- Email: [email protected]
DNA, The versatile vector of life: two-dimensional NMR studies
109
Journal ofMolecuhr Structure, 141(1986) 109-126 Elsevier Science publishers B.V., Amsterdam - printed in The Netherlands
DNA, THE VERSATILE VECTOR OF LIFE:
TWO-DIMENSIONALNMR STUDIES
C. ALTONA Gorlaeus
Laboratories,
State
University,
P.O.Box
9502,
2300 RA Leiden
(The Netherlands)
ABSTRACT Deoxyribonucleic acid (DNA), until recently regarded as a relatively stiff in reality displays a multitude of surprising and regularly built double helix, local structural variations. According to X-ray crystallographic findings, three different families of DNA structures exist: the right-handed duplexes A DNA and Modern 2D NMFt techniques now alB DNA as well as the left-handed duplex Z DNA. low for unequivocal assignment of base proton resonances and at least Hl’, H2’, Nuclear Overhauser Enhancement (NOE) intenH2” signals in intact DNA duplexes. sities then lead to a quick determination of the overall structure (B or Z DNA, the A form thus far has not been detected in aqueous solution). However, the study of finer structural details requires the determination and interpretation Examples of local structural variations of the of vicinal coupling constants. sugar ring in single-helical as well as in double-helical DNAs are given.
INTRODUCTION A DNA molecule backbone;
any of
tached
the
to
the
sugars
(or
In the
course
translated access sary
into
only for
of
to
background
its
a protein a small
of
own function.
is
off
the
genome at the cular to
biology:
recognize
proper
of
time.
what are faithfully
0022-2660166/$03.50
the
the
the
time All
refs
a cell
the
a specific
above location
two complementary
copied.
requires
call
that
the the
the
cell
the
on DNA in order
-
of
e.g., DNA
on the
in mole-
allow
block
neces-
of
place
problem that
to
has
part
expression
proper
a central level
0 1966 Elaevier Science Publishers B.V.
specialized
and finally
upon synthesis
at
illustrates at atomic
In the
namely,
means that
and must be activated
features
may pair
a double-helical
must be read
information, would
by
1-5.
faithfully
This
in-
characterized
to
contains
information
total
needed.
rise
at-
pyrimidines
equilibrium
is
two DNA molecules
Specialization
of
is
see
DNA is
coded
Moreover,
when it
most
the
which
C, giving
DNA polymer
are
A and G and the
stack
In addition,
sequence.
enzyme only
purines
diester
thereof)
in a conformational
information
fraction
a specific blocked
exists
T and G with
reproduction parts
the
a duplexed
sugar-phosphate
some modifications
single-helical
overlap.
Thus,
specific
cell,
solution)
A with
structure.
a deoxyribose (and
position,
and the
bonds:
For general
strands.
(in
of
bases
Cl’
base-base
hydrogen
duplex)
living
at the
random coil
nearest-neighbour through
1) consists different
Such a chain
C and T. volving
(Fig. four
it
a protein or
to
110
Fig, 1. Arbitrary sequence of a self-complementary deoxyribotetranucleoside triphosphate: d(TGCA). The standard numbering of base and sugar entities is shown. The residues themselves are numbered (l), (2) ... starting with the St-OH terminal (T(1) in this particular example). activate it. Most of these features are poorly understood and much further work remains necessary. Many questions concerning the structure and dynamics of protein-DNA complexes and of protein-free DNA are presently under investigation in many laboratories. Two powerful techniques, besides others, are currently applied in studies on DNA and DNA complexes:
(a) X-ray analysis of single crystals;
(b) nuclear mag-
netic resonance (NHR). The purpose of the present paper is twofold. First, a brief review of modern aspects of the conformational analysis of the deoxyribose ring in DNA constituents -
both single-helical as well as double-helical -
will be given, together with selected NMR techniques that have proven their worth. Emphasis will be laid on the use and interpretation of NMR coupling constants. Secondly, our results concerning B DNA will be contrasted to findings by other researchers.
CONFORMATIONAL ANALYSIS OF DEOXYRIBOSES In biochemical literature the two distinct B-D-deoxyribose conformations are commonly denoted as C3'-endo (3E) and C2'-endo (zE). Note that these terms tend to give the false impression that these sugar conformations are rigid, whereas in reality the pseudorotational movement
161 of the furanose ring constitutes
one of the major degrees of freedom of the backbone of B DNA. For this reason we prefer to distinguish between the N-type genus of forms (P = 0' f 90') and the S-type genus (P = 180' * 900), Fig. 2. The endocyclic torsion angles +j in an
111
H2
BASE
N-conformer
S-conformer
Fig. 2. N- and S-type conformations of furanose rings. Idealized forms are shown, whereas in fact N and S stand for large ranges of possible conformations [6,7] . For the sake of clarity, the HZ" proton has been omitted. The classical A-DNA form is characterized by an N-N-N-N sequence of sugars, the classical B-DNA by an S-S-S-S sequence, whereas the Z-DNA structure displays alternation: S-N-S-N .
-
approximately -
rameter equation
oj
=
equilateral S-membered ring are mutually related by a 2-pa-
[6] , es.(l).
$m cos[P * 4n(j-2) / 51
j = 0, 1, 2, 3, 4
in which 4, represents the maximum amplitude of pucker, P the phase angle and the endocyclic torsions are numbered clockwise, starting with C4'-04'-Cl'-C2' (j = 0). Knowledge of two or three endocyclic torsions, e.g., from X-ray crystallography or from proton NMR coupling constants, enables one to uniquely deduce the pseudorotation parameters P and $,, from which not only the remaining torsions but also the important backbone angle 6(C5'-C4'-C3'-03')can be calculated, eq. (2).
6
=
120.6 +
1.1 $m cos(P + 145.2)
Eq. (2) is based upon a corrected pseudorotation equation
(2) [S]
. The constants
of eq. (2) were determined from a least-squares fit to our crystal structure data set
[9]
.
Given the fact that in aqueous solution of nucleic acids the N- and S-type conformers most often occur side-by-side in fast equilibrium, the NMR spectroscopist is faced with the simultaneous determination of four geometrical parameters (PN, $,, Ps, 4~) and one equilibrium constant K. The Leiden group has spent considerable effort in order to provide a dependable and routine solution to this problem. Two keys to success were developed. First, de Leeuw et at. [9] derived a set of empirical equations which relate proton-proton torsion angles to the endocyclic furanose torsions. Eq. (3) applies to 8-D-deoxyribose:
112 =
121.4O
+
1.03 $m cos(P
- 144O)
Ua)
$1’2”
=
0.9O
+
1.02 +m cos(P
- 144O)
WI
$2,3,
=
2.40
+
1.06@mcosP
(3c)
=
122.9O
+
1.06$mcosP
(3d)
+
1.09 0, cos(P
%,2,
‘2”3
= -124.0”
$3’4’
Secondly,
Haasnoot et ~2.
equation
by the introduction
and orientation [4,
iterative
i.e.
acids
not be treated
couplings
curacy of the procedure
is greatly
displays
with temperature,
fact,
shift
the geometry of the sugars,
accuracy
here.
acid
fragments
that is at least
Karplus
of these findings it to say that an
yields
the pseudorotation
sugars,
and the N/S
along the bonds Cl’-C2’
,
are shown in Table I. The ac-
enhanced in case the N/S equilibrium as is usually often
constant
found for oligomers.
and thus the important in solution‘now
to
has been documented in
Suffice
set of 35HH couplings
. Some representative
of small nucleic
application
and prolines
geometry of the N- and S-type
given a complete
a strong
three-parameter
for the electronegativity
computer program PSEUROT [12]
the detailed
, C3’-C4’
The successful
of nucleic
and will
least-squares
molar ratio, C2’-C3’
analysis
11, 121
parameters,
extended the classical of terms which account
of substituents.
the conformational estenso
[lo]
+ 144O)
In
backbone angle 6 (Eq.2),
can be determined
equal to that of a high-resolution
with an
X-ray crystal
struc-
ture analysis. The determination the determination COSY spectra. ed
-
of the sugar geometry in intact
of signal
These signal
from the foregoing
pseudorotational each residue, prerequisite haviour
widths of Hl’ width contain equations
-
for each conformer
before
valid
from at least conclusions
can be drawn. In addition,
31-phosphorus
, H2’ and H2” from either sums of couplings
pathway, Table I and Fig. information
decoupled
spectra
three
concerning
analysis
(N/S) ratio
formational
species
with two conformational
P(S)
=
(31.7
-
(ii)
and
observables
(PS in B DNA). Clearly,
the population
ratio
C2”) / 10.7
(Hl’
or
for
, H2’ , H2”) is
the sugar conformational
two main degrees
be-
width in This
of freedom:
the phase angle P of the major conself-consistency
parameters
is best
along the
that,
should be attempted whenever possible.
(i)
upon
1D or from
encountered
of the H3’ resonance
that one has to determine
practice,
resonances
rests
which can be predict-
3. It should be stressed
stems from the fact the population
double helices
approximated p(N)
of three or four
lends credence
=
from
22”,
(22” - 21.0)
to the results. eq.
In
(4):
/ 10.7
(4)
113
TABLE I Calculateda
coupling
formations
constants
(Hz) of deoxyribose
running from N-type via Ol’-endo
principle
obtainable
from COSY spectra,
rings
to S-typeb.
are also
in DNAfor
sugar con-
sums of couplings,
in
presented.
Deoxy N
21T
ZT ZT !T
2E
3E
qE
OE
P
6c
1’2’
1’2”
2’3’
2”3’
3’4’
324 342 0 18 36 54 72 90
108 97 89 84 82 84 90 99
1.3 1.3 1.5 2.1 3.3 5.0 6.8 8.4
6.9 7.1 7.7 8.4 8.9 9.0 8.4 7.5
7.5 7.3 7.2 7.5 8.1 9.0 9.6 9.6
8.7 9.5 9.7 9.5 8.7 7.3 5.5 3.6
5.3 6.7 7.7 8.3 8.4 8.3 7.7 6.7
108 126 144 162 180 198 216
110 121 133 144 152 158 159
9.5 10.0 10.2 10.0 9.5 8.4 6.8
6.5 5.9 5.6 5.7 5.8 6.2 6.6
8.9 7.8 6.6 5.8 5.5 5.6 5.9
2.1 1.3 1.2 1.2 1.3 1.2 1.2
5.3 3.7 2.3 1.4 1.0 0.9 0.9
d
C2fe
12”f
E3’g
8.2 8.4 9.2 10.5 12.2 14.0 15.2 15.9
22.8 22.6 22.7 23.6 25.4 28.0 30.4 32.0
29.6 30.6 31.4 32.0 31.6 30.3 27.9 25.1
21.5 23.5 24.6 25.3 25.2 24.6 22.8 19.9
16.0 15.9 15.8 15.7 15.3 14.6 13.4
32.4 31.8 30.8 29.8 29.0 28.0 26.7
22.6 21.2 20.8 20.9 21.1 21.4 21.8
16.3 12.8 10.1 a.4 7.8 7.7 7.7
Zl’
Deoxy S !T TT
lE 2E
ZT 3B ZT
aThe calculations were carried out by use of the extended Karplus equation [lo] and eq. (3); the initial values were corrected for the Barfield through-space btransmission effect [13]. A mean puckering amplitude Ornof 35” was assumed[4]. See refs 6,7 for the definition of the phase angle of pseudorotation P and the relation between P and the various twist (T) and envelope (E) conformations. iThe backbone angle 6 (C5’-C4’-C3’-03’) was calculated from eq. (2). El’ = 51121 + Jl’2” , the line width of the Hl’ signal. eZ2’ = 51121 + J2~3t + ,J212” . An average value of 14:DHz was used for J2’211 . 52” = 51~2~~+ J2”3 + 5212” ; see footnote e. gz3 ’ = J213t + ~J2~~31+ 53’41 , the line width of the H3’ signal in 31-phosphorus decoupled spectra. Eq. (4) is valid Table VIII)
521211 coupling is known cation
for PN O-18’,
a similar
PS 126-l&?O’, and brn 35”.
equation
was presented
has been subtracted
for a given residue,
of X2’ gives
from eq.
Recently, ed [13] -
attempts sight
these distances was claimed
a,
Once the S/N ratio of the lo-
pathway. Alternatively,
by means of program PSEUROT[12]
. Once
species
is known, backbone angle 6
sugar puckerings
from NOEShave been publish-
(2). to determine
. Since NOE intensities
at first
4 (footnote
a good impression
on the pseudorotational
can be done automatically
the phase angle of the major conformational follows
In ref.
however, the geminal
from the sums of couplings.
analysis
of the sugar conformation
the calculation
in which,
-
(iteratively)
[13]
depend upon l/r6
be tempted to translate to derive
that distances
(r = H . . . H distance)
the DNA sugar conformation.
in an intact
one could
NOESinto H . . . H distances duplex could
be fitted
and use
In fact,
it
with an
114 r.m.s.
accuracy
of 0.2 A.
tered at Cl’-exo
(‘E,
P 126”).
cism,
however.
falls
such as different
ization
according
and local
protons
[14] ).
Two pitfalls
B DNAduplexes
second,
usually
analyses exhibit
remain constant
within
and that concerns
H . . . H distances
to Cl’-exo
the stated
the Hl’
(&de
(126”),
freedom
insensitive
H . . . H distances
going from the center
of the
occurs
distances,
to N-type sugar.
cannot be detected
the outcome of the iterative
all
toas
generally
One exception
(c 0.35 A). Moreover,
except HZ” . . . H4’ , do not change much on going from S-type it might influence
completely
deoxyribose
are quite
limit.
pit-
then loom up:
the H . . . H distances
[13] 0.2 A error
. . . H4’ distance
origin infra),
conformational
Fig.. 4 shows the endocyclic
This means that a 10 - 20% N-type population although
critiof magnet-
can be avoided
residual
of the phase angle P. It can be seen that,
normal Ps range (150 - 160”)
motions and relay
protons
of conformational
constant
most intrasugar
ward changes of sugar pucker.
molecular
or via solvent
to our coupling
sugars in intact
function
thus found cen-
Such an approach remains open to serious
overall
U&J neighbouring
(pN 6 20%):
of the sugar puckerings
Let us, for the sake of argument, grant that major technical
(which is doubtful first,
The majority
by a NOEanalysis,
structure
calculation.
Fig. 3. Predicted sums of couplings (see footnotes d, e, f of Table I) along the pseudorotational pathway of the deoxyribose ring. In this example Ornwas kept constant at 35”.
115
u.00
3.75
2
p.50
5
5.25
P
E3.00 '" 0 I t 2.75 x
2.50
2.25
Fig. 4. Endocyclic H . . . H distances in deoxyribose as function of the phase angle of pseudorotation P at constant puckering amplitude (4, 35’). (Courtesy of Dr J.-R. Mellema). For example, distance)
the presence
of a small percentage
would increase
terpreted
-
the observed
would result
H2”/H4’
in a calculated
by a NQE study
distance
of 0.29 - 0.31 A is found for all
Fig.
whereas it is stated
4),
(S-type
[15] of double-stranded
sugars).
H2”...H4’
improperly
in-
d(CGTACG). An apparent H2”...H4’
residues
[15] that this
The latter
- if
PS that is too low. A good example is
provided
structure
of N-type sugar (short NOEand this
(i.e.,
in the N-type range,
duplex adopts a conventional
implies
a H2”...H4’
distance
B-type
of > 3.8 h .
ASSIGNMENT OF RESONANCES The principles recently
by Bax [16].
information parameters interest
of two-dimensional In most
-but
(2D) NMRspectroscopy not all
is added, compared to classical into
two different
for NMRof larger
frequency
molecules
and Hl’,
in a duplex decamer [la],
dimensions powerful
H2’, H2” resonances
current
types of 2D experiments
1D NMR, but the separation
assignment
no new of NMR
F1 and F, is of particular
with many overlapping
the 1D NOEmethod was shown to be quite ments of base proton
-
have been summarized
resonances.
and actually
in a duplex octamer
strategies
Although
led to full
for nucleic
assign-
[17] and
acids
favour
116
0
INTRARESIDUE SCALAR
COUPLINGS
KEY
NOES
(COSY)
ACTORS
(NOESYI
H5/CH3-H6 Hl’l
Fig. 5. Intraresidue coupling networks which are revealed by COSY and intraresiwhich give rise to NOESYcross peaks (for the sake of due short H... H distances clarity some NOESYconnectivities have been omitted). the simultaneous 19-241. lar
study of both COSY and NOESYspectra
In brief,
cross
(J) couplings
the existence nectivities
peaks in a COSY contour
between protons,
The normal COSY experiment strand
or duplex)
array.
In practice,
volving nicity
of e.g.
peaks.
often
Problems
ly introduced [20].
Fig.
experiment,
causes (ii)
and (iii)
extension
20
H4’ resonances
(iii)
HOMONUCLERR
7 gives
5.
in single
cross
accidental
peaks inisochro-
the complete
as-
in the H4’,
the envelope
HS’,
of diagonal
by the employment of a recent-
termed homonuclear
double-RELAY
schemes of a normal COSY and of the double-relayed
dubbed DAYCOSYby the present Fig.
con-
sugar in the
of signals
into
can be circumvented
(i)
(ii)
prohibits
overcrowding
of the COSY experiment,
6 shows the pulse
network.
the noise;
of sca-
reflect
are shown in Fig.
to each individual
peaks to disappear
mixing time unambiguous subspectra plete)
into
[S,
The intraresidue
may be encountered:
disappear
involved; cross
the existence
(n = number of residues
that belong
difficulties
often
(< Q, 5 A).
the n sets
two or more H3’ and/or
signment of the networks H5” region
yields
several
distances
betray
thereof
peaks in a NOESYplot
from COSY and NOESYspectra
of seven signals
small couplings
plot
whereas cross
of short proton-proton that one may expect
or variants
author.
are obtained
By a judicious for
each complete
choice
of the
(or almost com-
an example of a DAYCOSYspectrum for the simple
CORRELRTION
SPECTROScOPY
Fig. 6. Pulse scheme of a normal COSY (left) ment [ZO].
20
HOMONUCLEAR COHERENCE
DOUBLE-RELAYED SPECTROSCOPY
and of a DAYCOSY(right) experi-
117
0
wm
Fig. 7. High-field region of the 300 MHz DAYCOSY spectrum of d(TA). Extra cross peaks [compared to a standard COSY experiment) are encircled in the lower righthand half of the contour plot only. Chemical shifts are referenced to the methyl peak of tetramethylammonium chloride (TMA, 3.18 ppm upfield from DSS or TMS). dimer d(TA).
The “extra”
cross
peaks (absent
is seen that HI’ to H4’ can be correlated region
offers
no particular
A similar
problems.
Drobny [ZO] on the trimer AZ’-5*A2’-5’A. tell
us which set of signals
e.g.
refs
dure rests
belongs
to which residue. [19].
was obtained
22-24)
upon the fact
located
on residue own residue
and will
that a base proton
n in a right-handed (Fig.
5) and Hl’,
of the 5’ terminal
spectroscopist
first
not be treated
Therefore,
stack,
here in
(H8 of purines
of the strand).
does not
the information
of NOESYin the matter
or H6 of pyrimidines),
n-l
It is for this
(See
The proce-
extenso.
is sandwiched between Hl’,
HZ’, HZ” of residue
turns his attention
by Bax and
experiment
The application
It
that the HS’, H5”
of A- and B-DNA fragments has become an almost routine 5, 13-15,
of its
direction
result
and also
A COSY or DAYCOSY
must be supplemented by a NOESYspectrum assignment
in a normal COSY) are encircled.
at a glance
“above”
(i.e.
HZ’, HZ” in the
reason that the NMR
to the NOEconnectivities
between the
118 residue n-l
5'
-
n -
n+l
3'
H8/H6 ZI=L. Hl' - HZ' - HZ" t----,
H8/H6
--Zc__-W--, Hl' - HZ' - HZ" a
-
_??-Hl' - HZ' - HZ" 1_ Scheme 1. NOESY cross peaks most used in assignment of A- and B-DNA lH NMR spectra. H8/H6 protons and the Hl', HZ', HZ" regions. The procedure is outlined in Scheme 1. It shows that, except at the 5' terminal, each H8 or H6 should display two connectivities to Hl' resonances, one intra-, the other interresidue. An independent check is possible through the H8/H6 - HZ'/H2" and Hl' - HZ'/HZ" NOESY cross peaks. It should be mentioned here that, loosely speaking, the NOE is a function of l/r6 (r = H...H distance), of the motional correlation time rc' and of the mixing time used. Near the condition wrc = 1.1, where w is the angular Larmor frequency, the classical (i.e. laboratory frame) NOE vanishes. Thus, for a slowly tumbling nucleic acid strand (.ccg O.S~lp-~s) it is often difficult to observe NOES on NMR spectrometers operating in the 200-300 MHz range. An interesting way out of this difficulty appears to be offered by the ROESY experiment, recently advocated by Bax and Davis [Zl]. The rotating-frame NOE (under spinlocked conditions) is always positive and monotonically increases for increasing values of 'c C’
CONFORMATIONAL ANALYSIS OF DNA FRAGMENTS Single helices A single-helical DNA fragment is stabilized solely by "vertical" base-base overlap interactions (stacking) [l-3]. In contrast, the DNA duplex, besides stacking, has an additional stabilization through the formation of hydrogen bonds between bases on opposite strands, as well as the possibility of interstrand steric and/or charge-charge interactions. In order to understand the structure and dynamics of DNA, it is therefore important to study the behaviour of single helices of varied sequence in conjunction with the behaviour of the corresponding duplexes. We have set out to answer the following questions: (i) what conformational changes, if any, occur in going from the single helix to the double helix? (ii) do single-helical and duplex regions display conformational purity of the sugar rings or is N/S ring flip still possible:
119 TABLE II Pseudorotation
parameters
and predicted
6 deviations
mers,
see
[6,7],
backbone angle 6 (CS’-C4’-C3’-03’)
from mean (141’)
of various
single-helical
and observed DNAoligo-
text.
Compound
Residue
d (TAAT)1
T(1) A(2) A(3) T(4)
160 167 153 143
A(2) T(3) A(4)
T(1)
pS
Temp. range (“C)
6S(o)
A60bs
*‘pred
35 36 40 32
143 147 141 132
+2 +6 0 -9
0 +5 0 -5
8-43 I, II II
158 160 149 166
35 36 35 32
142 143 136 144
+l +2 -5 +3
0 +5 -5 0
a-44 ,I II ,t
A(1) T(2) A(3) T(4)
173 150 170 148
36 36 36 31
150 138 149 134
+9 -3 +8 -7
+5 -5 +5 -5
8-35 II
d (‘ITA) 3
T(1) T(2) A(3)
155 155 162
34 34 34
140 140 143
-1 -1 +2
0 0 0
5-65 II II
d&W4
A(1) A(2) A(3)
170 157 153
36 36 34
149 142 138
+8 +l -3
+5 0 -5
11-25 ,1 11
d(AA)4
A(1) A(2)
167 153
36 35
147 139
+6 -3
+5 -5
o-25 II
d (TGTG)5
T(1) G(2) T(3) G(4)
150 158 149 166
37 37 35 33
138 142 137 144
-3 +l -4 +3
0 +5 -5 0
27 11 ,I 11
d (CAACTT)6
C(1) A(2) A(3) C(4) T(5) T(6)
158 171 153 162 159 -
34 36 38 35 35 -
141 149 140 144 142 -
0 +8 -1 +3 +l -
0 +5 0 -5 0 0
15-35 II 11 II 11 11
d (CGT)7
C(l) G(2) T(3)
154 163 146
35 37 33
140 145 134
-1 +4 -7
0 +5 -5
2-52 11 II
d(TCG)7
T(l1 C(2) G(3)
158 157 160
33 35 34
141 141 142
0 0 +l
0 0 0
12-52 11 II
d (TATA)1
d (ATAT]2
8 35
l) J.-R.Mellema, A.K.Jellema, C.A.G.Haasnoot , .J.H. van Boom, and C.Altona, Eur. J.Biochem. 141 (1984) 165-175. 2, Refs 25.26. 3, C.A.G.Haasnoot, J.-R.Mellema, and C.Altoncunpublished results. 4, C.S.M.Olsthoorn, L.J.Bostelaar, J.H. van Boom, and C.Altona, Eur. J. Biochem. 112 (1980) 95-110. 5, L.J.Rinkel, J.-R.Mellema, and C.Altona, to be pubzhed. 6, L.P.M.Orbons and C.Altona, to be published. 7, J.-R. Mellema, R. van der Noerd, G.A. van der Marel, J.H. van Boom, and C.Altona, Nucleic Acids Res. -12 (1984) 5061-5078.
120 Crystallographic
information
concerning
single-helical
and knowledge must be gained from NMRspectroscopy. ber of such fragments have been studied ings will
be summarized here.
and 6S of deoxyribose vicinal
coupling
sugar ring
constants,
by PS ‘L 150’. character
Closer
(purine
of roughly
sugar conformational particular
occur
variation
Cg proposed
simple additivity sequence.
error
of such a step, [27],
especially
Elellema et al.
the values
values
negative
concerned
Analogous
[25,
appear
values to the
261 devised
6 variation
a
with DNA
the values
+1, -1,
0, 0. The unit of sum function
Fig.
g6
8 shows the A60bs (= 6obs-1410)
61 residues.
for the observed
already
noted
concomitant
results
in an in-plane
similar
sliding
effect,
in the single
in a B-DNA single-helix
An overall
with an increase sliding
leading helix.
r.m.s.
Fig.
deviation expe-
to 133” slides
less
aligned
with the adenine C6-N6 dipole.
tion
of electrostatic of 6
has another
the purine
interesting
a change in 6 leads
of the Cl’-N
vectors
this
where the
and becomes more or
suggests
a.B,Y,e,<)
to an appreciable residues,
a dA-dT step
that optimiza-
role.
conformational
of successive
is
6 angle from the
a position
6-membered ring This result
for
6
that a
interaction,
principle
the T base into
(angles
angle
base-paired
It is proposed
stacking
of the thymidine
may play a certain
pathway of the phosphate backbone fixed,
of 6 of the opposite
9 illustrates Reduction
by computer
of a pyrimidine
of the base pair.
is positioned,below interaction
is suggested
to optimum base-base
conformation.
value in the monomer (152’)
6 variation
[27] that a decrease
C4-04 dipole
more or less
changes of
when the residue
us to correlate
angle difference.
purine,
entation
II shows that positive
in a dR-dR or dR-dY step;
incorporating
explanation
Dickerson
Variation
the important
on local
which is of the same order of magnitude as the estimated
in a B-DNA duplex,
general
into
Since the
in 6.
A possible modelling.
operative
to the sugar in ques-
now be centered
in Table
by Dickerson
at 5O of torsion is found,
in
to the
in the sequence.
of 6 is seen in dY-dR or dY-dY steps.
are assigned
of 16 sequences,
rimental
variability
is not related
The sugars at each dR-dR and dR-dY step are assigned
is fixed of 2.6’
bases
will
scheme I& which allows
dY-dR and dY-dY steps values
pattern
P and 4, can be translated
R residue
in the second residue
sum function
of
angle.
with the first
is dY. No large
PS, 0,
analysis
in the case of d(ATAT) the
Y) of the base attached
Z), the discussion
torsion
parameters
whereas the dT sugars are characterized
of two neighbouring
parameters
years a large num-
sequence-dependent
that this
Study of the column headed Mobs to correlate
A clear
170°,
reveals
R or pyrimidine
but to the character
the geometrical
261. For example,
is scanty
Some of the main find-
by means of pseudorotation
supra. [25,
scrutiny
backbone angle 6 (eq. this
vi&
geometry is evident
dA sugars have PS values
tion,
obtained
In recent
in our laboratory.
Table II lists
sugars,
DNA fragments
consequence.
If the
is assumed to remain change in relative
viewed in a direction
ori-
121
-+-c-rT--R--T--R 0
0 .I
-I 0
0
-1
0
-.
b*
0 .,
G-G 1, -1 _f
&.*I
.I
-I
1
&.
-I 0
0
-1
0
. 0 f,
0
Fig. 8. Correlation of the variation of the backbone angle 6 observed for a series of single-helical DNAfragments in aqueous solution with the expected variation according to the sum function C6. Experimental values are connected by full lines, predicted ones by broken lines. The unit of change in Lg corresponds to So of 6. (From ref. 26, see also ref. 25 and Table II). perpendicular ly identical
to the base planes. to the local
From the model depicted
twist in Fig.
The relative
orientation
so defined
is virtual-
angle tl as defined by Dickerson and Drew [28]. 9 it is seen that a change from 6 152” to 6 133”
122
Fig. 9. Schematic illustration of the effect of 6 variation on base-base overlap in a regular B-type single-helical dA-dT step, viewed in the 5’+3’ direction. The broken outline shows the expected position of the dT base for 6 152’, i.e. the approximate 6 value of the thymidine monomer. A decrease of the dT 6 angle to 133’ (Table II) causes an appreciable sliding of the dT base with respect to the overlying dA base (solid line), concomitant with a decrease of the local twist angle tl from about 42’ to about 25’: allows
the lower base to slide
about 25’,
i.e.,
helical
dependent adjustments stitutes
in such a way that tl
“untwisting”.
These considerations
of the deoxyribose
a mechanism for
fine
decreases
suggest
expressed
pucker,
tuning of the base-base
from about 42’
to
that sequence
as 6 variation,
con-
overlap.
Double helices DNAs can adopt several mental conditions DNAduplexes E,T),
double-helical
such as counterions,
are classified
the B family
this
that several
deoxyoligonucleotides
clearly
mode is S (2’-endo).
indicate
At this
point
sequence d(GGCCGGCC)[29,30]. in solution
indicated contrast
a major contribution to present
Thus far,
lines
the sole
is read off
Similarly, of B-like
representative
These duplexes
display
interesting
[33] to steric
the duplex formed by
It is well
to remember that
clashes
N- or S-type
RNA-DNAhybrid
forms in the deoxyribose
studies
[18]
strands,
in
[3]. of the B-type duplex in the single
is the dodecamer sequence d(CGCGAATTCGCG) and its by Calladine
as
compounds in aqueous
in the A form [32] whereas solution
of thought
to note
and characterized
from two criteria:
the covalent
(B,C,D,
whereas for
is of interest
This concerns
the self-complementary
[31].
the B-family
of one of these
A- or B-type character sugar and NOE intensities
it
have been crystallized
B-type character.
r(GCG)d(TATACGC) crystallizes
sequence and composition.
A-type,
mode for A DNA.is N (3’-endo),
DNA, whereas our NMRinvestigations
solution
depending upon environ-
water activity,
in three broad groups:
and Z DNAs. The sugar puckering
A-type
conformations,
local
C(9) brominated
changes of geometry
between adjacent
purines
crystal
derivative.
[27,28,34] in opposite
ascribed strands.
123
d(C A C A T G T Gl
d(C A C Al d(T G T G)
Fig. 10. Populations of S conformer (%, hatched areas) in two single-helical tetramers - d (CACA) and d (TGTG) - and in the composite octamer duplex d(CACATGTG), ref. 35. Note that the sugars in the core of the duplex appear to retain some conformational freedom. Obviously,
such clashes
can occur
posed to be exceptionally mations are concerned, (unimodal) (P 90°)
variation
tend to favour
and coworkers
of sugar puckers,
In the crystal
about Cl’-exo
(P 126’)
favour
a similar
picture,
prefer
albeit
d(CACATGTG), together
high 6 values.
Moreover,
with those
in the two constituent
single
[35] by means of line
The results
(Fig.
lo),
it
of the duplex existence
still
distribution). studies hybrid
retain
flip
duplex octamer d(GGm5Cm5C)2[17],
alternating
already
influence
pared to that observed
the geometrical a considerable offered
principle
parameters
since
a certain
found.
the marked decrease
RNA-DNA
amount of residual
that a limited Second (Fig. 6 variation,
fragments.
by the Calladine-Dickerson
[27,33],
(bimodal
our earlier
amount
at Zft resolution,
amount of additional single-helical
confor-
Thus, the
and on the covalent
in an X-ray analysis
in the constituent
to the rationale
anticomplementarity
freedom.
It must be mentioned in passing
duplex induces
on
the sugars in the core
duplex is indicated
revealed
12”
10 and 11.
towards the S-type
does not come as a surprise,
might remain undetected
though it could
contrast
(S Z N) in the intact
duplex mentioned above [IS],
freedom of the sugar rings. of ring
driven
tetramers,
some measure of conformational flip
This conclusion
on B-DNA
although
single-helical
fragments X2’,
in the section
are summarized in Figures
is noted that,
of dynamic ring
helical
widths Cl’,
with the aid of the method outlined
First,
[35]
dY-dR octamer duplex
conformational
mer compared to the constituent
(omitting
pyrimidines
one.
taken from COSY spectra, analysis.
to 04’-endo
Our NMRstudies
sugars in the alternating
d(CACA) and d(TGTG), were analyzed
(P 162’)
with a mean 6 = 125’
a more complicated
The puckers of the individual
a continuous
the sugar conformations
and a 6 range running from 155’ to 80’.
low 6 and purines
and are sup-
As far as the sugar confor-
[27,28,34]
running from C2’-endo
6 variation.
distributed
the 3’ terminals),
in the minor groove.
Dickerson
with a concomitant
are apparently
reveal
strong
only in dR-dY and in dY-dR steps
rules,
alll),
the com-
However, in i.e.,
the
in 6 of the pyrimidine
124
110-L
CACATGTG
Fig. 11. The sequence-dependent variation of backbone angle 6 of the major Stype conformation in two single-helical tetramers d(CACA) and d(TGTG), indicated deduced for the B-type double helix formed by +, compared to the 6 variation by the octamer d(CACATGTG), indicated by o. residues
dC and dT is not accompanied by a similar
residues
dA and dG (perhaps with the exception
the &-range seen in aqueous solution smaller
than the b-range
(155O to 80°, (134’) this
omitting
therefore point
of local
it
strand steric generalizations
(157” to 115’)
the 3’ terminals).
can be concluded
Moreover,
of the dodecamer
The value of Gmean in our octamer
than that found for the dodecamer (125’). that the NMRresults
of geometry in a B-DNA duplex,
effects.
in 6 of the purine
appears to be decidedly
deduced from X-ray crystallography
remains greater
variations
increase
of the dG(6) sugar).
More solution
studies
decidedly possibly
favour
At
the concept
mediated tria inter-
appear necessary,
however,
before
can be made.
ACKNOWLEDGEMENTS This research was supported by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). Dr J.-R.Mellema, Mr L.P.M.Orbons and Mr L.J.Rinkel kindly supplied some of the illustrations used in this paper. REFERENCES P.O.P. Ts’o, “Basic Principles in Nucleic Acid Chemistry”, vols 1-3, Academic Press, New York (1974). C.R. Cantor and P.R. Schimmel, “Biophysical Chemistry”, vols 1-3, Freeman, San Francisco (1980) . of Nucleic Acid Structure”, Springer, New York (1984). W. Saenger, “Principles C. Altona, Reel. Trav. Chim. Pays-Bas 101 (1982) 413-433. C. Altona, in Stud. Org. Chem. Vol. 20 “Natural Products Chemistry”, Elsevier, Amsterdam (1985) 285-304. C. Altona and M. Sundaralingam, J. Am. Chem. Sot., 94 (1972) 8205-8212; idem ibid. 95 (1973) 2333-2344. IUPAC-IUB Joint Commission on Biochemical Nomenclature, Eur.. J. Biochem., 131 (1982) 9-1s. F.A.A.M. de Leeuw, P.N. van Kampen, C. Altona, E. Diez, and A.L. Esteban, J. Mol. Struct ., 125 (1984) 67-88. H.P.M. de Leeuw, C.A.G. Haasnoot, and C. Altona, Isr. J. Chem.,20 (1980) 108-126.
125
10 C.A.G. Haasnoot, F.A.A.M. de Leeuw, and C. Altona, Tetrahedron, 36 (1980) 2783-2792. 11 C.A.G. Haasnoot, F.A.A.M. de Leeuw, H.P.M. de Leeuw, and C. Altona, Biopolymers, 20 (1981) 1211-1245. 12 F.A.A.M. de Leeuw and C. Altona, J. Comput. Chem., 4 (1983) 428-437; iciem, Quant. Chem. Progr. Exch. No 463 (1983); idem, J. Chem. Sot. Perkin II, (1982) 375-384. 13 G.M. Clore and A.M. Gronenborn, EMBO J., 4 (1985) 829-835; i&m, FEBS Lett., 179 (1985) 187-198 and references therein. 14 .J.W.Keepers and T.L. .James,J. Magn. Reson. 57 (1984) 404-426: M.S. Broido, T.L. James, G. Zon and J.W.Keepers, Eur. J. Biochem., 150 (1985) 117-128. 15 A.M. Gronenborn, G.M. Clore and B.J. Kimber, Biochem. J., 221 (1984) 723-736. 16 A. Bax, "Two-Dimensional Nuclear Magnetic Resonance in Liquids", Reidel, Dordrecht, Holland, 1982. 17 M.R. Sanderson, J.-R. Mellema, G.A. van der Marel, G. Wille, J.H. van Boom, and C. Altona, Nucleic Acids Res. 11 (1983) 3333-3346. 18 J.-R. Mellema, C.A.G. Haasnoot, G.A. van der Marel, G. Wille, C.A.A. van Boeckel, J.H. van Boom and C. Altona, Nucleic Acids Res. 11 (1983) 5717-5738. 19 COSY = Correlated Spectroscopy, A. Bax, R. Freeman and G.A. Morris, J. Magn. Reson.,42 (1981) 164-168; NOESY q Nuclear Overhauser Enhancement Spectroscopy, J. Jeener, B.H. Beier, P. Bachmann and R.R. Ernst, .J.Chem. Phys., 71 (1979) 4546-4553. 20 DAYCOSY = double-RELAY experiment, A. Bax and G. Drobny, J. Magn. Reson., 61 (1985) 306-320; G. Eich, G. Bodenhausen and R.R. Ernst, J. Am. Chem. Sot., 104 (1982) 3731-3733. 21 ROESY = Rotating-frame Overhauser Enhancement Spectroscopy, A. Bax and D.G. Davis, J. Magn. Reson., 63 (1985) 207-213; A.A. Bothner-By, R.L. Stephens, J. Lee, C.D. Warren and R.W. Jeanloz, J. Am. Chem. Sot., 106 (1984) 811-813. 22 J. Feigon, J.M. Wright, W. Leupin, W.A. Denny and D.R. Kearns, J. Am. Chem. sot., 104 (1982) 5540-5541. 23 D.G. Reid, S.A. Salisbury, S. Bellard, Z. Shakked and D.H. Williams, Biochemistry, 22 (1983) 2012-2025. 24 R.M. Scheek, N. Russo, R. Boelens, R. Kaptein and .J.H.van Boom, J. Am. Chem. sot., 105 (1983) 2914-2916. 25 J.-R. Mellema, J.M.L. Pieters, G.A. van der Marel, J.H. van Boom, C.A.G. Haasnoot and C. Altona, Eur. .J.Biochem. 143 (1984) 285-301. 26 J.-R. Mellema, Appendix in Thesis, Leiden, 1984. 27 R.E. Dickerson, J. Mol. Biol. 166 (1983) 419-441. 28 R.E. Dickerson and H.R. Drew, J. Mol. Biol. 149 (1981) 761-768. 29 A.H.-J. Wang, S. Fujii, J.H. van Boom and A. Rich, Proc. Natl. Acad. Aci. U.S.A., 79 (1982) 3968-3972. 30 L.J. Rinkel, M.R. Sanderson and C. Altona, to be published. 31 C.A.G. Haasnoot, H.P. Westerink, G.A. van der Marel, and J.H. van Boom, J. Biomol. Struct. Dyn., 2 (1984) 3445-3460. 32 A.H.-J. Wang, S. Fujii, J.H. van Boom, G.A. van der Marel, C.A.A. van Boeckel and A. Rich, Nature, 299 (1982) 601-604. 33 C.R. Calladine, J. Mol. Biol. 161 (1982) 343-352. 34 A.V. Fratini, M.L. Kopka, H.R. Drew and R.E. Dickerson, J. Biol. Chem., 257 (1982) 14686-14707. 35 L..J.Rinkel, J.-R. Mellema, L.P.H. Orbons, G.A. van der Marel, J.H. van Boom and C. Altona, Abstracts 4th Conversation in Biomolecular Stereodynamics, R.H. Sarma @d.)State University of New York at Albany, 1985, pp 126-127; L.J. Rinkel et al. to be published.
Journal ofMolecuhr Structure, 141(1986) 109-126 Elsevier Science publishers B.V., Amsterdam - printed in The Netherlands
DNA, THE VERSATILE VECTOR OF LIFE:
TWO-DIMENSIONALNMR STUDIES
C. ALTONA Gorlaeus
Laboratories,
State
University,
P.O.Box
9502,
2300 RA Leiden
(The Netherlands)
ABSTRACT Deoxyribonucleic acid (DNA), until recently regarded as a relatively stiff in reality displays a multitude of surprising and regularly built double helix, local structural variations. According to X-ray crystallographic findings, three different families of DNA structures exist: the right-handed duplexes A DNA and Modern 2D NMFt techniques now alB DNA as well as the left-handed duplex Z DNA. low for unequivocal assignment of base proton resonances and at least Hl’, H2’, Nuclear Overhauser Enhancement (NOE) intenH2” signals in intact DNA duplexes. sities then lead to a quick determination of the overall structure (B or Z DNA, the A form thus far has not been detected in aqueous solution). However, the study of finer structural details requires the determination and interpretation Examples of local structural variations of the of vicinal coupling constants. sugar ring in single-helical as well as in double-helical DNAs are given.
INTRODUCTION A DNA molecule backbone;
any of
tached
the
to
the
sugars
(or
In the
course
translated access sary
into
only for
of
to
background
its
a protein a small
of
own function.
is
off
the
genome at the cular to
biology:
recognize
proper
of
time.
what are faithfully
0022-2660166/$03.50
the
the
the
time All
refs
a cell
the
a specific
above location
two complementary
copied.
requires
call
that
the the
the
cell
the
on DNA in order
-
of
e.g., DNA
on the
in mole-
allow
block
neces-
of
place
problem that
to
has
part
expression
proper
a central level
0 1966 Elaevier Science Publishers B.V.
specialized
and finally
upon synthesis
at
illustrates at atomic
In the
namely,
means that
and must be activated
features
may pair
a double-helical
must be read
information, would
by
1-5.
faithfully
This
in-
characterized
to
contains
information
total
needed.
rise
at-
pyrimidines
equilibrium
is
two DNA molecules
Specialization
of
is
see
DNA is
coded
Moreover,
when it
most
the
which
C, giving
DNA polymer
are
A and G and the
stack
In addition,
sequence.
enzyme only
purines
diester
thereof)
in a conformational
information
fraction
a specific blocked
exists
T and G with
reproduction parts
the
a duplexed
sugar-phosphate
some modifications
single-helical
overlap.
Thus,
specific
cell,
solution)
A with
structure.
a deoxyribose (and
position,
and the
bonds:
For general
strands.
(in
of
bases
Cl’
base-base
hydrogen
duplex)
living
at the
random coil
nearest-neighbour through
1) consists different
Such a chain
C and T. volving
(Fig. four
it
a protein or
to
110
Fig, 1. Arbitrary sequence of a self-complementary deoxyribotetranucleoside triphosphate: d(TGCA). The standard numbering of base and sugar entities is shown. The residues themselves are numbered (l), (2) ... starting with the St-OH terminal (T(1) in this particular example). activate it. Most of these features are poorly understood and much further work remains necessary. Many questions concerning the structure and dynamics of protein-DNA complexes and of protein-free DNA are presently under investigation in many laboratories. Two powerful techniques, besides others, are currently applied in studies on DNA and DNA complexes:
(a) X-ray analysis of single crystals;
(b) nuclear mag-
netic resonance (NHR). The purpose of the present paper is twofold. First, a brief review of modern aspects of the conformational analysis of the deoxyribose ring in DNA constituents -
both single-helical as well as double-helical -
will be given, together with selected NMR techniques that have proven their worth. Emphasis will be laid on the use and interpretation of NMR coupling constants. Secondly, our results concerning B DNA will be contrasted to findings by other researchers.
CONFORMATIONAL ANALYSIS OF DEOXYRIBOSES In biochemical literature the two distinct B-D-deoxyribose conformations are commonly denoted as C3'-endo (3E) and C2'-endo (zE). Note that these terms tend to give the false impression that these sugar conformations are rigid, whereas in reality the pseudorotational movement
161 of the furanose ring constitutes
one of the major degrees of freedom of the backbone of B DNA. For this reason we prefer to distinguish between the N-type genus of forms (P = 0' f 90') and the S-type genus (P = 180' * 900), Fig. 2. The endocyclic torsion angles +j in an
111
H2
BASE
N-conformer
S-conformer
Fig. 2. N- and S-type conformations of furanose rings. Idealized forms are shown, whereas in fact N and S stand for large ranges of possible conformations [6,7] . For the sake of clarity, the HZ" proton has been omitted. The classical A-DNA form is characterized by an N-N-N-N sequence of sugars, the classical B-DNA by an S-S-S-S sequence, whereas the Z-DNA structure displays alternation: S-N-S-N .
-
approximately -
rameter equation
oj
=
equilateral S-membered ring are mutually related by a 2-pa-
[6] , es.(l).
$m cos[P * 4n(j-2) / 51
j = 0, 1, 2, 3, 4
in which 4, represents the maximum amplitude of pucker, P the phase angle and the endocyclic torsions are numbered clockwise, starting with C4'-04'-Cl'-C2' (j = 0). Knowledge of two or three endocyclic torsions, e.g., from X-ray crystallography or from proton NMR coupling constants, enables one to uniquely deduce the pseudorotation parameters P and $,, from which not only the remaining torsions but also the important backbone angle 6(C5'-C4'-C3'-03')can be calculated, eq. (2).
6
=
120.6 +
1.1 $m cos(P + 145.2)
Eq. (2) is based upon a corrected pseudorotation equation
(2) [S]
. The constants
of eq. (2) were determined from a least-squares fit to our crystal structure data set
[9]
.
Given the fact that in aqueous solution of nucleic acids the N- and S-type conformers most often occur side-by-side in fast equilibrium, the NMR spectroscopist is faced with the simultaneous determination of four geometrical parameters (PN, $,, Ps, 4~) and one equilibrium constant K. The Leiden group has spent considerable effort in order to provide a dependable and routine solution to this problem. Two keys to success were developed. First, de Leeuw et at. [9] derived a set of empirical equations which relate proton-proton torsion angles to the endocyclic furanose torsions. Eq. (3) applies to 8-D-deoxyribose:
112 =
121.4O
+
1.03 $m cos(P
- 144O)
Ua)
$1’2”
=
0.9O
+
1.02 +m cos(P
- 144O)
WI
$2,3,
=
2.40
+
1.06@mcosP
(3c)
=
122.9O
+
1.06$mcosP
(3d)
+
1.09 0, cos(P
%,2,
‘2”3
= -124.0”
$3’4’
Secondly,
Haasnoot et ~2.
equation
by the introduction
and orientation [4,
iterative
i.e.
acids
not be treated
couplings
curacy of the procedure
is greatly
displays
with temperature,
fact,
shift
the geometry of the sugars,
accuracy
here.
acid
fragments
that is at least
Karplus
of these findings it to say that an
yields
the pseudorotation
sugars,
and the N/S
along the bonds Cl’-C2’
,
are shown in Table I. The ac-
enhanced in case the N/S equilibrium as is usually often
constant
found for oligomers.
and thus the important in solution‘now
to
has been documented in
Suffice
set of 35HH couplings
. Some representative
of small nucleic
application
and prolines
geometry of the N- and S-type
given a complete
a strong
three-parameter
for the electronegativity
computer program PSEUROT [12]
the detailed
, C3’-C4’
The successful
of nucleic
and will
least-squares
molar ratio, C2’-C3’
analysis
11, 121
parameters,
extended the classical of terms which account
of substituents.
the conformational estenso
[lo]
+ 144O)
In
backbone angle 6 (Eq.2),
can be determined
equal to that of a high-resolution
with an
X-ray crystal
struc-
ture analysis. The determination the determination COSY spectra. ed
-
of the sugar geometry in intact
of signal
These signal
from the foregoing
pseudorotational each residue, prerequisite haviour
widths of Hl’ width contain equations
-
for each conformer
before
valid
from at least conclusions
can be drawn. In addition,
31-phosphorus
, H2’ and H2” from either sums of couplings
pathway, Table I and Fig. information
decoupled
spectra
three
concerning
analysis
(N/S) ratio
formational
species
with two conformational
P(S)
=
(31.7
-
(ii)
and
observables
(PS in B DNA). Clearly,
the population
ratio
C2”) / 10.7
(Hl’
or
for
, H2’ , H2”) is
the sugar conformational
two main degrees
be-
width in This
of freedom:
the phase angle P of the major conself-consistency
parameters
is best
along the
that,
should be attempted whenever possible.
(i)
upon
1D or from
encountered
of the H3’ resonance
that one has to determine
practice,
resonances
rests
which can be predict-
3. It should be stressed
stems from the fact the population
double helices
approximated p(N)
of three or four
lends credence
=
from
22”,
(22” - 21.0)
to the results. eq.
In
(4):
/ 10.7
(4)
113
TABLE I Calculateda
coupling
formations
constants
(Hz) of deoxyribose
running from N-type via Ol’-endo
principle
obtainable
from COSY spectra,
rings
to S-typeb.
are also
in DNAfor
sugar con-
sums of couplings,
in
presented.
Deoxy N
21T
ZT ZT !T
2E
3E
qE
OE
P
6c
1’2’
1’2”
2’3’
2”3’
3’4’
324 342 0 18 36 54 72 90
108 97 89 84 82 84 90 99
1.3 1.3 1.5 2.1 3.3 5.0 6.8 8.4
6.9 7.1 7.7 8.4 8.9 9.0 8.4 7.5
7.5 7.3 7.2 7.5 8.1 9.0 9.6 9.6
8.7 9.5 9.7 9.5 8.7 7.3 5.5 3.6
5.3 6.7 7.7 8.3 8.4 8.3 7.7 6.7
108 126 144 162 180 198 216
110 121 133 144 152 158 159
9.5 10.0 10.2 10.0 9.5 8.4 6.8
6.5 5.9 5.6 5.7 5.8 6.2 6.6
8.9 7.8 6.6 5.8 5.5 5.6 5.9
2.1 1.3 1.2 1.2 1.3 1.2 1.2
5.3 3.7 2.3 1.4 1.0 0.9 0.9
d
C2fe
12”f
E3’g
8.2 8.4 9.2 10.5 12.2 14.0 15.2 15.9
22.8 22.6 22.7 23.6 25.4 28.0 30.4 32.0
29.6 30.6 31.4 32.0 31.6 30.3 27.9 25.1
21.5 23.5 24.6 25.3 25.2 24.6 22.8 19.9
16.0 15.9 15.8 15.7 15.3 14.6 13.4
32.4 31.8 30.8 29.8 29.0 28.0 26.7
22.6 21.2 20.8 20.9 21.1 21.4 21.8
16.3 12.8 10.1 a.4 7.8 7.7 7.7
Zl’
Deoxy S !T TT
lE 2E
ZT 3B ZT
aThe calculations were carried out by use of the extended Karplus equation [lo] and eq. (3); the initial values were corrected for the Barfield through-space btransmission effect [13]. A mean puckering amplitude Ornof 35” was assumed[4]. See refs 6,7 for the definition of the phase angle of pseudorotation P and the relation between P and the various twist (T) and envelope (E) conformations. iThe backbone angle 6 (C5’-C4’-C3’-03’) was calculated from eq. (2). El’ = 51121 + Jl’2” , the line width of the Hl’ signal. eZ2’ = 51121 + J2~3t + ,J212” . An average value of 14:DHz was used for J2’211 . 52” = 51~2~~+ J2”3 + 5212” ; see footnote e. gz3 ’ = J213t + ~J2~~31+ 53’41 , the line width of the H3’ signal in 31-phosphorus decoupled spectra. Eq. (4) is valid Table VIII)
521211 coupling is known cation
for PN O-18’,
a similar
PS 126-l&?O’, and brn 35”.
equation
was presented
has been subtracted
for a given residue,
of X2’ gives
from eq.
Recently, ed [13] -
attempts sight
these distances was claimed
a,
Once the S/N ratio of the lo-
pathway. Alternatively,
by means of program PSEUROT[12]
. Once
species
is known, backbone angle 6
sugar puckerings
from NOEShave been publish-
(2). to determine
. Since NOE intensities
at first
4 (footnote
a good impression
on the pseudorotational
can be done automatically
the phase angle of the major conformational follows
In ref.
however, the geminal
from the sums of couplings.
analysis
of the sugar conformation
the calculation
in which,
-
(iteratively)
[13]
depend upon l/r6
be tempted to translate to derive
that distances
(r = H . . . H distance)
the DNA sugar conformation.
in an intact
one could
NOESinto H . . . H distances duplex could
be fitted
and use
In fact,
it
with an
114 r.m.s.
accuracy
of 0.2 A.
tered at Cl’-exo
(‘E,
P 126”).
cism,
however.
falls
such as different
ization
according
and local
protons
[14] ).
Two pitfalls
B DNAduplexes
second,
usually
analyses exhibit
remain constant
within
and that concerns
H . . . H distances
to Cl’-exo
the stated
the Hl’
(&de
(126”),
freedom
insensitive
H . . . H distances
going from the center
of the
occurs
distances,
to N-type sugar.
cannot be detected
the outcome of the iterative
all
toas
generally
One exception
(c 0.35 A). Moreover,
except HZ” . . . H4’ , do not change much on going from S-type it might influence
completely
deoxyribose
are quite
limit.
pit-
then loom up:
the H . . . H distances
[13] 0.2 A error
. . . H4’ distance
origin infra),
conformational
Fig.. 4 shows the endocyclic
This means that a 10 - 20% N-type population although
critiof magnet-
can be avoided
residual
of the phase angle P. It can be seen that,
normal Ps range (150 - 160”)
motions and relay
protons
of conformational
constant
most intrasugar
ward changes of sugar pucker.
molecular
or via solvent
to our coupling
sugars in intact
function
thus found cen-
Such an approach remains open to serious
overall
U&J neighbouring
(pN 6 20%):
of the sugar puckerings
Let us, for the sake of argument, grant that major technical
(which is doubtful first,
The majority
by a NOEanalysis,
structure
calculation.
Fig. 3. Predicted sums of couplings (see footnotes d, e, f of Table I) along the pseudorotational pathway of the deoxyribose ring. In this example Ornwas kept constant at 35”.
115
u.00
3.75
2
p.50
5
5.25
P
E3.00 '" 0 I t 2.75 x
2.50
2.25
Fig. 4. Endocyclic H . . . H distances in deoxyribose as function of the phase angle of pseudorotation P at constant puckering amplitude (4, 35’). (Courtesy of Dr J.-R. Mellema). For example, distance)
the presence
of a small percentage
would increase
terpreted
-
the observed
would result
H2”/H4’
in a calculated
by a NQE study
distance
of 0.29 - 0.31 A is found for all
Fig.
whereas it is stated
4),
(S-type
[15] of double-stranded
sugars).
H2”...H4’
improperly
in-
d(CGTACG). An apparent H2”...H4’
residues
[15] that this
The latter
- if
PS that is too low. A good example is
provided
structure
of N-type sugar (short NOEand this
(i.e.,
in the N-type range,
duplex adopts a conventional
implies
a H2”...H4’
distance
B-type
of > 3.8 h .
ASSIGNMENT OF RESONANCES The principles recently
by Bax [16].
information parameters interest
of two-dimensional In most
-but
(2D) NMRspectroscopy not all
is added, compared to classical into
two different
for NMRof larger
frequency
molecules
and Hl’,
in a duplex decamer [la],
dimensions powerful
H2’, H2” resonances
current
types of 2D experiments
1D NMR, but the separation
assignment
no new of NMR
F1 and F, is of particular
with many overlapping
the 1D NOEmethod was shown to be quite ments of base proton
-
have been summarized
resonances.
and actually
in a duplex octamer
strategies
Although
led to full
for nucleic
assign-
[17] and
acids
favour
116
0
INTRARESIDUE SCALAR
COUPLINGS
KEY
NOES
(COSY)
ACTORS
(NOESYI
H5/CH3-H6 Hl’l
Fig. 5. Intraresidue coupling networks which are revealed by COSY and intraresiwhich give rise to NOESYcross peaks (for the sake of due short H... H distances clarity some NOESYconnectivities have been omitted). the simultaneous 19-241. lar
study of both COSY and NOESYspectra
In brief,
cross
(J) couplings
the existence nectivities
peaks in a COSY contour
between protons,
The normal COSY experiment strand
or duplex)
array.
In practice,
volving nicity
of e.g.
peaks.
often
Problems
ly introduced [20].
Fig.
experiment,
causes (ii)
and (iii)
extension
20
H4’ resonances
(iii)
HOMONUCLERR
7 gives
5.
in single
cross
accidental
peaks inisochro-
the complete
as-
in the H4’,
the envelope
HS’,
of diagonal
by the employment of a recent-
termed homonuclear
double-RELAY
schemes of a normal COSY and of the double-relayed
dubbed DAYCOSYby the present Fig.
con-
sugar in the
of signals
into
can be circumvented
(i)
(ii)
prohibits
overcrowding
of the COSY experiment,
6 shows the pulse
network.
the noise;
of sca-
reflect
are shown in Fig.
to each individual
peaks to disappear
mixing time unambiguous subspectra plete)
into
[S,
The intraresidue
may be encountered:
disappear
involved; cross
the existence
(n = number of residues
that belong
difficulties
often
(< Q, 5 A).
the n sets
two or more H3’ and/or
signment of the networks H5” region
yields
several
distances
betray
thereof
peaks in a NOESYplot
from COSY and NOESYspectra
of seven signals
small couplings
plot
whereas cross
of short proton-proton that one may expect
or variants
author.
are obtained
By a judicious for
each complete
choice
of the
(or almost com-
an example of a DAYCOSYspectrum for the simple
CORRELRTION
SPECTROScOPY
Fig. 6. Pulse scheme of a normal COSY (left) ment [ZO].
20
HOMONUCLEAR COHERENCE
DOUBLE-RELAYED SPECTROSCOPY
and of a DAYCOSY(right) experi-
117
0
wm
Fig. 7. High-field region of the 300 MHz DAYCOSY spectrum of d(TA). Extra cross peaks [compared to a standard COSY experiment) are encircled in the lower righthand half of the contour plot only. Chemical shifts are referenced to the methyl peak of tetramethylammonium chloride (TMA, 3.18 ppm upfield from DSS or TMS). dimer d(TA).
The “extra”
cross
peaks (absent
is seen that HI’ to H4’ can be correlated region
offers
no particular
A similar
problems.
Drobny [ZO] on the trimer AZ’-5*A2’-5’A. tell
us which set of signals
e.g.
refs
dure rests
belongs
to which residue. [19].
was obtained
22-24)
upon the fact
located
on residue own residue
and will
that a base proton
n in a right-handed (Fig.
5) and Hl’,
of the 5’ terminal
spectroscopist
first
not be treated
Therefore,
stack,
here in
(H8 of purines
of the strand).
does not
the information
of NOESYin the matter
or H6 of pyrimidines),
n-l
It is for this
(See
The proce-
extenso.
is sandwiched between Hl’,
HZ’, HZ” of residue
turns his attention
by Bax and
experiment
The application
It
that the HS’, H5”
of A- and B-DNA fragments has become an almost routine 5, 13-15,
of its
direction
result
and also
A COSY or DAYCOSY
must be supplemented by a NOESYspectrum assignment
in a normal COSY) are encircled.
at a glance
“above”
(i.e.
HZ’, HZ” in the
reason that the NMR
to the NOEconnectivities
between the
118 residue n-l
5'
-
n -
n+l
3'
H8/H6 ZI=L. Hl' - HZ' - HZ" t----,
H8/H6
--Zc__-W--, Hl' - HZ' - HZ" a
-
_??-Hl' - HZ' - HZ" 1_ Scheme 1. NOESY cross peaks most used in assignment of A- and B-DNA lH NMR spectra. H8/H6 protons and the Hl', HZ', HZ" regions. The procedure is outlined in Scheme 1. It shows that, except at the 5' terminal, each H8 or H6 should display two connectivities to Hl' resonances, one intra-, the other interresidue. An independent check is possible through the H8/H6 - HZ'/H2" and Hl' - HZ'/HZ" NOESY cross peaks. It should be mentioned here that, loosely speaking, the NOE is a function of l/r6 (r = H...H distance), of the motional correlation time rc' and of the mixing time used. Near the condition wrc = 1.1, where w is the angular Larmor frequency, the classical (i.e. laboratory frame) NOE vanishes. Thus, for a slowly tumbling nucleic acid strand (.ccg O.S~lp-~s) it is often difficult to observe NOES on NMR spectrometers operating in the 200-300 MHz range. An interesting way out of this difficulty appears to be offered by the ROESY experiment, recently advocated by Bax and Davis [Zl]. The rotating-frame NOE (under spinlocked conditions) is always positive and monotonically increases for increasing values of 'c C’
CONFORMATIONAL ANALYSIS OF DNA FRAGMENTS Single helices A single-helical DNA fragment is stabilized solely by "vertical" base-base overlap interactions (stacking) [l-3]. In contrast, the DNA duplex, besides stacking, has an additional stabilization through the formation of hydrogen bonds between bases on opposite strands, as well as the possibility of interstrand steric and/or charge-charge interactions. In order to understand the structure and dynamics of DNA, it is therefore important to study the behaviour of single helices of varied sequence in conjunction with the behaviour of the corresponding duplexes. We have set out to answer the following questions: (i) what conformational changes, if any, occur in going from the single helix to the double helix? (ii) do single-helical and duplex regions display conformational purity of the sugar rings or is N/S ring flip still possible:
119 TABLE II Pseudorotation
parameters
and predicted
6 deviations
mers,
see
[6,7],
backbone angle 6 (CS’-C4’-C3’-03’)
from mean (141’)
of various
single-helical
and observed DNAoligo-
text.
Compound
Residue
d (TAAT)1
T(1) A(2) A(3) T(4)
160 167 153 143
A(2) T(3) A(4)
T(1)
pS
Temp. range (“C)
6S(o)
A60bs
*‘pred
35 36 40 32
143 147 141 132
+2 +6 0 -9
0 +5 0 -5
8-43 I, II II
158 160 149 166
35 36 35 32
142 143 136 144
+l +2 -5 +3
0 +5 -5 0
a-44 ,I II ,t
A(1) T(2) A(3) T(4)
173 150 170 148
36 36 36 31
150 138 149 134
+9 -3 +8 -7
+5 -5 +5 -5
8-35 II
d (‘ITA) 3
T(1) T(2) A(3)
155 155 162
34 34 34
140 140 143
-1 -1 +2
0 0 0
5-65 II II
d&W4
A(1) A(2) A(3)
170 157 153
36 36 34
149 142 138
+8 +l -3
+5 0 -5
11-25 ,1 11
d(AA)4
A(1) A(2)
167 153
36 35
147 139
+6 -3
+5 -5
o-25 II
d (TGTG)5
T(1) G(2) T(3) G(4)
150 158 149 166
37 37 35 33
138 142 137 144
-3 +l -4 +3
0 +5 -5 0
27 11 ,I 11
d (CAACTT)6
C(1) A(2) A(3) C(4) T(5) T(6)
158 171 153 162 159 -
34 36 38 35 35 -
141 149 140 144 142 -
0 +8 -1 +3 +l -
0 +5 0 -5 0 0
15-35 II 11 II 11 11
d (CGT)7
C(l) G(2) T(3)
154 163 146
35 37 33
140 145 134
-1 +4 -7
0 +5 -5
2-52 11 II
d(TCG)7
T(l1 C(2) G(3)
158 157 160
33 35 34
141 141 142
0 0 +l
0 0 0
12-52 11 II
d (TATA)1
d (ATAT]2
8 35
l) J.-R.Mellema, A.K.Jellema, C.A.G.Haasnoot , .J.H. van Boom, and C.Altona, Eur. J.Biochem. 141 (1984) 165-175. 2, Refs 25.26. 3, C.A.G.Haasnoot, J.-R.Mellema, and C.Altoncunpublished results. 4, C.S.M.Olsthoorn, L.J.Bostelaar, J.H. van Boom, and C.Altona, Eur. J. Biochem. 112 (1980) 95-110. 5, L.J.Rinkel, J.-R.Mellema, and C.Altona, to be pubzhed. 6, L.P.M.Orbons and C.Altona, to be published. 7, J.-R. Mellema, R. van der Noerd, G.A. van der Marel, J.H. van Boom, and C.Altona, Nucleic Acids Res. -12 (1984) 5061-5078.
120 Crystallographic
information
concerning
single-helical
and knowledge must be gained from NMRspectroscopy. ber of such fragments have been studied ings will
be summarized here.
and 6S of deoxyribose vicinal
coupling
sugar ring
constants,
by PS ‘L 150’. character
Closer
(purine
of roughly
sugar conformational particular
occur
variation
Cg proposed
simple additivity sequence.
error
of such a step, [27],
especially
Elellema et al.
the values
values
negative
concerned
Analogous
[25,
appear
values to the
261 devised
6 variation
a
with DNA
the values
+1, -1,
0, 0. The unit of sum function
Fig.
g6
8 shows the A60bs (= 6obs-1410)
61 residues.
for the observed
already
noted
concomitant
results
in an in-plane
similar
sliding
effect,
in the single
in a B-DNA single-helix
An overall
with an increase sliding
leading helix.
r.m.s.
Fig.
deviation expe-
to 133” slides
less
aligned
with the adenine C6-N6 dipole.
tion
of electrostatic of 6
has another
the purine
interesting
a change in 6 leads
of the Cl’-N
vectors
this
where the
and becomes more or
suggests
a.B,Y,e,<)
to an appreciable residues,
a dA-dT step
that optimiza-
role.
conformational
of successive
is
6 angle from the
a position
6-membered ring This result
for
6
that a
interaction,
principle
the T base into
(angles
angle
base-paired
It is proposed
stacking
of the thymidine
may play a certain
pathway of the phosphate backbone fixed,
of 6 of the opposite
9 illustrates Reduction
by computer
of a pyrimidine
of the base pair.
is positioned,below interaction
is suggested
to optimum base-base
conformation.
value in the monomer (152’)
6 variation
[27] that a decrease
C4-04 dipole
more or less
changes of
when the residue
us to correlate
angle difference.
purine,
entation
II shows that positive
in a dR-dR or dR-dY step;
incorporating
explanation
Dickerson
Variation
the important
on local
which is of the same order of magnitude as the estimated
in a B-DNA duplex,
general
into
Since the
in 6.
A possible modelling.
operative
to the sugar in ques-
now be centered
in Table
by Dickerson
at 5O of torsion is found,
in
to the
in the sequence.
of 6 is seen in dY-dR or dY-dY steps.
are assigned
of 16 sequences,
rimental
variability
is not related
The sugars at each dR-dR and dR-dY step are assigned
is fixed of 2.6’
bases
will
scheme I& which allows
dY-dR and dY-dY steps values
pattern
P and 4, can be translated
R residue
in the second residue
sum function
of
angle.
with the first
is dY. No large
PS, 0,
analysis
in the case of d(ATAT) the
Y) of the base attached
Z), the discussion
torsion
parameters
whereas the dT sugars are characterized
of two neighbouring
parameters
years a large num-
sequence-dependent
that this
Study of the column headed Mobs to correlate
A clear
170°,
reveals
R or pyrimidine
but to the character
the geometrical
261. For example,
is scanty
Some of the main find-
by means of pseudorotation
supra. [25,
scrutiny
backbone angle 6 (eq. this
vi&
geometry is evident
dA sugars have PS values
tion,
obtained
In recent
in our laboratory.
Table II lists
sugars,
DNA fragments
consequence.
If the
is assumed to remain change in relative
viewed in a direction
ori-
121
-+-c-rT--R--T--R 0
0 .I
-I 0
0
-1
0
-.
b*
0 .,
G-G 1, -1 _f
&.*I
.I
-I
1
&.
-I 0
0
-1
0
. 0 f,
0
Fig. 8. Correlation of the variation of the backbone angle 6 observed for a series of single-helical DNAfragments in aqueous solution with the expected variation according to the sum function C6. Experimental values are connected by full lines, predicted ones by broken lines. The unit of change in Lg corresponds to So of 6. (From ref. 26, see also ref. 25 and Table II). perpendicular ly identical
to the base planes. to the local
From the model depicted
twist in Fig.
The relative
orientation
so defined
is virtual-
angle tl as defined by Dickerson and Drew [28]. 9 it is seen that a change from 6 152” to 6 133”
122
Fig. 9. Schematic illustration of the effect of 6 variation on base-base overlap in a regular B-type single-helical dA-dT step, viewed in the 5’+3’ direction. The broken outline shows the expected position of the dT base for 6 152’, i.e. the approximate 6 value of the thymidine monomer. A decrease of the dT 6 angle to 133’ (Table II) causes an appreciable sliding of the dT base with respect to the overlying dA base (solid line), concomitant with a decrease of the local twist angle tl from about 42’ to about 25’: allows
the lower base to slide
about 25’,
i.e.,
helical
dependent adjustments stitutes
in such a way that tl
“untwisting”.
These considerations
of the deoxyribose
a mechanism for
fine
decreases
suggest
expressed
pucker,
tuning of the base-base
from about 42’
to
that sequence
as 6 variation,
con-
overlap.
Double helices DNAs can adopt several mental conditions DNAduplexes E,T),
double-helical
such as counterions,
are classified
the B family
this
that several
deoxyoligonucleotides
clearly
mode is S (2’-endo).
indicate
At this
point
sequence d(GGCCGGCC)[29,30]. in solution
indicated contrast
a major contribution to present
Thus far,
lines
the sole
is read off
Similarly, of B-like
representative
These duplexes
display
interesting
[33] to steric
the duplex formed by
It is well
to remember that
clashes
N- or S-type
RNA-DNAhybrid
forms in the deoxyribose
studies
[18]
strands,
in
[3]. of the B-type duplex in the single
is the dodecamer sequence d(CGCGAATTCGCG) and its by Calladine
as
compounds in aqueous
in the A form [32] whereas solution
of thought
to note
and characterized
from two criteria:
the covalent
(B,C,D,
whereas for
is of interest
This concerns
the self-complementary
[31].
the B-family
of one of these
A- or B-type character sugar and NOE intensities
it
have been crystallized
B-type character.
r(GCG)d(TATACGC) crystallizes
sequence and composition.
A-type,
mode for A DNA.is N (3’-endo),
DNA, whereas our NMRinvestigations
solution
depending upon environ-
water activity,
in three broad groups:
and Z DNAs. The sugar puckering
A-type
conformations,
local
C(9) brominated
changes of geometry
between adjacent
purines
crystal
derivative.
[27,28,34] in opposite
ascribed strands.
123
d(C A C A T G T Gl
d(C A C Al d(T G T G)
Fig. 10. Populations of S conformer (%, hatched areas) in two single-helical tetramers - d (CACA) and d (TGTG) - and in the composite octamer duplex d(CACATGTG), ref. 35. Note that the sugars in the core of the duplex appear to retain some conformational freedom. Obviously,
such clashes
can occur
posed to be exceptionally mations are concerned, (unimodal) (P 90°)
variation
tend to favour
and coworkers
of sugar puckers,
In the crystal
about Cl’-exo
(P 126’)
favour
a similar
picture,
prefer
albeit
d(CACATGTG), together
high 6 values.
Moreover,
with those
in the two constituent
single
[35] by means of line
The results
(Fig.
lo),
it
of the duplex existence
still
distribution). studies hybrid
retain
flip
duplex octamer d(GGm5Cm5C)2[17],
alternating
already
influence
pared to that observed
the geometrical a considerable offered
principle
parameters
since
a certain
found.
the marked decrease
RNA-DNA
amount of residual
that a limited Second (Fig. 6 variation,
fragments.
by the Calladine-Dickerson
[27,33],
(bimodal
our earlier
amount
at Zft resolution,
amount of additional single-helical
confor-
Thus, the
and on the covalent
in an X-ray analysis
in the constituent
to the rationale
anticomplementarity
freedom.
It must be mentioned in passing
duplex induces
on
the sugars in the core
duplex is indicated
revealed
12”
10 and 11.
towards the S-type
does not come as a surprise,
might remain undetected
though it could
contrast
(S Z N) in the intact
duplex mentioned above [IS],
freedom of the sugar rings. of ring
driven
tetramers,
some measure of conformational flip
This conclusion
on B-DNA
although
single-helical
fragments X2’,
in the section
are summarized in Figures
is noted that,
of dynamic ring
helical
widths Cl’,
with the aid of the method outlined
First,
[35]
dY-dR octamer duplex
conformational
mer compared to the constituent
(omitting
pyrimidines
one.
taken from COSY spectra, analysis.
to 04’-endo
Our NMRstudies
sugars in the alternating
d(CACA) and d(TGTG), were analyzed
(P 162’)
with a mean 6 = 125’
a more complicated
The puckers of the individual
a continuous
the sugar conformations
and a 6 range running from 155’ to 80’.
low 6 and purines
and are sup-
As far as the sugar confor-
[27,28,34]
running from C2’-endo
6 variation.
distributed
the 3’ terminals),
in the minor groove.
Dickerson
with a concomitant
are apparently
reveal
strong
only in dR-dY and in dY-dR steps
rules,
alll),
the com-
However, in i.e.,
the
in 6 of the pyrimidine
124
110-L
CACATGTG
Fig. 11. The sequence-dependent variation of backbone angle 6 of the major Stype conformation in two single-helical tetramers d(CACA) and d(TGTG), indicated deduced for the B-type double helix formed by +, compared to the 6 variation by the octamer d(CACATGTG), indicated by o. residues
dC and dT is not accompanied by a similar
residues
dA and dG (perhaps with the exception
the &-range seen in aqueous solution smaller
than the b-range
(155O to 80°, (134’) this
omitting
therefore point
of local
it
strand steric generalizations
(157” to 115’)
the 3’ terminals).
can be concluded
Moreover,
of the dodecamer
The value of Gmean in our octamer
than that found for the dodecamer (125’). that the NMRresults
of geometry in a B-DNA duplex,
effects.
in 6 of the purine
appears to be decidedly
deduced from X-ray crystallography
remains greater
variations
increase
of the dG(6) sugar).
More solution
studies
decidedly possibly
favour
At
the concept
mediated tria inter-
appear necessary,
however,
before
can be made.
ACKNOWLEDGEMENTS This research was supported by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). Dr J.-R.Mellema, Mr L.P.M.Orbons and Mr L.J.Rinkel kindly supplied some of the illustrations used in this paper. REFERENCES P.O.P. Ts’o, “Basic Principles in Nucleic Acid Chemistry”, vols 1-3, Academic Press, New York (1974). C.R. Cantor and P.R. Schimmel, “Biophysical Chemistry”, vols 1-3, Freeman, San Francisco (1980) . of Nucleic Acid Structure”, Springer, New York (1984). W. Saenger, “Principles C. Altona, Reel. Trav. Chim. Pays-Bas 101 (1982) 413-433. C. Altona, in Stud. Org. Chem. Vol. 20 “Natural Products Chemistry”, Elsevier, Amsterdam (1985) 285-304. C. Altona and M. Sundaralingam, J. Am. Chem. Sot., 94 (1972) 8205-8212; idem ibid. 95 (1973) 2333-2344. IUPAC-IUB Joint Commission on Biochemical Nomenclature, Eur.. J. Biochem., 131 (1982) 9-1s. F.A.A.M. de Leeuw, P.N. van Kampen, C. Altona, E. Diez, and A.L. Esteban, J. Mol. Struct ., 125 (1984) 67-88. H.P.M. de Leeuw, C.A.G. Haasnoot, and C. Altona, Isr. J. Chem.,20 (1980) 108-126.
125
10 C.A.G. Haasnoot, F.A.A.M. de Leeuw, and C. Altona, Tetrahedron, 36 (1980) 2783-2792. 11 C.A.G. Haasnoot, F.A.A.M. de Leeuw, H.P.M. de Leeuw, and C. Altona, Biopolymers, 20 (1981) 1211-1245. 12 F.A.A.M. de Leeuw and C. Altona, J. Comput. Chem., 4 (1983) 428-437; iciem, Quant. Chem. Progr. Exch. No 463 (1983); idem, J. Chem. Sot. Perkin II, (1982) 375-384. 13 G.M. Clore and A.M. Gronenborn, EMBO J., 4 (1985) 829-835; i&m, FEBS Lett., 179 (1985) 187-198 and references therein. 14 .J.W.Keepers and T.L. .James,J. Magn. Reson. 57 (1984) 404-426: M.S. Broido, T.L. James, G. Zon and J.W.Keepers, Eur. J. Biochem., 150 (1985) 117-128. 15 A.M. Gronenborn, G.M. Clore and B.J. Kimber, Biochem. J., 221 (1984) 723-736. 16 A. Bax, "Two-Dimensional Nuclear Magnetic Resonance in Liquids", Reidel, Dordrecht, Holland, 1982. 17 M.R. Sanderson, J.-R. Mellema, G.A. van der Marel, G. Wille, J.H. van Boom, and C. Altona, Nucleic Acids Res. 11 (1983) 3333-3346. 18 J.-R. Mellema, C.A.G. Haasnoot, G.A. van der Marel, G. Wille, C.A.A. van Boeckel, J.H. van Boom and C. Altona, Nucleic Acids Res. 11 (1983) 5717-5738. 19 COSY = Correlated Spectroscopy, A. Bax, R. Freeman and G.A. Morris, J. Magn. Reson.,42 (1981) 164-168; NOESY q Nuclear Overhauser Enhancement Spectroscopy, J. Jeener, B.H. Beier, P. Bachmann and R.R. Ernst, .J.Chem. Phys., 71 (1979) 4546-4553. 20 DAYCOSY = double-RELAY experiment, A. Bax and G. Drobny, J. Magn. Reson., 61 (1985) 306-320; G. Eich, G. Bodenhausen and R.R. Ernst, J. Am. Chem. Sot., 104 (1982) 3731-3733. 21 ROESY = Rotating-frame Overhauser Enhancement Spectroscopy, A. Bax and D.G. Davis, J. Magn. Reson., 63 (1985) 207-213; A.A. Bothner-By, R.L. Stephens, J. Lee, C.D. Warren and R.W. Jeanloz, J. Am. Chem. Sot., 106 (1984) 811-813. 22 J. Feigon, J.M. Wright, W. Leupin, W.A. Denny and D.R. Kearns, J. Am. Chem. sot., 104 (1982) 5540-5541. 23 D.G. Reid, S.A. Salisbury, S. Bellard, Z. Shakked and D.H. Williams, Biochemistry, 22 (1983) 2012-2025. 24 R.M. Scheek, N. Russo, R. Boelens, R. Kaptein and .J.H.van Boom, J. Am. Chem. sot., 105 (1983) 2914-2916. 25 J.-R. Mellema, J.M.L. Pieters, G.A. van der Marel, J.H. van Boom, C.A.G. Haasnoot and C. Altona, Eur. .J.Biochem. 143 (1984) 285-301. 26 J.-R. Mellema, Appendix in Thesis, Leiden, 1984. 27 R.E. Dickerson, J. Mol. Biol. 166 (1983) 419-441. 28 R.E. Dickerson and H.R. Drew, J. Mol. Biol. 149 (1981) 761-768. 29 A.H.-J. Wang, S. Fujii, J.H. van Boom and A. Rich, Proc. Natl. Acad. Aci. U.S.A., 79 (1982) 3968-3972. 30 L.J. Rinkel, M.R. Sanderson and C. Altona, to be published. 31 C.A.G. Haasnoot, H.P. Westerink, G.A. van der Marel, and J.H. van Boom, J. Biomol. Struct. Dyn., 2 (1984) 3445-3460. 32 A.H.-J. Wang, S. Fujii, J.H. van Boom, G.A. van der Marel, C.A.A. van Boeckel and A. Rich, Nature, 299 (1982) 601-604. 33 C.R. Calladine, J. Mol. Biol. 161 (1982) 343-352. 34 A.V. Fratini, M.L. Kopka, H.R. Drew and R.E. Dickerson, J. Biol. Chem., 257 (1982) 14686-14707. 35 L..J.Rinkel, J.-R. Mellema, L.P.H. Orbons, G.A. van der Marel, J.H. van Boom and C. Altona, Abstracts 4th Conversation in Biomolecular Stereodynamics, R.H. Sarma @d.)State University of New York at Albany, 1985, pp 126-127; L.J. Rinkel et al. to be published.