(Received for publication, April 14, 1995; and in revised form, July 20, 1995)
From the
Molar [K] induces aggregate formation
in d(CGG)
, as evidenced by absorbance, circular dichroic
(CD), and gel measurements. The kinetics of this transformation are
extremely slow at pH 8 but are found to be greatly facilitated in
acidic conditions. Kinetic profiles via absorbance or CD monitoring at
single wavelength resemble those of autocatalytic reacting systems with
characteristic induction periods. More than 0.8 M KCl is
needed to observe the onset of aggregation at 20 °C and pH 5.4
within the time span of 1 day. Time-dependent CD spectral
characteristics indicate the formation of parallel G-tetraplexes prior
to the onset of aggregation. Despite the evidence of
K
-induced parallel G-quadruplex and higher molecular
weight complex formation, both d(TGG)
and
d(CGG)
T fail to exhibit the observed phenomenon, thus
strongly implicating the crucial roles played by the terminal G and
base protonation of cytosines. A plausible mechanism for the formation
of a novel self-assembled structure is speculated. Aided by the
C
C base pair formation, parallel quadruplexes
are initially formed and subsequently converted to quadruplexes with
contiguous G-tetrads and looped-out cytosines due to high
[K
]. These quadruplexes then vertically
stack as well as horizontally expand via inter-quadruplex
C
C base pairing to result in dendrimer-type
self-assembled super structures.
Fragile X syndrome is the most common cause of inherited mental retardation(1) . Individuals affected by this disorder have an X chromosome in which the tip of its long arm is attached by only a slender thread of DNA. A gene designated as FMR-1 contains about 60 or fewer tandem repeats of CGG trinucleotide sequence in normal individuals. Healthy carriers of this disease may have as many as 200 tandem copies. In sick individuals, however, the tandem repeat region is dramatically larger(2) . Recently, amplifications of trinucleotide repeats have also been shown to be associated with several other disorders, including Kennedy and Huntington diseases(3) . Although the mechanism of this unusual trinucleotide amplification is still unknown, it would not be surprising if there were structural bases for such remarkable amplifications.
Guanine is unique among the four DNA bases by virtue of its four hydrogen bonding sites being strategically distributed in such a way that four G bases can readily form 8 hydrogen bonds to result in a cyclic base-quartet (see Fig. 1). Thus, a DNA sequence with a stretch of G bases can form a four-stranded helical structure called G-quadruplex which is of current intense interest. This interest has been further stimulated by the possible relevance of such structures in the recombinational events at the immunoglobulin switching regions as well as in telomeric functions(4) .
Figure 1:
Upper panel, schematic
representation of base pair formation in G-quartet of parallel strand
orientations. Lowerpanel, CC
base pairing scheme.
Telomeres are specialized DNA-protein complexes comprising the
chromosomal termini and are essential for the stability and integrity
of chromsomes. The telomeric DNA consists of a simple tandemly repeated
sequence characterized by clusters of G residues in one strand with a
3` overhang of 12-16 nucleotides in length(5) . Effects
of monovalent cations on the G-quadruplex structural formation of
telomeric DNA sequences have been extensively studied in recent years
(see reviews in (6, 7, 8, 9) ).
Evidence suggests that due to its optimal size, K is
much more effective in stabilizing G-quadruplex formation. The ion is
found to be sandwiched between two G-tetrads to form an
octa-coordination complex with the carbonyl groups of guanines. It was
also found that for a contiguous guanine oligomer, the parallel strand
orientation is thermodynamically more favorable than the anti-parallel
orientation in the G-quadruplex formation(4, 10) . A
G-quadruplex with parallel strand orientation is further characterized
by a strong positive CD band near 265
nm(11, 12, 13) .
Cytosine is also unusual
in that it can form three hydrogen bonds with its protonated
counterpart (see also Fig. 1). A tract of C bases can, thus,
form a parallel duplex via CC base pairing in
acidic solutions. The ability of oligomers containing contiguous
guanines to form quadruplex G-DNA and the recent findings indicating
that cytosine base protonation can facilitate such a quadruplex
formation (14, 15) suggest that a physicochemical
study on oligomers containing CGG repeats will be of considerable
value.
This report describes the observation of an interesting
K-induced CD intensity enhancement and aggregate
formation of dodecamer d(CGG)
and proposes a plausible
mechanism for the formation of super molecular assemblies of
G-quadruplexes via C
C base pairing.
Oligonucleotides were purchased from Integrated DNA
Technologies, Coralville, IA and used without further purification.
Experiments were carried out in either 10 mM HEPPS ()buffer solutions of pH 8 containing 0.1 M NaCl
and 1 mM MgCl
, or 10 mM sodium citrate
buffer of pH 5.4 containing 0.1 M NaCl and 1 mM EDTA.
Concentrations of these oligomers (per nucleotide) were determined by
measuring the absorbances at 260 nm after melting, with use of
extinction coefficients obtained via nearest-neighbor approximation
using mono- and dinucleotide values tabulated by Fasman(16) .
Absorption spectra were measured with a Cary 1E spectrophotometric
system. Thermal denaturation experiments were carried out with 1-cm
semimicro cells by monitoring absorbances at various wavelengths. A
heating rate of 0.5 °C/min was maintained by the temperature
controller accessory. Absorbance kinetic measurements were made with a
stirrer accessory.
CD (circular dichroic) spectra were measured with a Jasco J-500A recording spectropolarimeter using water-jacketed cylindrical cells of 2-cm pathlength. CD kinetic measurements were made by monitoring ellipticity changes at appropriate wavelengths. Electrophoretic measurements were made on a Pharmacia Phast System using 20% polyacrylamide native gels at 200 V with appropriate pre- and post-loading run times at different temperatures. PhastGel buffer strips containing 0.25 M Tris of pH 8.8 were used, and the gels were developed by silver staining.
Figure S1:
Figure 2:
A, toppanel, CD spectra
before (dottedcurve with 10-fold amplification) and
at 1, 2, 4, and 8 days after the addition of 2 M KCl to a 40
µM (per nucleotide) d(CGG) solution in pH
8/0.1 M NaCl buffer. Each spectrum was measured at room
temperature after rigorous manual shaking of the solution. B, middlepanel, CD spectra of 40 µM d(CGG)
solution in acidic buffer of pH 5.4/0.1 M NaCl before (dottedcurve) and after 40 min (squares) and 1 week (solidcurve) of 1 M KCl addition. C, bottompanel,
time-dependent CD spectra of 40 µM d(CGG)
solution of pH 5.4/2 M KCl after cooling the solution
from 95 °C to 40 °C. Immediately (dots), 10 (solidcurve), 20 (+), 30 (
), and 50 (squares) min after 40 °C is
reached.
In contrast to pH 8, addition of molar concentration of
KCl to an acidic d(CGG) solution results in immediately
noticeable CD intensity changes. The initial CD spectral alteration
after the addition of 1 M KCl to a d(CGG)
solution
of pH 5.4 is shown in Fig. 2B. Of interest is the
initial development of a maximum at 265 nm. Subsequent intensity
enhancement slowly changes to a
-type spectrum with a maximum
near 300 nm.
In addition to acidity, it was also found that the
kinetics of aggregation can further be accelerated by a slight increase
in temperature and/or melting and cooling the oligomer in the presence
of molar [K] (see below). Taking advantage
of these facts, the large K
-induced CD intensity
enhancement can be observed in a reasonably short time. Fig. 2C shows the time-dependent CD spectral
characteristics of a post-melt d(CGG)
solution in pH 5.4/2 M KCl buffer at 40 °C. As is apparent, significant
intensity enhancement is already evident at 10 min after cooling the
solution back from 95 °C to 40 °C. More dramatic spectral
enhancements occur during the next 20 min to result in
-type CD
characteristics with a maximum near 300 nm and large intensities well
beyond 350 nm. The equilibrium is seen to be approached in slightly
over 1 h. Notice the gradual red shift of the long wavelength maximum
as time progresses and the presence of a CD maximum at 265 nm prior to
the onset of
-type CD spectra.
Figure 3:
A,
CD kinetic traces of pH 5.4 d(CGG) solution with 263 nm
ellipticity monitoring at 20 °C (triangles), 30 °C (circles), and 40 °C (squares). B,
time-dependent CD intensity enhancements at 40 °C via 300 nm (pH
5.4, squares) and 290 nm (pH 8, triangles)
monitoring. The process was initiated by adding solid KCl to a 40
µM nucleotide solution of appropriate temperature to
result in 2 M salt concentration. Less than 10 s is needed to
dissolve the added solid via rigorous manual shaking. Cooling curve
from 95 °C to 40 °C for the pH 8 solution in the presence of 2 M KCl (circles) is also included for comparison, with t = 0 corresponding to 95
°C.
Kinetic profiles
for the K-induced aggregation of d(CGG)
at
40 °C in pH 5.4 and 8 via respective 300 and 290 nm monitoring are
compared in Fig. 3B. Despite the absence of initial
slow phase intensity enhancement at these wavelengths, the onset of
aggregation for the acidic solution commences near 30 min, in agreement
with the 263 nm monitoring. In contrast, the pH 8 solution exhibits the
first sign of aggregation only after about 2 h. These kinetic patterns
resemble those of autocatalytic reacting systems, exhibiting
characteristic induction periods and subsequent rapid rate
accelerations(19) . Facilitation of aggregate reformation via
prior melting in the presence of KCl is also included for comparison
with a pH 8 solution (see the following section).
Figure 4:
Representative absorption spectra at 20
°C of 40 µM d(CGG) in pH 5.4 buffer at: 0 (1), 5 (2), 10 (3), 15 (4), and 20 (5) h after the addition of 1 M KCl. Curve6 corresponds to that of 30 h and after rigorous manual
shaking. Inset, time-dependent absorbance changes at 255 (squares), 267 (circles), 285 (triangles),
and 320 nm (diamonds).
Figure 5:
Comparison of melting profiles for 40
µM d(CGG) solutions of pH 5.4 containing 1.4 (squares), 1.8 (circles), and 2.2 M (triangles) KCl with heating (open symbols) and
cooling (solid symbols).
Figure 6:
Comparisons of CD spectra of 40 µM nucleotide solutions of pH 5.4 for d(TGG) in the
absence (+) and in the presence (squares) of 2 M KCl for 4 days and for d(CGG)
T before (dotted
curve) and after 4 days of 2 M KCl addition (solid
curve).
Figure 7:
Comparison of gel electrophoretic mobility
patterns at 4 °C (A) and 14 °C (B) for
d(CGG), d(TGG)
, and d(CGG)
T of pH
5.4 in the absence (lanes2, 4, and 6, respectively) and in the presence of 2 M KCl (lanes3, 5, and 7, respectively).
Dodecamer d(TGGGGGGGGGGT) (lane1) of pH 5.4/2 M KCl and self-complementary dodecamer d(CCGCCGCGGCGG) (lane8) of pH 8/0.1 M NaCl in the presence (A) and in the absence (B) of 2 M KCl are
included to serve as references. Measurements were made after 3 months
of KCl additions.
It is instructive to follow the progression of gel
electrophoretic mobility patterns during the course of aggregate
formation. Fig. 8compares the gel patterns at 4 °C after 1 (panel A) and 6 (panel B) days of 2 M KCl
additions (even-numbered lanes) to solutions of
d(TGGGGGGGGGGT) (lane1), d(CGG) (lane3), d(TGG)
(lane5), and d(CGG)
T (lane7).
It is evident that the prominent presence of slow moving tails is
already apparent for all oligomers after 1 day of KCl additions but
becomes more so after 6 days. Of particular interest is the observation
that for d(CGG)
(lane 4) the quadruplex conformation (band
IV) is predominantly induced after day 1 (panelA)
but complexes with molecularities higher than 16 become apparent after
day 6 (panelB), as indicated by the appearance of a
much slower band with a accompanied trailing tail. This, however, is
accomplished via concomitant intensity reduction of band IV, suggesting
that the quadruplex formation precedes the higher molecular weight
aggregation.
Figure 8:
Comparison of gel electrophoretic mobility
patterns after 1 (panel A) and 6 (panel B) days of 2 M KCl additions for d(TGGGGGGGGGGT), d(CGG),
d(TGG)
, and d(CGG)
T of pH 5.4 at 4 °C in
the absence (lanes 1, 3, 5, and 7,
respectively) and in the presence of 2 M KCl (lanes2, 4, 6, and 8,
respectively).
Although G-quadruplex formation in oligomers containing a
large number of guanine is to be expected, the large
K-induced CD intensity enhancement of d(CGG)
is somewhat surprising. The kinetic facilitation of such a
process in acidic solutions and the absence of
-type CD
characteristics in d(TGG)
suggest a crucial role played by
cytosines in this oligomer, likely via the C
C
base pair formation. The presence of a terminal G appears to be also
important, since d(CGG)
T does not aggregate in molar KCl
solutions. Thus, the ability of d(CGG)
to form
-type
aggregates appears to be the consequence of the simultaneous presence
of C bases and a terminal G in the strand.
As stated earlier, a
G-quadruplex with parallel strand orientation is characterized by a
strong positive CD band at 265
nm(11, 12, 13) . The observations that
d(TGG) or d(CGG)
T in the presence of molar
concentration of KCl exhibits a strong positive CD maximum at 265 nm
(see Fig. 6) and that an initial intensity enhancement near this
wavelength was also observed for d(CGG)
prior to the onset
of
-type CD appearance (see Fig. 2B and Fig. 3A) strongly support the notion that aggregate
formation in d(CGG)
is preceded by parallel G-quadruplex
formation. This is further strengthened by the time-dependent gel
mobility measurements of d(CGG)
on the effect of 2 M KCl, which indicate an initial prominent presence of quadruplex
band that subsequently diminishes as the much slower moving tail
becomes progressively more important (Fig. 8). Such speculation
appears to be consistent with observations by others, indicating that
the presence of cytosines (15) or high monocation concentration (22) facilitates the parallel G-quadruplex formation and the
most recent report on the observation of stable tetraplex formation of
oligomers with CGG repeats in 0.2 M KCl, especially those with
5-methylated cytosines(23) .
CC base
pairing had been shown to result in a greatly enhanced positive CD at
wavelengths above 280 nm (24, 25) and to form parallel
duplexes(26, 27) . CD spectral studies on poly(dC-dT)
even led to the proposal that this polynucleotide forms a structure
consisting of a core of C
C
base pairs and
individually looped-out thymidyl residues in acidic
solutions(28, 29, 30) . Furthermore,
self-assembly via branching of parallel C
C
duplex formation has recently been proposed(31) . These
observations, thus argue strongly for the involvement of
C
C base pairing in the observed phenomenon in
d(CGG)
.
DNA oligomers containing guanine clusters and a
terminal guanine are known to generate, in addition to tetramers,
higher order products via quadruplex stacking in the presence of
K(20, 21) . Since both d(TGG)
and d(CGG)
contain terminal G at the 3`-end,
formation of these higher order products are possible. Although the
absence of K
-induced
-type CD in d(TGG)
suggests that the remarkable CD intensity enhancement observed in
d(CGG)
is not due to the sole presence of these higher
order products, they may play important roles in furthering the
observed aggregate formation. Indeed, the inability of oligomer
d(CGG)
T to exhibit
-type CD suggests that the mere
presence of C bases is not sufficient and testifies to the important
role played by terminal G in the aggregation process, possibly via
vertical end stacking.
Based on these spectral observations, a
mechanism of self-assembly may be envisioned. Aided by the
CC base pair formation, parallel quadruplexes
are initially formed. Driven by favorable K
complexation and purine stacking interactions, they further
convert to quadruplexes with contiguous G-tetrads and looped-out
cytosines (see Fig. 9). These quadruplexes can expand vertically
via stacking and horizontally via inter-quadruplex
C
C base pairing to link with additional
quadruplexes and the process continues to result in dendrimer-type
self-assembled super structures. The structures formed by branching
covalent connections have been termed dendrimers ( (32) and
references therein). The proposed self-assembly structures of
G-quadruplexes can thus be regarded as G-DNA dendrimers with the novel
feature of branching via vertical stacking of G-quartets and lateral
expansion via C
C base pairing rather than
covalent formation. The role of C
C base pairing
in the proposed model is, thus, two-fold: to facilitate the initial
G-quadruplex formation via parallel dimeric duplex formation and
subsequent inter-quadruplex association via looped out cytosine base
pairing.
Figure 9:
Schematic representation of the
K-driven formation of quadruplexes with contiguous
G-quartets and looped-out cytosines.
Interestingly, chemical probing by Kohwi et al.(33) has revealed that under physiological salt and pH
conditions, Zn or Co
ions induce
AGC repeats to adopt a novel non-B DNA structure where all cytosines
but no adenine residues in either strand become unpaired. Looping-out
of thymidines has also been speculated in the G-DNA structural studies
on d(TGTGGGTGTGTGTGGG) (34) . These results lend further
credence to our proposed looping out of cytosines for inter-quadruplex
C
C base pairing. The fact that poly(dC) was
shown to form a self-complex via C
C base pair
formation with a pK
of 7.4 at 0.05 M NaCl (35) further suggests that such a process is not impossible at
pH 8 and accounts for its extremely slow kinetics. Unfortunately, such
slow kinetics have prevented us from carrying out a detailed pH
titration.
The fact that the observed kinetic behaviors exhibit
characteristics of autocatalytic reactions with induction periods (19) gives additional support to the proposed mechanism, as
each product provides further stacking and cytosine binding sites
analogous to that of chain branching polymerization. The failure of
concentrated Na to induce similar phenomenon is also
consistent with the proposed model, as this ion is too small to form a
stable octa-coordinated complex with two G-quartets. The facilitation
of aggregate formation via melting in the presence of molar
[K
] most likely is the consequence of
freeing the kinetically or thermodynamcally trapped conformers for
ready participation in the parallel quaruplex formation upon cooling.
Although the proposed mechanism seems plausible, other possibilities
cannot be ruled out, such as: extensive concatemers stabilized by
stacking and protonation, some geometrical arrangements with crossover
of strands, formation of linked G-quadruplexes and C-tetraplexes
(i-DNAs) (36, 37) via interdigitation of inter
G-quadruplex CC duplexes to result in a network
of linked G-tetraplexes with alternating tetraplex polarities. Most
recently, Marsh and Henderson (38) observed the self-assembly
of a telomeric oligonucleotide d(GGGGTTGGGG) into a superstructure,
which they termed ``G-wire.'' This structure is formed by
slipped tetraplex association to result in a long vertical extension
consisting of parallel G-4 DNA domains punctuated by T nodes (see also (43) ). A model incorporating lateral extension via
inter-G-wire C
C base pairing would not be
inconsistent with our observed aggregation phenomenon in
d(CGG)
.
The observed molar K-induced
aggregation phenomenon is the more remarkable when it is realized that
in optical measurements one is dealing with rather dilute solutions of
µM strand concentrations. Thus, facilitation of
inter-quadruplex assembly via C
C base pair
formation is eminently reasonable. However, the role of H
may not simply be the base protonation but also the
neutralization of phosphate groups to reduce the interchain repulsive
effect(39) . It is also of interest to note that the gel formed
by 8-bromoguanosine exhibits a strong positive CD maximum near 290 nm
and a shoulder around 265 nm but without the presence of extended long
wavelength tail(40) . The x-ray structural determination of
this gel had indicated a right-handed helical formation of
tetraplex(41) . The gross similarity with the observed
-type CD characteristics in this report suggests that the
quadruplexes of d(CGG)
most likely are also of right-handed
helical form.
In their studies on the contribution of light
scattering to the CD of DNA films with twisted structures,
DNA-polylysine complexes, and condensed DNA aggregates, Maestre and
Reich (42) showed that -type CD spectra are a
manifestation of superorganization of the DNA in these films,
particles, and aggregates. The sense of twist or superhelix can be
determined from the sign of the CD bands. A right-handed helix gives
positive CD signals and vice versa. The periodicity is given
by the Bragg law. The CD tail would be a property of the size of the
particle since it is caused by birefringence dispersion. Thus, a
(+) CD maximum near 300 nm in our aggregates would suggest a
right-handed helical periodicity on the order of 1500 Å. However,
the elucidation of structural details of the aggregates must await the
availability of other techniques. It is interesting to note in passing
that the observed progressive red-shift of the
-CD maximum (see Fig. 2C) is consistent with the formation of
progressively larger complexes during the aggregation process.
Although this report has focused only on d(CGG), similar
results have also been found with d(CGG)
,
d(CGG)
, and other cytosine-containing sequences. In
addition, Sr
, which has been shown to facilitate
parallel G-quadruplex formation(12, 40) , is also
found to be capable of inducing self-assembly of oligomers with CGG
repeats.
Note Added in Proof-A study with
d(CGG) (Mitas, M., Yu, A., Dill, J., & Haworth, I.
S.(1995) Biochemistry, in press) has indicated that this
oligomer exists predominantly in the hairpin confirmations at
[K
]
0.75 M. This, however,
does not alter our interpretation on the observed aggregation phenomena
induced by [K
]
1 M.