From the Division of Medicinal Chemistry, College of
Pharmacy, University of Texas, Austin, Texas 78712, the
§ College of Pharmacy, University of Arizona, Tucson,
Arizona 85721, and the ¶ Arizona Cancer Center,
Tucson, Arizona 85724
Received for publication, July 7, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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A major control element of the human
c-myc oncogene is the nuclease-hypersensitive
purine/pyrimidine-rich sequence. This double-stranded DNA fragment,
corresponding to the 27-base pair segment in the nuclease-hypersensitive element of the c-myc promoter
region, forms a stable Watson-Crick double helix under physiological
conditions. However, this duplex DNA can be effectively converted to
G-quadruplex DNA by a small molecular weight ligand. Both
intermolecular and intramolecular G-quadruplex forms can be induced by
this ligand. Similar transitional changes are also observed with the
duplex telomeric sequence from the Oxytricha species. These
results provide additional support to the idea that G-quadruplex
structures may play structural roles in vivo and also
provide insight into novel methodologies for rational drug design.
These structurally altered DNA elements might serve as regulatory
signals in gene expression or in telomere dynamics and hence are
promising targets for drug action.
The protein product of the c-myc protooncogene plays a
vital role in the process of cellular growth and differentiation (1). Deregulation of c-myc expression has been detected in many
cancers and is believed to be an important step in tumorigenesis (2). The control of c-myc gene expression is a complex process
and occurs at various steps of transcription, such as initiation, elongation, and attenuation, as well as during the post-transcriptional stages. Although the mechanisms involved in this regulation are not yet
completely understood, a major control element of the human
c-myc oncogene has been localized. This is a
purine/pyrimidine-rich region located 115 bases upstream from the P1
promoter, which controls up to 95% of the total c-myc
transcription (3, 4). This DNA segment is highly sensitive to DNase I
and S1 nuclease (5, 6) and is termed the nuclease-hypersensitive
element (NHE).1 The
appearance of this hypersensitive site is coupled with transcription activation of the c-myc oncogene. Structural variations in
the NHE can influence the binding of transcription factors.
Transcription factors such as heterogenous nuclear ribonucleoprotein K
and nucleoside diphosphate kinase B (7) bind sequence specifically to
the pyrimidine-rich strand of the NHE and activate c-myc
transcription (8). The transacting factors heterogenous nuclear
ribonucleoprotein A/B (9) and cellular nucleic acid-binding protein
(10) bind to the NHE and are shown to augment c-myc
expression in vitro. Apart from protein factors, antisense
oligonucleotides bind to the NHE and repress c-myc
transcription in vitro (11, 12). Formation of a colinear
triplex between the synthetic oligonucleotide and the NHE was proposed
to cause this observed repression in transcription.
The NHE has a high potential to form atypical DNA structures under
superhelical stress. It was proposed to be in a slow equilibrium between a Watson-Crick base-paired double helix and an atypical DNA
structure (6). Many models have been suggested to explain the
conformational changes observed in the NHE. The marked disparity in the
nucleotide composition of the two strands of the NHE prompted suggestion of an H-DNA structure as a model for the noncanonical NHE
structure (13). This H-DNA structure is an intramolecular pyrimidine-purine-pyrimidine triplex. Alternatively, a
purine-pyrimidine-purine triplex has also been proposed as an
explanation for the observed nuclease hypersensitivity (11). Both of
these structures require nonphysiological conditions to be stable,
either low pH for the pyrimidine triplex or very high magnesium
concentration for the purine triplex, and are therefore highly unlikely
to form in vivo.
Recently the NHE fragment of DNA has been shown to adopt an intrastrand
fold-back DNA tetraplex under physiological conditions (14). According
to the proposed model, an interconversion of the NHE between a normal
B-DNA conformation and a very stable atypical G-quadruplex DNA
conformation can recruit transcription factors and activate
c-myc transcription. Involvement of G-quadruplex structures
in the regulation of c-myc transcription opens up an interesting area for the design of small molecules that can selectively interact with the G-quadruplex structure.
A number of G-quadruplex-interactive agents, exemplified by porphyrins
(15), anthraquinones (16), perylenes (17), and carbocyanines (18), have
been developed and are shown to promote and/or stabilize these
secondary DNA structures.
N,N'-Bis[2-(1-piperidino)ethyl]-3,4,9,10-perylenetetracarboxylic diimide (PIPER) is a small molecule from the class of perylene compounds that is well characterized for G-quadruplex interactions (17). For example, it is shown to act as a "driver" in accelerating the assembly of G-quadruplex structures from single-stranded DNA (19).
In this respect, the role of PIPER is very analogous to the In this study we demonstrate that a small molecule such as PIPER
can facilitate the formation of G-quadruplex structures not only from
G-rich single-stranded DNA but also from the Watson-Crick base-paired
G-rich duplex DNA. This is the first evidence that a small ligand can
cause the transition from Watson-Crick base-paired duplex DNA to
G-quadruplex DNA under physiological conditions.
Polynucleotide and PIPER Preparation--
The 27- and 24-base
pair duplex DNA oligomers (for gel electrophoresis experiments) NHE-27,
mutated NHE-27, OT-24 (OT = Oxytricha telomeric),
mutated OT-24, and HT-24 (HT = human telomeric), shown in Scheme
I, were synthesized on a DNA synthesizer (PerSeptive Biosystems
Expedite 8909) and purified using 20% denaturing polyacrylamide gel
electrophoresis. About 400 ng of DNA was 5'-end-labeled with 32P using T4 polynucleotide kinase (New England Biolabs)
and subsequently annealed with the complementary strand. The duplex was
purified using 20% native polyacrylamide gel electrophoresis, diluted
to 40 µM, and dispensed into small aliquots. The duplex
DNAs for NMR experiments, d(TAGGGTTA)-d(TAACCCTA) and
d(TTGGGGTT)-d(AACCCCAA), were synthesized on a DNA synthesizer,
purified by reverse phase high performance liquid chromatography on a
C18 column (Dynamax 300A), and dialyzed extensively. Solid supports and
phosphoramidites were purchased from Glen Research and PerSeptive
Biosystems. PIPER (Scheme ID) was synthesized and purified
as described previously (17). A 1 mM stock solution of
PIPER prepared in distilled water was stored at Electrophoretic Mobility Shift
Assays--
32P-end-labeled duplex DNA at a concentration
of 4 µM was heated to 95 °C for 10 min in a 1 × KCl/TE buffer (10 mM Tris·HCl, 1 mM EDTA, 100 mM KCl, pH 8.0). After the DNA was cooled to room temperature, 2 µl of stock PIPER was dispensed to each sample to
obtain the concentrations specified in the figures at a total volume of
20 µl. For the concentration-dependent experiments, the
incubation time was 10 h at either 28 °C or 55 °C. For the time-course experiments, the samples were taken at the times specified in the figures and the incubation was carried out at either 28 °C or
55 °C. The incubation was terminated by the addition of 5 µl of
gel loading buffer (30% glycerol, 0.1% bromphenol blue, and 0.1%
xylene cyanol). Eight microliters of each sample was then loaded onto a
16% native polyacrylamide gel that was prerun for 30 min. The samples
were electrophoresed for 15 h at 4 °C in a 1× TBE/KCl running
buffer (90 mM Tris borate, 90 mM boric acid, 2 mM EDTA, pH 8.3, and 100 mM KCl). Gels were
dried, exposed to a phosphor screen, and visualized on a PhosphorImager
(Molecular Dynamics model 445SI).
NMR Spectroscopy--
NMR experiments were performed on a Varian
UNITY Plus 500-MHz spectrometer. All titration experiments were carried
out in a 90% H2O, 10% D2O solution containing
100 mM KCl, 25 mM
KH2PO4, and 1 mM EDTA (pH 7.0). A
standard 1-1 echo pulse sequence with a maximum excitation centered at
12.0 ppm was used for water suppression.
The role of PIPER in inducing the formation of G-quadruplex
structures from Watson-Crick base-paired duplex DNA was examined using
electrophoretic mobility shift assays and NMR studies. The double-stranded DNA fragment corresponding to the 27-base pair segment
in the NHE of the c-myc promoter region was used in the first part of this study (see Scheme
IA). The purine-rich strand of
this double-stranded fragment has the ability to form at least two
types of G-quadruplex DNA, namely, the tetramolecular G-quadruplex and
the intramolecular foldover G-quadruplex (Scheme
II). To provide additional insight into
dynamic requirements for PIPER-driven conversion of Watson-Crick
base-paired duplex to G-quadruplex, two more duplexes, namely, the
ciliate telomeric sequence from the Oxytricha species and
the human telomeric sequence, were compared for their propensity to
form G-quadruplex DNA in the presence of PIPER.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit
of the Oxytricha telomere-binding protein, altering the
dimerization kinetics from second to first order. This implies that
PIPER may elicit biological effects by promoting G-quadruplex structures analogous to the chaperone proteins. In addition, once the
G-quadruplex structure is formed, PIPER prevents G-quadruplex unwinding
by Sgs1, a G-quadruplex-specific helicase (20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C and diluted
to required concentrations immediately before use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Scheme I.
View larger version (15K):
[in a new window]
Scheme II.
All the experiments were carried out at 100 mM concentrations of potassium. Experiments carried out in the absence of potassium showed patterns similar to those seen in the presence of potassium; however, the intensity of the G-quadruplex bands was weaker in the absence of potassium.
Incubation of the 27-mer NHE Duplex with PIPER Results in
Conversion to G-quadruplex Structures--
Watson-Crick base-paired
duplex NHE-27 DNA was incubated with increasing concentrations of PIPER
at 55 °C. In this experiment the 5'-end of the G-rich strand of the
duplex DNA was radiolabeled. As the concentration of PIPER was
increased to 32 µM, there was increased formation of a
slow migrating species, indicative of a higher order structure
(corresponding to T in Fig.
1A). It is significant that
the intensity of only a single band is increased, indicating that PIPER
may be promoting a specific type of structure, which may be a
G-quadruplex. The diffuse bands that occur in the lanes
labeled Isomeric forms in Fig. 1A are
most likely to be tetramolecular G-quadruplex structures in which there
is misalignment of one or more of the G-rich strands. Parallel
experiments in which the 5'-end of the C-rich strand of the
Watson-Crick base-paired duplex DNA was radiolabeled were performed
under identical conditions. The intensity of the band corresponding to
single-stranded C-rich DNA (SS in Fig. 1B)
increased at elevated concentrations of PIPER. Therefore, a higher
order structure is formed by the G-rich strand, leaving an excess of
single-stranded C-rich DNA. Previous studies with G-rich
single-stranded DNA indicated that PIPER binds tightly and specifically
to G-quadruplexes and acts as a driver in the assembly of G-quadruplex
structures (19). Hence, the structure formed by the G-rich strand in
our experiments is most likely to be a G-quadruplex structure, which is
consistent with the results from the DMS chemical footprinting
experiments.2
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Besides the tetramolecular G-quadruplex, PIPER induces the formation of an intramolecular foldover G-quadruplex structure. This was deduced from the experiments carried out at 28 °C using the Watson-Crick base-paired duplex NHE-27 DNA in which the 5'-end of either the G-rich or the C-rich DNA was radiolabeled (Fig. 1, C and D). These experiments exhibited the formation of tetramolecular G-quadruplex (T in Fig. 1C) and another species with a higher mobility (M in Fig. 1C). In accordance with the literature (21), the compact intramolecular G-quadruplex has faster mobility than either the double-stranded DNA or the nonstructured single-stranded DNA. Results from the DMS footprinting experiments were consistent with the idea that the M band is a G-quadruplex structure.2 That the intramolecular G-quadruplex structure was not observed in the experiments carried out at 55 °C suggests that the intramolecular G-quadruplex is less stable than the tetramolecular G-quadruplex.
A Mutant 27-mer NHE Duplex That Is Incapable of Adopting
G-quadruplex Structures Does Not Form Higher Order Structures in the
Presence of PIPER--
The specificity of PIPER to unwind G-rich
Watson-Crick base-paired duplexes was determined by modifying the
NHE-27 duplex used in the previous experiments. It was modified such
that neither strand had runs of two or more consecutive guanines and
was thus incapable of forming G-quadruplex structures. Incubation of
this mutated NHE-27 Watson-Crick base-paired duplex (Scheme
IA) under conditions similar to those of the nonmutant
NHE-27 neither affected the duplex bands nor indicated the appearance
of new faster or slower migrating species (Fig.
2, A and B). This
demonstrates that PIPER may be specific in promoting the formation of a
G-quadruplex DNA structure from the Watson-Crick base-paired G-rich
duplexes.
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PIPER Facilitates the Formation of G-quadruplex Structures and
Interconversion between Different G-quadruplex Structures from the
27-mer NHE Duplex DNA--
PIPER has been shown to play a driver role
in the formation of G-quadruplex structures from single-stranded DNA
(19). It promotes the formation of both dimeric and tetrameric
G-quadruplexes from DNA oligomers containing two repeats of telomeric
DNA from the Oxytricha species, while converting the
reaction kinetics of oligomer dimerization from second to first order
(19). To investigate the ability of PIPER to induce interconversion
between different types of G-quadruplex structures in the presence of a
complementary DNA strand, the 27-mer NHE Watson-Crick base-paired duplex DNA was incubated with 8 µM PIPER at 28 °C for
32 h. At incubation times of up to 12 h, the tetramolecular
G-quadruplex is the predominant structure; however, at times greater
than 24 h, the intramolecular foldover G-quadruplex becomes the
predominant structure (Fig.
3B). A comparison of
panels A and B in Fig. 3 shows that
the formation of the tetrameric species and the subsequent G-quadruplex
interconversion reactions require the presence of PIPER. Under these
experimental conditions, the intramolecular foldover G-quadruplex
is the most stable structure thermodynamically, but it is kinetically
unfavorable compared with the tetrameric G-quadruplex. Similar results
were observed at 55 °C.2
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PIPER-mediated Interconversion of Tetramolecular G-quadruplex to the Intramolecular G-quadruplex Structure Does Not Occur in the Absence of the Complementary C-rich Strand-- The involvement of a G-rich single strand as an intermediate in the PIPER-mediated interconversion was evaluated. The design of this experiment was analogous to that for Fig. 3 (A and B), except that single-stranded G-rich DNA was substituted for Watson-Crick base-paired duplex DNA. The G-rich single strand of NHE-27 was incubated in the absence (Fig. 3C) and in the presence (Fig. 3D) of PIPER for 32 h at 28 °C. As expected, the results show that the presence of 8 µM PIPER does facilitate the formation of G-quadruplex structures. However, somewhat surprisingly, the tetramolecular G-quadruplex was the only structure formed over the 32-h period, suggesting that the presence of a complementary strand affects the overall transition of the Watson-Crick base-paired duplex to G-quadruplex structures.
A graphical representation comparing the formation of different G-quadruplex structures from the Watson-Crick base-paired duplex 27-mer NHE and from the single-stranded G-rich 27-mer NHE over extended periods of time is shown in Fig. 3 (E and F, respectively). With Watson-Crick base-paired duplex 27-mer NHE, there is a concomitant loss of the tetrameric G-quadruplex with an increase in the monomeric G-quadruplex over time (Fig. 3E). In the single-strand experiment, the only species formed is the tetrameric G-quadruplex, which shows a biphasic increase with incubation time (Fig. 3F).
Differential Effect of PIPER on Duplex Regions of the Telomeric DNA from Varied Sources-- The effect of PIPER on other G-rich genomic regions apart from the 27-mer NHE duplex was also investigated. The intrinsic ability of PIPER to drive the conversion of human and Oxytricha Watson-Crick base-paired duplexes to G-quadruplex was evaluated.
Ciliate Telomeric Duplex Sequences Can Also Be Effectively
Converted to G-quadruplex Structures in the Presence of PIPER--
The
Watson-Crick base-paired duplex DNA OT-24 (Scheme IB),
containing four repeats of the Oxytricha telomeric sequence,
was incubated with increasing concentrations of PIPER at 55 °C.
Experiments were carried out in which either the 5'-end of the G-rich
strand (Fig. 4A) or the 5'-end
of the C-rich strand (Fig. 4B) was radiolabeled. As the
concentration of PIPER increased, the formation of a slow mobility
species corresponding to the tetramolecular G-quadruplex structure
(T in Fig. 4A) was observed. However, there was
no effect on a mutated OT-24 (Fig. 4C), which did not
contain two or more runs of guanines.
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Parallel 1H NMR titration experiments were carried out
using the Watson-Crick base-paired duplex sequence
d(TTGGGGTT)-d(AACCCCAA) (containing one repeat of the
Oxytricha telomeric sequence) by titrating the duplex
against PIPER. The one-dimensional spectrum of the Watson-Crick
base-paired duplex is shown in Fig.
5A. Inspection of the imino
proton region (10-14 ppm) indicates the presence of equilibrium
between the Watson-Crick base-paired duplex and G-quadruplex
structures. The Watson-Crick base-paired imino protons are observed
between 12.5 and 13.2 ppm, and the imino protons involved in the
G-quadruplex Hoogsteen base pairing are observed in the region of 10.6 to 11.6 ppm. Upon titration with increasing concentrations of PIPER
(Fig. 5B), the signals from the Watson-Crick base-paired
imino protons are reduced and imino protons involved in the
G-quadruplex predominate at the 1:1 DNA:PIPER concentrations. It is
important to note that the Hoogsteen base-paired imino protons are
shifted upfield, indicating the probable interaction of PIPER with the
G-quadruplex.
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Human Telomeric Duplex DNA Cannot Be Converted to G-quadruplex,
Even in the Presence of PIPER--
The Watson-Crick base-paired human
telomeric duplex DNA d(TAGGGTTA)-d(TAACCCTA) (containing one human
telomeric repeat) was titrated against PIPER under conditions similar
to those of the previous NMR experiment. The one-dimensional NMR
spectra are shown in Fig. 6A.
Unlike the Oxytricha duplex sequence, the human telomeric duplex was predominantly in Watson-Crick base-paired duplex form. These
Watson-Crick base-paired duplex DNA resonances were unaffected upon
titration with PIPER, even at a 1:1 DNA:PIPER concentration. This
suggests that the human telomeric G-quadruplex may be less stable than
the Watson-Crick base-paired duplex and hence is not observed.
Additionally, since the 1H NMR resonance signals of the
Watson-Crick base-paired imino protons are unchanged, this indicates
the high selectivity of PIPER for its interaction with G-quadruplex
versus Watson-Crick base-paired duplex DNA. Electrophoretic
mobility shift assays carried out with the four-repeat sequence of the
human telomere HT-24 (see Scheme IC) also indicated that there were no
transitional changes with increasing concentrations of PIPER (Fig.
6B), a result that is consistent with the
1H NMR data in Fig. 6A.
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DISCUSSION |
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Structural heterogeneity in DNA is evidenced by its capability to adopt several types of conformation (22). These include cruciform DNA, Z-DNA, triplex DNA, and G-quadruplex DNA, apart from the usual B-DNA. Repetitive sequences in the telomeric and promoter regions, as well as in the coding regions of the eukaryotic genome, have a high potential to form unusual structures (23). Although the biological significance of these unorthodox structures is not yet completely understood, they are often associated with functional roles in transcription, gene regulation (24), and tumor-associated genetic changes (25). These alternative structures may be stabilized in vivo by the presence of a specific protein or a small peptide. The promoter regions are often guanine-rich on one strand and exhibit nuclease hypersensitivity, emphasizing the dynamic nature of the DNA structure (26).
For this study, we chose the NHE fragment of the c-myc promoter on the basis of the observation that NHE can be in a conformational equilibrium between two states, a nuclease-sensitive state and a nuclease-insensitive state. Moreover, it was proposed that in vivo endogenous RNA molecules could bind to the regulatory region of c-myc and affect this conformational equilibrium (6). This prompted the idea that factors stabilizing or destabilizing the S1 nuclease-sensitive conformer of NHE might have an effect on the expression of the c-myc oncogene. Indeed, switching of DNA secondary structures is shown to affect the transcription response of many genes (24, 27, 28).
The characterization of NHE under physiological conditions showed that
it could adopt a G-quadruplex structure (14). It was observed that
potassium ions stabilized the G-quadruplex structure. In isolation, the
formation of G-quadruplex structures is a slow process. In fact, the
inability of plasmid NHE, but not the single-stranded NHE (G-rich), to
form G-quadruplex structures was attributed to the slow kinetics of
G-quadruplex formation as well as to the competitive binding of the
complementary strand (14). In this study we have demonstrated that
PIPER is able to facilitate the formation of two secondary structures
from Watson-Crick base-paired duplex DNA (Scheme
III). On the basis of the electrophoretic
mobility, sequence variation, and DMS footprinting results, the lower
mobility species is assigned as a tetramolecular G-quadruplex, and the structure with higher mobility is more likely to be a foldover G-quadruplex. Although the tetramolecular G-quadruplex is the kinetically favored species (Fig. 3B), the foldover
G-quadruplex structure is thermodynamically favored. As a possible
explanation for this observation, we propose that early on in the
incubation there is sufficient PIPER to bind to and stabilize the
tetramolecular structure (III in Scheme III), but as the
concentration of this species increases, there is insufficient PIPER to
bind to and stabilize this species. The tetramolecular species then
dissociates, releasing ligand-free single-stranded DNA, which can fold
into the thermodynamically stable foldover structure (upper
pathway in Scheme III). Significantly, only the
tetramolecular G-quadruplex structure is formed from the
single-stranded DNA in the presence of excess PIPER. This is because
less PIPER is bound up in the disordered single-stranded DNA-PIPER
conjugates (lower pathway in Scheme III) and is
therefore available to stabilize the tetramolecular structure, which
favors an equilibrium in which little of the free tetramolecular
G-quadruplex is found. Consequently, little, if any, of the foldover
structure is formed. Alternatively, it can be speculated, based on this
model, that at low concentrations of DNA the parallel species will be
the predominant structure, while at higher DNA concentrations at longer
periods of time, the intramolecular foldover structure will
predominate. This model also identifies a mechanism for the
facilitation of G-quadruplex formation. Based on this model,
ordered PIPER molecules stack in between parallel duplex molecules to
facilitate formation of G-G parallel duplex molecules, which dimerize
to form the G-quadruplex structures with dissociation of the ordered
stacked PIPER molecules (I II
III in Scheme III).
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The induction of duplex to G-quadruplex by PIPER cannot be generalized to all G-rich-containing duplex sequences capable of forming G-quadruplexes, as indicated by the experiments using telomeric DNA. The ciliate telomeric duplex DNA is in a state of equilibrium between the duplex and G-quadruplex structure (Figs. 4 and 5), and PIPER can cause a transition from the duplex to G-quadruplex. However, the human telomeric duplex DNA (Fig. 6) is present predominantly in the Watson-Crick base-paired duplex form. Thus, it appears that the human telomeric G-quadruplex may be less stable than the duplex and PIPER cannot effectively induce the transition of the duplex to a G-quadruplex structure. This indicates that the inherent ability of certain duplexes to be in equilibrium with a G-quadruplex may be a requirement for PIPER to cause a transition from a duplex to a G-quadruplex structure.
The role of PIPER in promoting and stabilizing G-quadruplex structures
is analogous to the chaperone action observed with the -subunit of
the Oxytricha telomere binding protein (29). Hence it is
conceivable that PIPER can mimic the activity of a G-quadruplex binding
protein in vivo. PIPER, therefore, can be used to probe for
regions in the genome that have a potential to be in equilibrium
between normal B-DNA and G-quadruplex DNA. Such regions include the
immunoglobulin switch region, triplet repeats of the fragile X
syndrome, and the promoter regions of oncogenes such as
bcl-2 and c-fos, in addition to c-myc
and telomeric regions mentioned here.
Besides its potential to identify genomic structural perturbances, the specificity of PIPER to recognize a 27-base pair sequence suggests a possible application in therapeutics. Regulatory proteins exhibit a high level of selectivity toward target DNA sequences by recognizing either the structure or the sequence. Artificial gene regulation would require DNA-binding molecules to exhibit sequence specificity similar to that for proteins. In this regard, DNA sequence-specific agents have been developed that rely on the recognition of specific duplex base sequences. So far, the direct readout approach has been easier to realize for drug design. Triplex-forming oligonucleotides (30, 31) and polyamides (32) have been designed to achieve high sequence selectivity. These approaches hold significant promise for clinical success, but the conversion of this technology to therapeutic agents still remains elusive. In this study we achieved the specific recognition and folding of a 27-base pair sequence by a small molecule. In contrast, if we relied on the direct duplex sequence recognition approach, a molecule the size of PIPER could possibly recognize and interact with a trinucleotide sequence at most.
Studies are in progress to evaluate the effect of G-quadruplex driver
molecules on in vitro transcription of the c-myc
oncogene. These studies will provide insight into the possible roles of G-quadruplex structures in c-myc transcription regulation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Brent Iverson (University of Texas, Austin, TX) for important insights into the effects of limiting concentrations of PIPER on G-quadruplex transitions and for the development of the model for conversion of Watson-Crick base-paired duplex to G-quadruplex by PIPER. We are grateful to Dr. David Bishop for proofreading, editing, and preparing the final version of the manuscript and figures.
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Note Added in Proof |
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Recently, Simmonson et al. (33) have shown that the C-rich strand of the c-myc NHE can form an intramolecular i-motif structure. We have also recently demonstrated that G-quadruplex-interactive compounds such as TMPyP4 can also facilitate the formation of i-motif DNA and bind to the resulting structure by a nonintercalative mechanism (34). This provides an additional mechanism for intervention with small molecules in the conversion of c-myc NHE between duplex and alternative secondary structures.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA49751.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Arizona Cancer
Center, 1515 N. Campbell Ave., Tucson, AZ 85724. Tel.: 520-626-5622; Fax: 520-626-5623; E-mail: hurley@pharmacy.arizona.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M005962200
2 A. Rangan, O. Yu. Fedoroff, and L. H. Hurley, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: NHE, nuclease-hypersensitive element; PIPER, N,N'-bis[2-(1-piperidino)ethyl]-3,4,9,10-perylenetetracarboxylic diimide; DMS, dimethyl sulfate.
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