structure of a DNA Duplex Containing an a -Anomeric Adenosine: Insights into Substrate
Recognition by Endonuclease IV. J. Mol. Biol. (2004), 338, 77-91.
Aramini, J. M., Mujeeb, A., Ulyanov, N. B. & Germann, M. W.: Conformational Dynamics in Mixed
a /b- Oligonucleotides Containing Polarity Reversals: A Molecular Dynamics Study using
Time-averaged Restraints. J. Biomol. NMR, (2000), 18, 287-303.
Aramini, J. M. & Ge rmann, M. W. NMR solution structure of a DNA/RNA hybrid c ontaining an
alpha 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 the
Sugar Puckers of the DNA. J. Am. Chem. Soc. 120, 821- 822
Iwahara J, Wojciak JM, Clubb RT. (2001), An efficient NMR experiment for analyzing sugarpuckering in unlabeled DNA: application to the 26-kDa dead ringer-DNA complex. J Magn
Reson. 2001, 153, 262
Multinuclear experiments, DNA/RNA
Pardi, A. and Nikonowicz, E.P. (1992) Simple procedure for resonance assignment of the sugar
protons 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 1H
resonances 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–31P
triple 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 of
adenine 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 resonances
in 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 tripleresonance HCNCH experiment for direct correlation of ribose H1 and Base H8, H6 protons
in 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 and
sugar 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 H
Kessler (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 glycosidic
torsion angles in uniformly 13C-labeled nucleic acids from vicinal coupling constants
3J(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,15NLabeled 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}-Spinecho-difference Constant-time [ 13C,1 H]-HMQC Experiment for Simultaneous
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Magn. Reson. 140, 491 -494.
H. Schwalbe, W. Samstag, J. W. Engels, W. Bermel, and C. Griesinger, (1993) "Determination of
3J(C,P) and 3J(H,P) Coupling Constants in Nucleotide Oligomers", J. Biomol. NMR 3, 479486
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)
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