| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, April 14, 1995; and in revised form, July 20, 1995) From the
Molar [K
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, C
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 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 C This report describes the observation of an interesting
K Oligonucleotides were purchased from Integrated DNA
Technologies, Coralville, IA and used without further purification.
Experiments were carried out in either 10 mM HEPPS ( 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)
In contrast to pH 8, addition of molar concentration of
KCl to an acidic d(CGG) 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
Figure 3:
A,
CD kinetic traces of pH 5.4 d(CGG)
Kinetic profiles
for the K
Figure 4:
Representative absorption spectra at 20
°C of 40 µM d(CGG)
Figure 5:
Comparison of melting profiles for 40
µM d(CGG)
Figure 6:
Comparisons of CD spectra of 40 µM nucleotide solutions of pH 5.4 for d(TGG)
Figure 7:
Comparison of gel electrophoretic mobility
patterns at 4 °C (A) and 14 °C (B) for
d(CGG)
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)
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)
Although G-quadruplex formation in oligomers containing a
large number of guanine is to be expected, the large
K 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) C 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 Based on these spectral observations, a
mechanism of self-assembly may be envisioned. Aided by the
C
Figure 9:
Schematic representation of the
K
Interestingly, chemical probing by Kohwi et al.(33) has revealed that under physiological salt and pH
conditions, Zn 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 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 C The observed molar K 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 Although this report has focused only on d(CGG) Note Added in Proof-A study with
d(CGG)
Volume 270,
Number 39,
Issue of September 29, pp. 23090-23096, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
(*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
] 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.
C
base pairing scheme.
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) .
C 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.-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 CC base pairing.
)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.
Multi-conformational States of
d(CGG)
Although dodecamer
d(CGGCGGCGGCGG) is not entirely self-complementary, some monomeric and
dimeric conformations involving conventional Watson-Crick base pairings
are possible (see Fig. S1). Thus, the single-stranded form (I)
can coexist with dimeric duplex conformations (II and III) having 8
Gin SolutionsC base pairs with 4 or 3 GG mismatches and with hairpin
conformations (IV and V) having duplex stems of 3 GC base pairs
with 4- or 3-base loop. In addition, the presence of a large number of
guanine bases may also result in possible triplex or quadruplex DNA
conformations (not shown). Indeed, the multi-conformational state of
this oligomer in a pH 8 buffer containing 0.1 M NaCl is
reflected by a very weak and diffuse CD spectrum, a non-monophasic
melting profile, and a multi-band electrophoretic pattern with smear
background (see below).
CD Enhancement and Spectral Characteristics of the
K
A CD
intensity enhancement of nearly 2 orders of magnitude is observed upon
additions of molar concentration of KCl to a d(CGG)-induced Aggregation
solution of pH 8, resulting in a positive maximum near 290 nm, a
shoulder around 265 nm, and a large tail extending well beyond 350 nm.
A very slight turbidity is also discernible in such a solution. An
intense CD with extended long wavelength tail had previously been
coined as -type CD (17, 18) and is usually
associated with aggregate formation. The kinetics of this enhancement
process, however, are extremely slow at pH 8 and take about 10 days to
reach equilibrium in the presence of 2 M KCl at room
temperature (Fig. 2A). In fact, no appreciable CD
intensity changes were apparent within the first 2 h of KCl addition.
Interestingly, molar quantity of NaCl failed to induce a similar
enhancement.
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.
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.
] (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.
Kinetics of Aggregate Formation Resemble Those of
Autocatalytic Systems
The kinetics of aggregate formation
were investigated by monitoring CD intensity enhancements at several
wavelengths. Fig. 3A shows the time-dependent CD
intensity changes at 263 nm for d(CGG) of pH 5.4 at three
different temperatures with 2 M KCl additions. Although
aggregates are barely formed at 20 °C after 2 h, the multiphasic
nature of the kinetics are clearly evident at higher temperatures. At
40 °C, for example, the initial slow CD intensity enhancement is
overtaken by a much greater intensity increase due to aggregation
around 30 min, which eventually levels off and commences a slight
decrease near 100 min, consequence of the progressive spectral red
shift (see Fig. 2C). At 30 °C, both the initial
enhancement and the aggregation process are significantly slower so
that no leveling effect is apparent even after 2 h.
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.
-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).Melting of DNA and Cooling in the Presence of Molar
[K
Melting of aggregates was also
investigated by CD monitoring at 300 nm to yield a melting temperature
of approximately 65 °C in pH 5.4/2 M KCl. Interestingly,
melting of DNA in the presence of molar concentration of KCl appears to
facilitate the reformation of aggregates upon cooling. Time-dependent
ellipticity changes at 300 nm were monitored when d(CGG)] Facilitates the Kinetics of
Aggregate Formation
solutions of pH 5.4/2 M KCl were first melted and cooled
back from 95 °C to three different temperatures (not shown). The
onsets of aggregation are seen to be rather abrupt and occur on or
before 30 min. In view of the fact that about 20 min are needed to cool
the solutions from 95 °C to 40 °C, it is apparent that the
kinetics of aggregate formation have been significantly accelerated.
This is dramatically illustrated for the 20 °C cooling, as
considerable aggregation has already occurred by the time it reached
this temperature. This is to be contrasted with the lack of apparent
aggregate formation after 2 h of adding KCl directly (see Fig. 3A). The corresponding pH 8 solution after melting
and cooling to 30 °C exhibits negligible intensity enhancement in 2
h, confirming the much slower kinetics in non-acidic solutions.K
Time-dependent absorption spectral
measurements also provide evidence of aggregate formation, as typified
by a pH 5.4 solution of d(CGG)-induced Aggregate Formation as
Evidenced by Absorption Spectral
Measurements
with 1 M KCl
induction at 20 °C (Fig. 4). Initial spectral changes
consist of absorbance increases around 285 nm and decreases near 255 nm
with an isosbestic point at 267 nm. Beyond 10 h, aggregate formation
becomes progressively more important, as evidenced by the prominent
presence of a long wavelength tail. This is seen more clearly by
comparing the time-dependent absorbance changes at four different
wavelengths, as shown in the inset. The multiphasic nature of
the process is quite evident in the 285 nm plot, in which an initial
slow intensity increase is followed by a more rapid enhancement and
then a decrease (due to the slow sedimentation of aggregates). The
absorbance changes at 255 nm exhibit a slight progressive decrease
initially and then a more rapid decrease starting around 13 h. In
contrast, no discernible absorbance changes are evident for the initial
15 h when one monitors at the isosbestic wavelength of 267 nm. The
aggregate formation starting near 13 h is further supported by the
significant absorbance increases at 320 nm, where a regular DNA
solution exhibits negligible absorbance. The slow sedimentation of
aggregates is also evidenced by the observed larger absorbances for all
four wavelengths after shaking at 30 h. Similar results were observed
with solutions containing higher KCl concentrations except the kinetics
of aggregate formation become somewhat more rapid.
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).
Minimum [K
Effects of
K] Required for
the d(CGG)
Aggregation concentration on the aggregate formation of
d(CGG)
at 20 °C were investigated by similar
time-dependent absorbance measurements. Spectra were taken for
d(CGG) solutions containing KCl concentrations ranging from
0.4 to 1.2 M at 1-h intervals for 24 h and thereafter at 24-h
intervals for 3 days. Absorbances at 320 nm were then plotted versus time, and the results (not shown) indicate that in the
time span of 24 h, only solutions containing 1.0 and 1.2 M KCl
exhibit significant absorbance increases at 320 nm, signifying the
onset of aggregation at 14 and 9 h, respectively, after the salt
additions. Although there is a discernible absorbance increase after 3
days for the 0.8 M KCl solution, these results suggest that at
40 µM nucleotide concentration, greater than 0.8 M KCl is needed to observe aggregation within the time span of 1
day.[K
Melting profiles of aggregates were
also obtained by absorbance monitoring at 320 nm. Consistent with the
CD results, the aggregates in a pH 5.4/2 M KCl solution melt
near 65 °C but exhibits a gross hysteresis. The apparent hysteresis
exhibited by the cooling profile testifies to the slow kinetics of this
self-assembly process. To investigate the
[K]-dependent Melting
Profiles of Aggregates
] dependence on the stability of
aggregates, melting measurements were made with solutions containing
various KCl concentrations and the results are shown in Fig. 5for the 1.4, 1.8, and 2.2 M KCl solutions. The
enhanced stability of aggregates at higher
[K
] can be more clearly seen via cooling
hysteresis. In particular, the onset of reaggregation for the 1.4 M KCl solution does not occur until approaching 20 °C.
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).
Absence of
To ascertain the
role played by cytosines in the observed aggregate formation of
d(CGG)-type CD in d(TGG)
or
d(CGG)T Solutions, similar experiments were carried out with
d(TGG). Additions of 2 M KCl to a 40 µM d(TGG) solution of either pH 8 or 5.4 failed to induce
-type CD characteristics after 4 days. Instead, the only
significant CD intensity enhancement was observed near 265 nm. No
appreciable CD spectral changes were evident with the addition of molar
quantities of NaCl. Similarly, aside from the presence of a large CD
maximum at 265 nm and a shoulder near 290 nm, no aggregation was
observed for an acidic d(CGG)
T solution in the presence of
2 M KCl. These spectral features are compared in Fig. 6.
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).
Gel Electrophoretic Mobility
Patterns
Electrophoretic mobility patterns of
d(CGG), d(TGG), and d(CGG)T in the
absence and in the presence of 2 M KCl along with a G-rich
dodecamer and a self-complementary oligomer of the same size are
compared in Fig. 7. As expected, the self-complementary
dodecamer d(CCGCCGCGGCGG) at 14 °C (panelB, lane8) is dominated by the dimeric duplex form, but
a faster moving band of apparent hairpin conformation is also barely
discernible. Band locations for the dimeric duplex and the monomeric
hairpin form of a dodecamer, designated as II and IA, respectively,
have also been established by other oligomers that are capable of
forming such conformations (results not shown). The electrophoretic
pattern of dodecamer d(TGGGGGGGGGGT) (lane1 of A and B) in 2 M KCl is seen to consist of a band
with a mobility similar to that of the dimeric duplex reference and a
considerably slower band of somewhat higher intensity (designated as
band IV). The slower moving band can reasonable be attributed to the
G-quadruplex structure, in view of the ability of K to
facilitate the formation of such a conformation in oligomers containing
a stretch of contiguous guanines. Consistent with the
multi-conformational state of d(CGG)
, a complex gel
mobility pattern is apparent in 0.1 M NaCl (lane2). At 4 °C (panelA), bands
corresponding to monomeric hairpin (IA), single strand (IB),
and dimeric duplex (II) conformations as well as a slow moving blob can
be discerned. Aside from conforming with the dodecameric reference, the
assignment of II as the dimeric duplex band is further supported by the
14 °C mobility pattern (panelB, lane2) where the intensity reduction of this band is clearly
apparent. This is consistent with the destabilizing effect of GG
mismatches (see Fig. S1) and the lower thermal stability of the
dimeric duplex as compared to the monomeric hairpin. Three diffuse
bands moving slower than band II are also discernible in this lane,
which can reasonably be attributed to complexes with molecularities of
4, 8, and >16, respectively. In the presence of 2 M KCl (panel A, lane3), however, a striking
appearance of a much slower moving tail with concomitant diminution of
the faster moving bands is clearly evident. The prominent presence of a
much slower moving smear of molecularities estimated to be higher than
16 is consistent with the aggregate formation of polydispersive nature
for d(CGG) in 2 M KCl.The effect of 2 M KCl on d(TGG) and d(CGG)T is the
appearance of slow moving smears capped with bands having
molecularities near 16 but without the much slower trailing tails
(compare lanes4versus5 and 6versus7). The presence of
K-induced slow moving smears for these two oligomers
most likely is the consequence of vertical stackings of G-quadruplexes (20, 21) and/or G-wire type of structural formation (38) .
, 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.
(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.
,
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).
-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 CC
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.
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) .C 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 CC 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
CC base pairing in the observed phenomenon in
d(CGG).(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.
C 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 CC base pairing rather than
covalent formation. The role of CC 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.
-driven formation of quadruplexes with contiguous
G-quartets and looped-out cytosines.
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 CC 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. 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.
C 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 CC base pairing would not be
inconsistent with our observed aggregation phenomenon in
d(CGG).-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.-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.
, 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.
(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.
)
I thank C. Liu for running the gel and D. Dunson, T.
Krugh, and M. Stone for helpful comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Z.-y. Kan, Y. Lin, F. Wang, X.-y. Zhuang, Y. Zhao, D.-w. Pang, Y.-h. Hao, and Z. Tan G-quadruplex formation in human telomeric (TTAGGG)4 sequence with complementary strand in close vicinity under molecularly crowded condition Nucleic Acids Res., June 28, 2007; 35(11): 3646 - 3653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Amrane, B. Sacca, M. Mills, M. Chauhan, H. H. Klump, and J.-L. Mergny Length-dependent energetics of (CTG)n and (CAG)n trinucleotide repeats Nucleic Acids Res., July 21, 2005; 33(13): 4065 - 4077. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Fojtik, I. Kejnovska, and M. Vorlickova The guanine-rich fragile X chromosome repeats are reluctant to form tetraplexes Nucleic Acids Res., January 12, 2004; 32(1): 298 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Jasinska, G. Michlewski, M. de Mezer, K. Sobczak, P. Kozlowski, M. Napierala, and W. J. Krzyzosiak Structures of trinucleotide repeats in human transcripts and their functional implications Nucleic Acids Res., October 1, 2003; 31(19): 5463 - 5468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Weisman-Shomer, E. Cohen, I. Hershco, S. Khateb, O. Wolfovitz-Barchad, L. H. Hurley, and M. Fry The cationic porphyrin TMPyP4 destabilizes the tetraplex form of the fragile X syndrome expanded sequence d(CGG)n Nucleic Acids Res., July 15, 2003; 31(14): 3963 - 3970. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Weisman-Shomer, E. Cohen, and M. Fry Interruption of the fragile X syndrome expanded sequence d(CGG)n by interspersed d(AGG) trinucleotides diminishes the formation and stability of d(CGG)n tetrahelical structures Nucleic Acids Res., April 1, 2000; 28(7): 1535 - 1541. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Weisman-Shomer, Y. Naot, and M. Fry Tetrahelical Forms of the Fragile X Syndrome Expanded Sequence d(CGG)n Are Destabilized by Two Heterogeneous Nuclear Ribonucleoprotein-related Telomeric DNA-binding Proteins J. Biol. Chem., January 21, 2000; 275(3): 2231 - 2238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Fry and L. A. Loeb Human Werner Syndrome DNA Helicase Unwinds Tetrahelical Structures of the Fragile X Syndrome Repeat Sequence d(CGG)n J. Biol. Chem., April 30, 1999; 274(18): 12797 - 12802. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Helbecque, J. Dallongeville, V. Codron, D. Arveiler, J.-B. Ruidavets, A. Evans, F. Cambien, J.-C. Fruchart, and P. Amouyel The Role of a Triplet Repeat Sequence of the Very Low Density Lipoprotein Receptor Gene in Plasma Lipid and Lipoprotein Level Variability in Humans Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 2759 - 2764. [Abstract] [Full Text]
|
|
|
|
|
|
H. Deissler, M. Wilm, B. Genc, B. Schmitz, T. Ternes, F. Naumann, M. Mann, and W. Doerfler Rapid Protein Sequencing by Tandem Mass Spectrometry and cDNA Cloning of p20-CGGBP. A NOVEL PROTEIN THAT BINDS TO THE UNSTABLE TRIPLET REPEAT 5'-d(CGG)n-3' IN THE HUMAN FMR1 GENE J. Biol. Chem., July 4, 1997; 272(27): 16761 - 16768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Weitzmann, K. J. Woodford, and K. Usdin The Development and Use of a DNA Polymerase Arrest Assay for the Evaluation of Parameters Affecting Intrastrand Tetraplex Formation J. Biol. Chem., August 23, 1996; 271(34): 20958 - 20964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Uliel, P. Weisman-Shomer, H. Oren-Jazan, T. Newcomb, L. A. Loeb, and M. Fry Human Ku Antigen Tightly Binds and Stabilizes a Tetrahelical Form of the Fragile X Syndrome d(CGG)n Expanded Sequence J. Biol. Chem., October 13, 2000; 275(42): 33134 - 33141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Kamath-Loeb, L. A. Loeb, E. Johansson, P. M. J. Burgers, and M. Fry Interactions between the Werner Syndrome Helicase and DNA Polymerase delta Specifically Facilitate Copying of Tetraplex and Hairpin Structures of the d(CGG)n Trinucleotide Repeat Sequence J. Biol. Chem., May 4, 2001; 276(19): 16439 - 16446. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||
