NMR solution structure of the major G-quadruplex
structure formed in the human BCL2 promoter region
Jixun Dai1, Ding Chen1, Roger A. Jones2, Laurence H. Hurley1,3,4and Danzhou Yang1,3,4,*
1College of Pharmacy, The University of Arizona, 1703 E. Mabel Street, Tucson, AZ 85721, USA,
2Department of Chemistry and Chemical Biology, Rutgers University, 610 Taylor Road, Piscataway,
NJ 08854, USA,3Arizona Cancer Center, 1515 N. Campbell Avenue, Tucson, AZ 85724, USA and
4BIO5 Institute, The University of Arizona, 1140 E. South Campus Dr, Tucson, AZ 85721, USA
Received March 22, 2006; Revised and Accepted August 3, 2006Protein Data Bank accession no. PDB ID 2F8U
BioMagResBank accession no. 6975
BCL2 protein functions as an inhibitor of cell
apoptosis and has been found to be aberrantly
expressed in a wide range of human diseases.
A highly GC-rich region upstream of the P1 promoter
plays an important role in the transcriptional regu-
lation of BCL2. Here we report the NMR solution
structure of the major intramolecular G-quadruplex
formed on the G-rich strand of this region in
antiparallel-stranded G-quadruplex structure con-
tains three G-tetrads of mixed G-arrangements,
which are connected with two lateral loops and one
side loop, and four grooves of different widths. The
three loops interact with the core G-tetrads in a
specific way that defines and stabilizes the overall
G-quadruplex structure. The loop conformations are
in accord with the experimental mutation and
footprinting data. The first 3-nt loop adopts a lateral
loop conformation and appears to determine the
overall folding of the BCL2 G-quadruplex. The third
1-nt double-chain-reversal loop defines another
example of a stable parallel-stranded structural
motif using the G3NG3sequence. Significantly, the
distinct major BCL2 promoter G-quadruplex struc-
ture suggests that it can be specifically involved in
gene modulation and can be an attractive target for
pathway-specific drug design.
The BCL2 (B-cell CLL/lymphoma 2) gene product is a mito-
chondrial membrane protein that plays an essential role in
cell survival. The BCL2 protein exists in delicate balance
with other apoptosis-related proteins and functions as an
inhibitor of cell apoptosis (1,2). Deregulation of the BCL2
gene is associated with aberrant cell growth in many human
diseases. For example, BCL2 has been found to be aberrantly
overexpressed in a wide range of human tumors, including
B-cell and T-cell lymphomas (3,4) and breast (5), prostate
(6), cervical (7), colorectal (8), and non-small cell lung
carcinomas (9). Moreover, BCL2 overexpression has also
been associated with poor prognosis and has been found to
interfere with traditional cancer therapeutics (10,11). Inhibi-
tion of BCL2 expression by small molecules (12,13), pep-
tidomimetics (14), or antisense oligonucleotides (15,16) has
been shown to reduce cellular proliferation and to enhance
chemotherapy efficacy. In contrast, aberrantly reduced BCL2
expression is associated with cardiovascular diseases and
neurological disorders, such as ischemia/reperfusion injury
of cardiac and renal tissues (17,18), multiple sclerosis (19),
Alzheimer’s and Parkinson’s diseases (20), and tissue dam-
age related with stroke (21) and spinal cord injuries (22).
Thus, BCL2 has also emerged as an attractive target for
neuroprotective and tissue-protective therapies.
There is accumulating in vitro evidence of G-quadruplex
structures formed in promoter regions of several genes as
transcriptional regulators, such as the transcriptional repres-
sors reported in the human MYC (c-MYC) gene (23,24),
the chicken b-globulin gene (25) and several muscle-specific
genes (26,27), as well as a transcriptional enhancer reported
in the human insulin gene (28,29). The human BCL2 gene
has two promoters, P1 and P2. The major promoter, P1,
located 1386–1423 bp upstream of the translation start site,
is a TATA-less, GC-rich promoter containing multiple tran-
scriptional start sites and positioned within a nuclease hyper-
sensitive site (30,31). A highly GC-rich 39-bp region located
58–19 bp upstream of the P1 promoter has been implicated in
playing a major role in the regulation of BCL2 transcription
(Figure 1A) (32). Multiple transcription factors have been
reported to bind to or regulate BCL2 gene expression through
this region, including CREB (33), WT1 (34), Sp1 (30),
E2F (35), NF-kB (36), and NGF (37). For example, CREB
functions to activate BCL2 expression (33), while WT1
functions to repress BCL2 expression (34).
*To whom correspondence should be addressed: Tel: +1 520 626 5969; Fax: +1 520 626 6988; Email: email@example.com
? 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Published online 22 September 2006Nucleic Acids Research, 2006, Vol. 34, No. 185133–5144
Our previous results have shown that the 39mer guanine-
rich strand (bcl2Pu39, Figure 1A) in the BCL2 promoter
can forma mixture of three distinct
G-quadruplexes in K+-containing solution, and that the
G-quadruplex formed on the middle four consecutive runs
of guanines is the most stable one (38,39), which adopts a
novel folding of mixed parallel/antiparallel-stranded structure
(39). In this paper we report the NMR solution structure
for the predominant G-quadruplex structure formed in the
BCL2 promoter region. The solution structure provides not
only the molecular details of this G-quadruplex but also
important insights for its loop conformations and interactions
with the core tetrad structures.
MATERIALS AND METHODS
b-cyanoethylphosphoramidite solid-phase chemistry on an
DNA oligonucleotideswere synthesizedusing
Expedite? 8909 Nucleic Acid Synthesis System (Applied
Biosystem, Inc.) in DMT-on mode, and were purified using
C18 reverse-phase HPLC chromatography, as described pre-
viously (39–41). Deprotection was carried out using 80%
AcOH for 1 h, followed by ether extraction. DMT-off DNA
was further purified by HPLC followed by successive dialysis
against 150 mM NaCl and H2O. 6% 1,2,7-15N, 2-13C-labeled
guanine phosphoramidite (42) was used for site-specific
labeled DNA synthesis. Samples in D2O were prepared
by repeated lyophilization and final dissolution in 99.96%
D2O. Samples in water were prepared in 10%/90%
D2O/H2O solution. The final NMR samples contained
0.2–2 mM DNA oligonucleotides in 20 mM K-phosphate
buffer (pH 7.0) and 40 mM KCl.
NMR experiments were performed on a Bruker DRX-600
spectrometer as described before (39–41,43). At the deter-
mined melting point (61?C), stoichiometric titration of the
melted and the folded strands as a function of total strand
concentration from 0.01 to 0.1 mM was performed (43).
Assignment of guanine imino and H8 protons were obtained
by 1D15N-filtered experiments using site-specific labeling.
Standard 2D NMR experiments, including NOESY, TOCSY,
and DQF-COSY, were used at 1, 7, 15, 20, 25, 30 and 35?C to
obtain complete proton resonance assignment. The NMR
experiments for samples in water solution were performed
with Watergate or Jump-and-Return water suppression tech-
niques. Relaxation delays were set to 3 s. The acquisition
data points were set to 2048 · (350?512) (complex points).
The 45?or 60?shifted sine-squared functions were applied
to NOESY and TOCSY spectra. The fifth-order polynomial
functions were employed for the baseline corrections. The
final spectral sizes are 2048 · 1024. Peak assignments and
integrations were achieved using the software Sparky (UCSF).
Only relatively isolated peaks were used for NOE-restrained
structure calculation. Severely overlapping peaks were
discarded. The NOE peaks were integrated using the peak
fitting function and volume integration of Sparky. We have
manually checked every peak to make sure the fitted
lineshape agrees with the experimental data. The average
linewidth of NMR peaks is 10–15 Hz, and the digitized res-
olution of the NOESY spectra is sufficient for the line-fitting
and accurate volume integration in Sparky. Distances
between non-exchangeable protons were estimated based on
the NOE cross-peak volumes at 50–300 ms mixing times,
with the upper and lower boundaries assigned to ±20% of
the estimated distances. Distances between exchangeable
protons were assigned with looser boundaries (±1–1.5 s).
The cytosine base proton H5–H6 distance (2.45 s) was used
as a reference. The distances involving the unresolved
protons, e.g. methyl protons, were assigned using pseudo-
atom notation to make use of the pseudo-atom correction
automatically computed by X-PLOR.
The31P NMR spectra were collected on a DNA sample at
1.5 mM in D2O (20 mM potassium-phosphate buffer, 40 mM
KCl, pH 7.0) at 25?C and were referenced to an external stan-
dard of 85% H3PO4, including the 1D proton-decoupled phos-
phorus spectrum, and 2D heteronuclear31P–1H Correlation
Spectroscopy (COSY) and Heteronuclear Single Quantum
Figure 1. (A) The promoter sequence of the BCL2 gene and its modifications.
Bcl2Pu39 is the wild-type BCL2 39mer sequence; bcl2MidG4 is the
BCL2 23mer sequence containing the middle four consecutive G-runs,
which forms the most stable G-quadruplex structure; bcl2Mid is the mutant
23mer with G-to-T mutations at positions 15 and 16, bcl2Midm2 is the
mutant 23mer with G-to-T mutations at positions 15 and 19, and bcl2Midm3
is the mutant 23mer with G-to-T mutations at positions 18 and 19. The six G-
runs are underlined and numbered using Roman numerals; the BCL2 23mer is
numbered using Arabic numerals. (B) The imino regions of 1D1H NMR
spectra of bcl2Mid samples at 0.1 mM (upper) and 1.5 mM (lower) strand
concentrations. Conditions: 25?C, 20 mM K-phosphate, 40 mM KCl, pH 7.0.
5134Nucleic Acids Research, 2006, Vol. 34, No. 18
Correlation Spectroscopy (HSQC). A series of31P–1H HSQC
spectra were collected at spectral widths of 10 p.p.m. (1H) ·
5 p.p.m. (31P) with 2048 · 128 complex points, using various
heteronuclear INEPT transfer delays corresponding to
J-couplings of 5, 10, 15, 20 and 25 Hz. The non-selective
and H30-selective31P–1H COSY experiments (44) were car-
ried out in States-TPPI mode using the same spectral width
with 2048 · 128 complex points and 256 scans. Assignments
of the individual
combination of 2D
31P resonance were accomplished by a
1H/1H NOESY, COSY, TOCSY and
Distance geometry and simulated annealing
Metric matrix distance geometry (MMDG) calculations were
carried out using X-PLOR (45) to embed and optimize 100
initial structures. An arbitrary extended conformation was
first generated for the single-stranded bcl2Mid sequence.
Substructure embedding was performed to produce a family
of 100 DG structures. The embedded DG structures were
then subjected to simulated annealing regularization. The
experimentally obtained distance restraints and G-tetrad
hydrogen-bonding distance restraints were included during
the calculation. All distance restraints were specified with
the SUM averaging option in X-PLOR (45). After simulated
annealing, 97% of the molecules were folded in the correct
topology, whereas 3% of the molecules were misfolded,
e.g. in left-handed folding topology.
NOE-distance restrained molecular dynamics
All ofthe 100 molecules obtained from the DGSAcalculations
ment in XPLOR (45) with a distance-dependent dielectric con-
stant. Atoms participating in hydrogen bonds in the G-tetrad
planes were restrained with distances corresponding to ideal
hydrogen bond geometry. Each individual hydrogen bond
was restrained using two distance restraints (heavy atom–
heavy atom and heavy atom–proton). Hydrogen-bonding dis-
tance restraints were also applied to A10:T15 bp, with larger
restraints were specified ambiguously with the sum-averaging
option. The force constants were scaled at 30 and 100 kcal
mol?1A˚?2for NOE and hydrogen bond distance restraints,
respectively. A total of 476 NOE-distance restraints, of which
into the NOE-restrained structure calculation.
Dihedral angle restraints were used to restrict the glyco-
sidic torsion angle (c) for the experimentally assigned syn
configuration, i.e. G1, G7, G8, G17 and G21, tetrad-guanines
[60(±30)?], and A13 and A14 in the 30-loop [60(±60)?],
as well as for some of the experimentally assigned anti con-
figuration bases, i.e. C4, G5 and C6 in the 50-loop
[220(±40)?]. Dihedral angle restraints were also used to
restrain the sugar backbone torsion angles b, g and e (46).
Based on the J-coupling constants of31P(n)–H50/H500(n) and
H30(n ? 1)–31P(n) obtained from
HSQC experiments with various J-couplings, the b angles
were restrained to the b?t conformation at 180(±90)?for
all the residues, except for G21 whose b angle was restrained
31P–1H COSY and
to ?60(±20)?. The e angles were restrained to 80(±20)?for
G19, and to 90(±40)?for T15 and C6. Based on the relative
intensities of H30–H50/H500and H40–H50/H500, the g angles of
the majority of residues with resolved H50/H500are in the
regular g+ conformation (?60?) or sometimes in the g? con-
formation (??60?), because for each residue, the H30–H50
(or H30–H500) NOE is clearly stronger than the H30–H500(or
H30–H50) NOE, except for A14 and T15 which show similar
intensities for H30–H50and H30–H500, and thus fall in the g?t
region (46). Thus only the g angles of A14 and T15 were
restrained to 170(±90)?. The force constants of dihedral
angle restraints were 10 kcal mol?1rad?2for c and 5 kcal
mol?1rad?2for b, g and e.
NOE-restrained simulated annealing refinement calcula-
tions were initiated at 300 K. The temperature was gradually
increased to 1000 K in 4 ps. The system was equilibrated at
1000 K for 20 ps, and was then slowly cooled to 300 K in
10 ps. The 20 best molecules were selected based both on
their minimal energy terms and number of NOE violations,
and were further subjected to NOE-restrained molecular
dynamics calculations at 300 K for 25 ps. The coordinates
saved every 0.1 ps during the last 2.0 ps of NOE-restrained
molecular dynamics calculations were averaged, and the
resulting averaged-structure was subjected to minimization
until the energy gradient of 0.1 kcal·mol?1was achieved. A
soft planarity restraint of 1 kcal mol?1A˚?2was imposed
on the tetrads before the heating process and was removed
at the beginning of the equilibration stage. The time steps
for all processes of heating, cooling, and equilibration were
equal to 0.5 fs. The 10 best molecules were selected based
both on their minimal energy terms and number of NOE vio-
lations and have been deposited in the Protein Data Bank
(accession no. 2F8U).
RESULTS AND DISCUSSION
Basis for selection of the bcl2Mid sequence with its dual
G-to-T mutations as the predominant G-quadruplex
in the BCL2 promoter
In a recently published study (38), we have demonstrated that
the six runs of G-tracts containing three or more contiguous
guanines in the wild-type sequence (bcl2Pu39, Figure 1A)
can form three overlapping G-quadruplex structures, i.e.,
50G4, midG4, and 30G4 (see Supplementary Table S1). Of
these three overlapping sequences, midG4, or bcl2MidG4
(Figure 1A), is by far the most stable. Since this sequence
contains a run of five guanines, it is possible to form three
loop isomers in which the three guanines involved in the
G-tetrads are either at the 30or the 50end or in the middle
of the five continuous guanines (Supplementary Table S1
and Figure 1A bottom three sequences). The three possible
loop isomers can be isolated by dual G-to-T mutations as
shown in Figure 1A. The bcl2MidG4 sequence gives rise
to an unambiguous DMS cleavage pattern in which there is
a clear preference for the loop isomer as formed in bcl2Mid
(Figure 1A). The bcl2Mid sequence also forms the most sta-
ble G-quadruplex structure, as demonstrated by NMR (Sup-
plementary Figure S1). This sequence gave a well-defined
spectrum that has many overlapping1H NMR resonance sig-
nals with the wild-type sequence (Supplementary Figure S1)
Nucleic Acids Research, 2006, Vol. 34, No. 18 5135
(39). In addition this same mutated sequence was previously
shown to be the most stable using polymerase stop assay (see
figure 6 in Ref. 38). Therefore the bcl2Mid sequence with the
dual (G-to-T) mutant at the 50-end of bcl2MidG4 (positions
15 and 16, Figure 1A) was chosen as the sequence for
NMR structure determination. The reason why the other pos-
sible contiguous runs of three guanines in Figure 1A are
unstable is that these mutational sequences eliminate the
G3NG3single-nt double-chain-reversal loop which provides
stability to the quadruplex. Thus it is clear that the dual
50-(G-to-T) mutation selected can give rise to a specific and
stable fold containing this G3NG3 single-nt double-chain-
reversal motif. Importantly, the two mutated guanines (G15
and G16, Figure 1A) give rise to the predominant DMS
cleavage pattern found in the wild-type sequence (38). Fur-
thermore, the bcl2Mid sequence gives rise to a CD spectrum
very similar to that of the wild-type sequence (39). We are
therefore confident that the G-quadruplex from this sequence
is not only the predominant one found in a single-stranded
BCL2 promoter DNA template, but its dual G-to-T mutations
force the sequence into the predominant loop isomer. This is
an important point because mutants that change the folding
pattern or result in isolation of the least stable species are
less likely to be biologically relevant.
bcl2Mid forms a monomeric G-quadruplex structure
The bcl2Mid molecule exhibits a stronger propensity to
aggregate than other G-quadruplex forming sequences we
have worked with. Minor conformations are also present as
indicated by the presence of weak resonances (Figure 1B),
whose intensities are <5% of the major species and thus
do not interfere with the unambiguous structural analysis of
the predominant BCL2 G-quadruplex structure. A sample of
bcl2Mid in potassium solution with a concentration over
3 mM, as has been routinely used for other G-quadruplex
structures (41,43,47,48), shows a markedly increased back-
ground after a week and readily aggregates to a gel form
after annealing from 95?C. We therefore used a lower
concentration of 1.5 mM of bcl2Mid in potassium for our
NMR structure determination (Figure 1B, lower panel). The
sample of 1.5 mM bcl2Mid is stable in potassium solution
for over 1 month which is sufficient for multiple 2D experi-
ments. The two 1D1H spectra of the 1.5 mM bcl2Mid NMR
sample, freshly made or in NMR solution for 5 weeks, are
shown in Supplementary Figure S2A&B. No noticeable dif-
ferences in 1D NMR spectra were observed for this sample
after 5 weeks in solution. However, after 5 months in NMR
solution, this sample gave rise to an observable higher back-
ground (Supplementary Figure S2C). After over 1 year in
NMR solution, a significantly increased level of broad reso-
nance background in 1D NMR spectra is observed for this
sample, even though the sharp peaks from the major species
are still clearly observable (Supplementary Figure S2D).
Figure 2. (A) Determination of stoichiometry by NMR titration for bclMid in
K+solution. The slope of the fitted line is 0.99, meaning that the quadruplex
structure existing in solution is unimolecular. (B) The variable-temperature
study of bcl2Mid by NMR. The peak intensities of two resolved peaks at
61?C (the one belonging to the melted form of bcl2Mid is labeled with
asterisk and the one belonging to the folded forms is labeled with cross) were
used for the calculation. (C) The extended region of 1D1H NMR spectra of
bcl2Mid at various concentrations showing the two peaks from the folded and
the melted forms. Conditions: 20 mM K-phosphate, 40 mM KCl, pH 7.0.
Figure 3. (A) (Left) Schematic drawing of the folding topology of bcl2Mid.
Red, guanine (anti); light red, guanine (syn); green, adenine; yellow, thymine;
blue, cytosine. (Right) A G-tetrad with H1–H1 and H1–H8 connectivity
pattern detectable in NOESY experiments. (B) Imino and aromatic regions of
the 1D1H NMR spectrum of bcl2Mid. The imino and aromatic protons are
assigned over the resonances. Conditions: 25?C, 20 mM K-phosphate, 40 mM
KCl, pH 7.0, 1.5 mM DNA.
5136Nucleic Acids Research, 2006, Vol. 34, No. 18
1D proton spectra of bcl2Mid at various temperatures
(VTs) indicate that the melting temperature for the quadru-
plex structure in 60 mM K+is around 65?C (Supplementary
Figure S3). The melting process of the G-quadruplex struc-
ture of bcl2Mid appears to be rather homogeneous throughout
the G-tetrad core structure. As described in our previous
report (39), bcl2Mid forms a monomeric intramolecular
G-quadruplex structure, as demonstrated by the independence
of the melting temperature from the concentration, the sharp
NMR spectral line widths (Figure 1B), and the EMSA
(electrophoretic mobility shift assay) results. The formation
of a unimolecular structure in bcl2Mid is unambiguously con-
firmed by the NMR stoichiometry titration experiment at
the melting temperature using DNA concentrations from
0.01 to 0.1 mM (43,49) (Figure 2). The NMR spectra of
bcl2Mid at a strand concentration of 1.5 mM (for 2D NMR
experiments and structure determination) and 0.1 mM (for
1D experiments and the titration experiment) are the same
(Figure 1B, upper and lower panels), indicating that the
same unimolecular G-quadruplex structure is formed in all
the conditions used for our NMR analysis. In addition,
DOSY (Diffusion Ordered Spectroscopy) experiments on
bcl2Mid also confirmed that the sharp NMR signals are
from a molecular weight equivalent to a monomeric structure.
(The DOSY results will be published elsewhere.)
Proton resonance assignments of bcl2Mid
The major G-quadruplex formed on the central BCL2 pro-
moter sequence has been shown to adopt a mixed parallel/
antiparallel-stranded folding (Figure 3A) (39). The imino
NH1 and base aromatic H8 protons of guanine residues of
this bcl2Mid were unambiguously assigned by the site-
specific low-concentration (6%) incorporation of 1, 2, 7-15N,
2-13C-labeled guanine nucleoside at each guanine position of
the sequence, one base at a time (Figure 3B and Supple-
mentary Figure S4) (39). The base H6 proton resonances of
thymines and cytosines were unambiguously assigned by sub-
stituting deoxyuridine (dU) for dT/dC one at a time at each
thymine/cytosine position of the sequence. Multiple 2D
experiments, including 2D-NOESY, TOCSY and COSY,
were carried out for bcl2Mid at a strand concentration of
1.5 mM in 20 mM pH 7.0 K-phosphate and 40 mM KCl at
various temperatures. Standard DNA sequential assignment
procedure was utilized for the assignment of the proton reso-
nances of bcl2Mid (Figure 4). The assignment of the aromatic
protons allowed the direct assignment of H10and H20/H200
resonances, which was then extended to other regions, includ-
ing those of H30, H40and H50/H500. The proton chemical
shifts at 25?C are listed in Table 1. All proton resonances
have been unambiguously assigned, except some H50/H500
protons which cannot be differentiated. The ambiguity of
H50/H500protons should not affect the NMR structure calcu-
lation, since the NOE intensities associated with H50or H500
do not vary much regardless whether a resonance is assigned
as H50or H500. In addition, very few NOE intensities associ-
ated with H50or H500were used (see below). For all the resi-
dues with resolved H20and H200resonance, the H10–H30NOE
is weaker than both the H10–H20and H10–H200NOEs, while
the H10–H200NOE is stronger than the H10–H20NOE, indicat-
ing a C20-endo sugar pucker conformation.
Phosphorus resonance assignments of bcl2Mid
We have carried out31P experiments on bcl2Mid, including
proton-decoupled 1D phosphorus spectroscopy, as well as
1D31P spectrum at 25?C in D2O is shown in Figure 5A, refer-
enced to the phosphoric acid standard. While the majority of
?1 ± 0.3 p.p.m., which has been shown to be the chemical
shifts for31P resonances of regular right-handed B-DNAs,
there are a number of31P resonances that are shifted outside
this region. Using 2D
proton assignments, we were able to assign all of the31P reso-
nances of bcl2Mid(Table 1). Every phosphorus resonance was
assigned by using the assignments of sugar protons H30, H40,
and H50/H500, and correlating it to its 50-coupled H30proton
and 30-coupled H40and H50/H500protons. Moreover, the phos-
phorus assignments confirmed the proton assignments of
H30(n ? 1), H40(n), and H50/H500(n) for each step. Very inter-
estingly, the phosphorus resonances that are observed out of
the ?1 ± 0.3 p.p.m. region are all from the residues in the
downfield-shifted (from ?1 p.p.m.) G21P (at C20–G21 step,
~P 1.5 p.p.m.), T15 (~P 0.88 p.p.m.), C4 (~P 0.54
p.p.m.), and C6 (~P 0.43 p.p.m.), and the upfield-shifted
G19 (~P ?0.93 p.p.m.), C20 (~P ?1.12 p.p.m.), and T16
(~P ?1.61 p.p.m.). In particular, those phosphorus reso-
nances associated with the unique double-chain-reversal
single-nt loop (G19, C20, G21, see Figure 3A) and with the
unique conformation at T15–T16 (Figure 3A) are the most
31P–1H COSY and
31P–1H HSQC. The
31P resonances of bcl2Mid are clustered around
31P–1H COSY (Figure 5B) and the
Figure 4. The expanded H8/H6–H10region with assignments of the non-
exchangeable 2D-NOESY spectrum of bcl2Mid.The sequential assignment
pathway is shown. Missing connectivities are labeled with asterisks. The H8–
H10NOEs of the nucleotides with syn configuration are labeled by residue
names, while the H5–H6 NOEs of cytosines are also labeled for reference.
The H8–H10NOE crosspeak of G21 has a large integration value; however,
the H8 and H10of G21 are much broader than those of other guanines and
give rise to a much broader H8–H10NOE crosspeak. The characteristic
G(i)H8/G(i+1)H10NOEs for the syn G(i)s are labeled as a–b for G1–G2 and
G7–G8. The NOE between C6H10and G19H8 is labeled as c. Conditions:
25?C, 20 mM K-phosphate, 40 mM KCl, pH 7.0, 1.5 mM DNA.
Nucleic Acids Research, 2006, Vol. 34, No. 185137
31P–1H COSY and
H30(n)–31P(n + 1) were obtained from
confirmed by HSQC experiments with various J-couplings.
The31P–H50and31P–H500coupling constants can define the
b torsion angle quite well (46). Except for G21, all other
10 Hz, indicating the b torsion angles fall in the common
trans-region, 180(±90)?. G21 gives rise to a31P–H50coupling
constant of ?12 Hz and no observable
indicating a b torsion angle in the b? region, ?60(±20)?.
Most H30-31P couplings are <10 Hz, indicating that the
e angles fall in the common trans-/gauche- conformation
(170?–300?), at which range the e angles can not be accu-
rately determined by the H30(n)–31P(n + 1) coupling constants
due to several possible e angles for one H30-31P coupling in
this region (46). However, when the H30(n)–31P(n + 1) cou-
pling constants are larger than 10 Hz, it is possible to more
accurately determine the e angles. The H30(n)–31P(n + 1) cou-
pling constantsfor G19(?C20P),
C6(?G7P) are 15, 12 and 12 Hz, respectively, indicative of
e angles in the gauche+/trans+ region (centered ?80–90?C).
It is again interesting to note that the residues exhibiting
unusual large coupling constants are all associated with the
loop regions, especially with the single-nt double-chain-
reversal loop. It may be noted that for non-B-DNA structures,
the31P chemical shifts show a very poor correlation with the
e angle values (50).
31P(n)–H50/H500(n) coupling constants are below
NOE interactions within G-tetrads regions in the
2D-NOESY and are summarized in Figure 6. Only one
inter-residue NOEcrosspeaks areobserved in
NOE intensity is associated with H50or H500. An expanded
NOESY spectrum of base and sugar H10protons is shown
in Figure 4. Five guanine residues of the core G-tetrads are
in the syn-configuration, including G1, G7, G8, G17 and
G21 (Figure 3A), as indicated by the very strong H8–H10
NOE intensities (Figure 4). A characteristic downfield shift
is observed for the H20/H200sugar protons of the syn-guanines
(Table 1). The sequential NOE crosspeak connectivities of
the base H8 protons to the 50-flanking residue sugar
H10/H20/H200protons, typical for right-handed DNA twist,
are observed for bcl2Mid (Figure 4). Bcl2Mid adopts a
mixed parallel/antiparallel-stranded G-quadruplex structure,
with the first, third and fourth G-strands being parallel with
each other, and the second G-strand being antiparallel with
the rest of the strands (Figure 3A) (39). The top two G-tetrads
have the same anti/syn distribution and are connected by
guanines with the same sugar configurations, while the third
G-tetrad has a reversed anti/syn distribution and is connected
with the middle G-tetrad by guanines with different sugar
configurations (Figure 3A). In accord with the topology, the
sequential NOE connectivities are indeed either missing
or very weak at the N(i)–synG(i + 1) steps, i.e. C6–G7,
G7–G8, T16–G17 and C20–G21. The characteristic G(i)H8/
G(i + 1)H10NOEs are observed when G(i) is in the syn con-
figuration, such as G1–G2, G7–G8, G8–G9 and G17–G18
(Figure 4). The same configurations of guanines (syn–syn
or anti–anti steps) connecting the top two G-tetrads are
reflected by clear NOE interactions of adjacent guanine
base protons, e.g. G2H1/G3H1, G7H1/G8H1, G18H1/G19H1
and G22H1/G23H1 (Supplementary Figure S5). The reversed
configurations of guanines connecting the bottom two
G-tetrads are clearly reflected by strong inter-tetrad NOE
interactions of guanine imino protons, such as G2H1/G9H1,
Table 1. Proton chemical shifts for the bcl2Mid at 25?Ca
aThe chemical shifts are measured in 20 mM K-phosphate, 40 mM KCl, pH 7.0 referenced to DSS.
bThese chemical shifts are measured at 1?C.
cThe31P chemical shifts are referenced to H3PO4.
dThe difference from ?1 p.p.m.
eGuanines in syn glycosidic conformation are in bold.
5138Nucleic Acids Research, 2006, Vol. 34, No. 18
G8H1/G17H1, G18H1/G21H1 and G22H1/G1H1 (Supple-
mentary Figure S5). Furthermore, the right-handedness of
the DNA backbone of the G-quadruplex is clearly indicated
by inter-tetrad NOE interactions, including G3H1/G8H8,
G7H1/G18H8, G19H1/G22H8 and G23H1/G2H8 (Figure 7).
NOE interactions within loop regions in the
The sequential NOE connectivities are interrupted at the
loop regions, including C4–G5–C6, A10–G11–G12–A13–
A14–T15–T16, and G19–C20–G21 (Figure 4), indicating a
poorly stacked loop conformation. All residues in the loop
regions are in the anti conformation except A13 and A14,
(Figure 4). A number of interesting NOE interactions are
observed for both the lateral loops, whereas the sequential
NOE connectivities are almost all missing for the double-
chain-reversal single-nucleotide C loop, except a couple
of NOEs between the sugar protons of G19 and C20
(Figure 6). For the 50-CGC lateral loop, sequential connectivi-
ties are clearly observable within G5–C6–G7. Interestingly,
clear NOEs are observed between G5/C6 and the imino pro-
tons of the top tetrad, such as G7H1/C6H6, H5&H10, G19H1/
C6H5&H6, G3H1/G5H8&H10, and G23H1/G5H10(Figure 4,
complete summary in Figure 6), indicating that the G5 and
C6 residues are stacking over the top G-tetrad. On the other
hand, unusual NOEs are observed within the G3–C4–G5
region. Strong NOEs are observed for C4 base protons
H5&H6 with G3 sugar protons H10&H40, e.g. C4H5/G3H40
and C4H6/G3H10(strong), C4H5/G3H10and C4H6/G3H40
(medium strong), but not with G3H30or H20. No NOEs are
observed between C4H6/H5 and G3H8 for base stacking.
For the C4–G5 step, clear NOEs are observed for G5H8
with C4H30(strong), H20(strong) and H200(medium strong),
but not with C4H10or H40. These NOE data suggest an
unusual structure adopted within this region, where the C4
base is not stacked over the top G-tetrad but is positioned
into the groove that is close to the H10/H40side of the G3
sugar. This will be discussed in more detail later.
Figure 5. (A) 1D proton-decoupled
phosphorus assignments. (B) 2D heteronuclear31P–1H Correlation Spectro-
scopy (COSY) of bcl2Mid with peak assignments. The NnP-N(n-1)H30
crosspeaks are labeled as Nn-N(n-1). The spectra were referenced to external
H3PO4. Conditions: 25?C, 20 mM K-phosphate, 40 mM KCl, pH 7.0, 1.5 mM
31P NMR spectrum of bcl2Mid with
Figure 6. Schematic diagram of inter-residue NOE connectivities of bcl2Mid.
The guanines in syn configuration are represented using gray circles. The
NOE connectivities clearly define the quadruplex conformation and provide
distance restraints for structure calculation.
Nucleic Acids Research, 2006, Vol. 34, No. 18 5139
For the middle 7-nt lateral loop, A10–G11–G12–A13–
A14–T15–T16, A10 is very well stacked with G9 and
the bottom G-tetrad, as is evident by the clear NOE inter-
actions, such as A10H2/G9H1&G17H1 (Figures 6 and 7).
Very interestingly, clear NOEs are observed between
T15, instead of T16, and the bottom G-tetrad, such as
and 7), indicating that T15, but not T16, is stacked right on
the bottom G-tetrad. Furthermore, a clear imino proton is
observed for T15H3, which exhibits a very strong NOE inter-
action with A10H62 (Figure 7), indicating that T15 is likely
to be involved in a stable H-bonded conformation with A10.
Mutational analysis for loop segments
We have carried out systematic analysis of BCL2 promoter
sequences with mutated residues in the loop regions to deter-
mine their functional role in BCL2 G-quadruplex formation
and stability. We incorporated one mutation at a time for
each loop residue, using various substitutions as listed in
Table 2, and then collected NMR spectra on each sample.
Mutations that do not induce changes in the NMR spectra
are marked with a ‘?’ symbol, whereas those that do
induce clear spectral changes are marked with a ‘+’ symbol.
Interestingly, the most sensitive positions are C4 and A10,
which cannot tolerate any substitutions.
NOE-restrained structure calculation
NOE data (Figure 6) define the overall topology of this
mixed antiparallel/parallel G-quadruplex and were used for
NOE-restrained structure calculation of the major BCL2
G-quadruplex formed with bcl2Mid. Solution structures of
this G-quadruplex were calculated using a NOE-restrained
distance geometry (DGSA) and molecular dynamics (RMD)
approach, starting from an arbitrary extended single-stranded
DNA model. A total of 476 NOE distance restraints, of
which 168 are from inter-residue NOE interactions, were
Figure 7. The expanded H1–H8/H6 and H1–H10regions with assignments of
the exchangeable proton 2D-NOESY spectrum of bcl2Mid. For NOEs
involved in G-tetrads: intra-tetrad NOEs are labeled in red, sequential NOEs
are labeled in green, and inter-tetrad NOEs are labeled in blue. NOEs
involved in the first C4–G5–C6 lateral loop region are labeled in purple, and
NOEs involved in the second A10–G11–G12–A13–A14–T15–T16 lateral
loop region are labeled in brown. Conditions: 1?C, 20 mM K-phosphate, 40
mM KCl, pH 7.0, 1.5 mM DNA.
Table 2. Effect of single base substitutions on the G-quadruplex formation of
the Bcl2Mid sequence
?:The G-quadruplex formationis notaffected;+:the G-quadruplexformation
aA stable G-quadruplex with some minor changes in proton chemical shifts.
Table 3. Structural statistics for the Bcl2Mida
NMR distance and dihedral constraints
Sequential (|i ? j| ¼ 1)
Non-sequential (|i ? j| > 1)
Total dihedral angle restraints
Violations (mean and s.d.)
Distance constraints (A˚)
Dihedral angle constraints (?)
Max. dihedral angle violation(?)
Max. distance constraint violation (A˚)
Deviations from idealized geometry
Bond length (A˚)
Bond angle (?)
Average pairwise r.m.s.d. of heavy atoms (A˚)
0.03 ± 0.003
0.81 ± 0.63
0.007 ± 0.0001
1.40 ± 0.01
1.05 ± 0.01
1.01 ± 0.12
1.14 ± 0.14
1.04 ± 0.12
1.13 ± 0.14
2.55 ± 0.65
aThe ensemble of 10 structures is selected based both on the minimal energy
terms and number of NOE violations.
5140Nucleic Acids Research, 2006, Vol. 34, No. 18
incorporated into the NOE-restrained structure calcula-
tion (Table 3). Dihedral angle restraints were used for the
glycosidic torsion angle (c) for all the syn residues, and for
the three anti residues, i.e. C4, G5 and C6, in the 50-loop.
Dihedral angle restraints were also used to restrain the sugar
backbone torsion angles b, g, and e (Materials and Methods).
The superimposition of the 10 lowest energy structures
produced by the refinement is shown in Figure 8 (PDB ID
2F8U). The structure statistics are listed in Table 3. An aver-
age of ?23 restraints per residue was used for the solution
structure calculation, including experimentally observed
H-bond interactions for the G-tetrads, whereas no planarity
constraints were used for RMD calculations. Remarkably,
out of the 10 lowest energy structures, the RMS deviation
for distance violations is only 0.03 s (Table 3). The bcl2Mid
G-quadruplex structure is very well defined, with a RMSD of
1.01 s for the three G-tetrads. The 50-CGC lateral loop is also
well defined, as the RMSD of the three G-tetrads and the
50-CGC loop is 1.04 s. The RMSDs are 1.14 s and 1.13 s,
respectively, when including the C20 single-nt double-chain-
reversal loop or the A10/T15 of the middle 7-nt lateral loop.
The RMSD for all residues is 2.55 s, indicating a much less-
defined middle 7-nt loop.
Molecular structure of the major G-quadruplex
structure in the BCL2 promoter
A representative model of the bcl2Mid G-quadruplex structure
is shown in different views in Figure 9 and Supplementary
Figure S6. The G-quadruplex consists of three G-tetrads
linked with mixed parallel/antiparallel right-handed G-strands
that are connected by the first two lateral loops (CGC and
AGGAATT) and a third single-nucleotide (nt) double-chain-
reversal side loop (C), as also shown in our previous report
(39). The first, third and fourth G-strands of this G-quadruplex
are parallel with each other, while the second G-strand is
antiparallel with the rest of the G-strands. The bcl2Mid
G-quadruplex contains one wide groove (groove I, between
the first and second antiparallel G-strands), one narrow groove
(groove II, between the second and third antiparallel
G-strands), and two intermediate grooves (groove III and
IV, between the third, fourth, and first parallel G-strands)
(Supplementary Figure S6). All the d torsion angels fall in
Figure 9. A representative model of the NMR-refined bcl2Mid G-quadruplex structure from two different views. (A) is prepared using PyMOL. (B) is prepared
using GRASP (57) (guanine, yellow; adenine, red; thymine, blue; and cytosine, magenta).
Figure 8. The superimposed 10 lowest energy structures of the bcl2Mid
G-quadruplex by NOE-restrained structure refinement.
Nucleic Acids Research, 2006, Vol. 34, No. 18 5141
the range of 110–150?C, consistent with the C20?sugar pucker
conformations indicated by the NMR data.
The three G-tetrads are well defined, with the top two
G-tetrads having the same arrangement of guanine configura-
tion (anti:syn:anti:anti) and the bottom G-tetrad having the
reversed arrangement (syn:anti:syn:syn). Extensive stacking
between the guanine five-membered rings is observed
for adjacent guanines of the bottom two G-tetrads with the
alternate guanine glycosidic configurations (Figure 10A),
while only partial stacking is observed for adjacent guanines
of the top two G-tetrads with the same guanine glycosidic
configurations (Figure 10B).
Loop conformation and functional role of loop residues
The NMR solution structures indicate that the three loop
regions interact with the core G-tetrads in a specific way
that defines and stabilizes the unique BCL2 G-quadruplex
structure. The first C4–G5–C6 linker that connects the first
and second antiparallel G-strands forms a unique lateral
loop conformation which is very well defined (Figures 9A
and 10C). G5 and C6 stack right on the top G-tetrad, where
G5 stacks very well with G3 and C6 is positioned above the
center of G7 and G19 (Figure 10C). Interestingly, the C4
residue adopts a unique conformation in which its base is
positioned in the wide groove I and is perpendicular to the
G-tetrad planes (Figure 9A), covering the outside of the
G3 residue of the top tetrad. This unique conformation of
C4 appears to be rather favored, as over 90% of the 100 struc-
tures from our DGSA calculation are in this conformation.
The conformation of the C4–G5–C6 loop is well defined as
indicated by the small root mean square deviation of this
loop region (Figure 8 and Table 3) (see above). The unique
conformation of C4 explains the unusual NOE interactions
observed between C4 and G3/G5 as discussed before,
e.g. strong NOEs between the C4 H5/H6 and G3 H10/H40,
and the lack of NOE between the G5H8 and the C4 H10
The second 7-nt A10–G11–G12–A13–A14–T15–T16 lin-
ker that connects the second and third antiparallel G-strands
also forms a lateral loop conformation in the NMR structure
(Figure 9). A10 stacks very well with G9 and the bottom
tetrad, while T15, which is the second residue from the
other end of the loop, stacks well with the bottom G-tetrad.
Potential reversed Watson–Crick hydrogen bonds could be
formed between A10 and T15 (Figure 10D), as the imino pro-
ton H3 of T15 can be detected at 1?C (Figure 7). T16, which
is sequentially adjacent to G17, however, is looped out from
the bottom G-tetrad (Figure 9). The remaining four residues
of this long lateral loop are not very well defined (Figure 8)
and are mostly exposed to the solvent (Figure 9).
Significantly, bcl2Mid contains a G3NG3sequence motif,
which adopts a single-nucleotide (C20) double-chain-reversal
(Figure 3A), representing another example of a very stable
parallel-stranded structural motif, in addition to the one first
observed in the MYC promoter sequence (41,51,52). The
conformation of this single-nucleotide double-chain-reversal
loop (C20) is very similar to those of the MYC G-quadruplex
(41). The right-handed DNA backbone twist brings the ends
of the two adjacent parallel G-strands very close to each other
spatially so that the single-nucleotide double-chain-reversal
loop conformation is rather favored (Figure 9B and Supple-
mentary Figure S6).
The loop conformations are defined by the experimental
NOEs and agree well with the experimental data (Figure 6
and Table 2). For the first 3-nt CGC lateral loop, C4, which
is positioned in groove I, cannot even tolerate a substitution
of uridine, which is the closest nucleotide to cytosine, indicat-
ing that the amino group of cytosine is clearly more favored
for the groove positioning, as reported previously for DNA
groove-binding ligands (53,54). On the other hand, G5 and
C6, which appear to be able to tolerate various substitutions,
can presumably still retain the stacking interactions with
different residues. For the second 7-nt AGGAATT lateral
loop, in accord with the NMR structure in which A10
forms a well-defined H-bonded conformation that stacks
extensively with G9 of the bottom tetrad, the A10 residue
cannot tolerate any substitution in our mutational analysis.
Interestingly, T15 and T16, which are the two mutated resi-
dues, display much higher tolerance and can be substituted
with uridines and adenosines, indicating a greater conforma-
tional flexibility of these two positions. The rest of the loop
residues display much higher flexibility and can tolerate
most substitutions. Indeed, G11 and G12, which adopt an
extended loop conformation, show enhanced cleavage in
the DMS footprinting study (38). Finally, the C20 single-
nucleotide double-chain-reversal loop appears to be able to
tolerate various substitutions.
Figure 10. Stacking interactions between (A) the middle (magenta) and
bottom (green) G-tetrads, which have reversed anti:syn:anti:anti and
syn:anti:syn:syn arrangements, and (B) the top (cyan) and middle (magenta)
G-tetrads, which have the same anti:syn:anti:anti arrangements; and stacking
interactions between (C) the first lateral loop C4–G5–C6 (cyan) and the top
G-tetrad (pink), and (D) the bottom G-tetrad (orange) and the A10:T15 bp
(green) from the second lateral loop, A10–G11–G12–A13–A14–T15–T16.
Figures are prepared using PyMOL.
5142 Nucleic Acids Research, 2006, Vol. 34, No. 18
The BCL2 G-quadruplexes and their
relationship to transcriptional control and other
G-quadruplex-containing promoter elements
The G-quadruplexes in the BCL2 promoter element represent
a more complex architecture than those found in other
promoters, such as the c-Myc NHE III1. In this contribution
we have characterized the major loop isomer from the
bcl2MidG4 sequence that overlaps with two other G-
quadruplexes (50G4 and 30G4). In principle there is a total
of 15 different possible loop and structural isomers in
the BCL2 promoter element. Since this element (bcl2Pu39,
Figure 1A) also overlaps with a binding site for WT1 tran-
scriptional factor, which is a suppressor protein, it may be
that formation of one or more of the G-quadruplexes results
in transcriptional activation. In addition to the possible tran-
scriptional activation role of the G-quadruplexes in this
region, it is also possible that the emergence or elimination
of individual G-quadruplexes in this region may differentially
regulate gene expression by selectively interacting with tran-
scriptional factors that either activate or suppress BCL2
A common feature of the most stable BCL-2 loop isomer
studied and the c-Myc promoter G-quadruplex (41,51,52),
and likely the VEGF and HIF-1a promoter G-quadruplexes
(55,56), is the single-nt G3NG3double-chain-reversal loop.
Only this isomer of the bcl2MidG4Pu23 retains this feature
that is essential for stability of these G-quadruplexes. The
single less stable G3–TTA–G3 double-chain-reversal loop
found in the human telomeric G-quadruplex (43) may be
important for the inherent more facile reversibility of the
G-quadruplex to alternative folding structures.
Last, the diversity in sequences and resulting folding pat-
terns found within the BCL2 promoter gives rise to differen-
tial drug binding (38), which may mimic protein recognition
differences. From a drug design perspective, this diversity in
drug binding provides opportunities for selectivity. Further-
more, the major G-quadruplexes formed in BCL2 and MYC
promoter sequences are different (41), with distinct folding
patterns, G-tetrad conformations, loop conformations, and
groove conformations, suggesting that such regions can be
differentially targeted by G-quadruplex-interactive agents.
The structural diversity of the G-quadruplexes formed in
oncogene promoter regions makes such regions attractive
targets for pathway-specific drug design.
Supplementary Data is available at NAR Online.
This research was supported by the National Institutes of
Health (1K01CA83886 and 1S10 RR16659) and Arizona
Biomedical Research Commission (05-002A). We thank
Dr Megan Carver for technical help and proofreading the
manuscript. Funding to pay the Open Access publication
charges for this article has been waivered by Oxford
Conflict of interest statement. None declared.
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