©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Acid-facilitated Supramolecular Assembly of G-quadruplexes in d(CGG)(*)

(Received for publication, April 14, 1995; and in revised form, July 20, 1995)

Fu-Ming Chen

From the Department of Chemistry, Tennessee State University, Nashville, Tennessee 37209-1561

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Molar [K] induces aggregate formation in d(CGG)(4), 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)(4) and d(CGG)(4)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 CbulletC 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 CbulletC base pairing to result in dendrimer-type self-assembled super structures.


INTRODUCTION

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, CbulletC 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 CbulletC 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)(4) and proposes a plausible mechanism for the formation of super molecular assemblies of G-quadruplexes via CbulletC base pairing.


MATERIALS AND METHODS

Oligonucleotides were purchased from Integrated DNA Technologies, Coralville, IA and used without further purification. Experiments were carried out in either 10 mM HEPPS (^1)buffer solutions of pH 8 containing 0.1 M NaCl and 1 mM MgCl(2), 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.


RESULTS

Multi-conformational States of d(CGG)(4)in Solutions

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 GbulletC base pairs with 4 or 3 GbulletG mismatches and with hairpin conformations (IV and V) having duplex stems of 3 GbulletC 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).


Figure S1:


CD Enhancement and Spectral Characteristics of the K-induced Aggregation

A CD intensity enhancement of nearly 2 orders of magnitude is observed upon additions of molar concentration of KCl to a d(CGG)(4) 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.


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)(4) 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)(4) 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)(4) solution of pH 5.4/2 M KCl after cooling the solution from 95 °C to 40 °C. Immediately (dots), 10 (solidcurve), 20 (+), 30 (times), 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)(4) solution results in immediately noticeable CD intensity changes. The initial CD spectral alteration after the addition of 1 M KCl to a d(CGG)(4) 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)(4) 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)(4) 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.


Figure 3: A, CD kinetic traces of pH 5.4 d(CGG)(4) 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)(4) 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] Facilitates the Kinetics of Aggregate Formation

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)(4) 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-induced Aggregate Formation as Evidenced by Absorption Spectral Measurements

Time-dependent absorption spectral measurements also provide evidence of aggregate formation, as typified by a pH 5.4 solution of d(CGG)(4) 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.


Figure 4: Representative absorption spectra at 20 °C of 40 µM d(CGG)(4) 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] Required for the d(CGG)(4)Aggregation

Effects of K concentration on the aggregate formation of d(CGG)(4) at 20 °C were investigated by similar time-dependent absorbance measurements. Spectra were taken for d(CGG)(4) 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]-dependent Melting Profiles of Aggregates

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] 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.


Figure 5: Comparison of melting profiles for 40 µM d(CGG)(4) 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 -type CD in d(TGG)(4)or d(CGG)(4)T Solutions

To ascertain the role played by cytosines in the observed aggregate formation of d(CGG)(4), similar experiments were carried out with d(TGG)(4). Additions of 2 M KCl to a 40 µM d(TGG)(4) 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)(4)T solution in the presence of 2 M KCl. These spectral features are compared in Fig. 6.


Figure 6: Comparisons of CD spectra of 40 µM nucleotide solutions of pH 5.4 for d(TGG)(4) in the absence (+) and in the presence (squares) of 2 M KCl for 4 days and for d(CGG)(4)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)(4), d(TGG)(4), and d(CGG)(4)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)(4), 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 GbulletG 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)(4) in 2 M KCl.The effect of 2 M KCl on d(TGG)(4) and d(CGG)(4)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) .


Figure 7: Comparison of gel electrophoretic mobility patterns at 4 °C (A) and 14 °C (B) for d(CGG)(4), d(TGG)(4), and d(CGG)(4)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)(4) (lane3), d(TGG)(4) (lane5), and d(CGG)(4)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)(4) (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)(4), d(TGG)(4), and d(CGG)(4)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).




DISCUSSION

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)(4) is somewhat surprising. The kinetic facilitation of such a process in acidic solutions and the absence of -type CD characteristics in d(TGG)(4) suggest a crucial role played by cytosines in this oligomer, likely via the CbulletC base pair formation. The presence of a terminal G appears to be also important, since d(CGG)(4)T does not aggregate in molar KCl solutions. Thus, the ability of d(CGG)(4) 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)(4) or d(CGG)(4)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)(4) prior to the onset of -type CD appearance (see Fig. 2B and Fig. 3A) strongly support the notion that aggregate formation in d(CGG)(4) is preceded by parallel G-quadruplex formation. This is further strengthened by the time-dependent gel mobility measurements of d(CGG)(4) 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) .

CbulletC 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 CbulletC base pairs and individually looped-out thymidyl residues in acidic solutions(28, 29, 30) . Furthermore, self-assembly via branching of parallel CbulletC duplex formation has recently been proposed(31) . These observations, thus argue strongly for the involvement of CbulletC base pairing in the observed phenomenon in d(CGG)(4).

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)(4) and d(CGG)(4) 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)(4) suggests that the remarkable CD intensity enhancement observed in d(CGG)(4) 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)(4)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 CbulletC 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 CbulletC 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 CbulletC base pairing rather than covalent formation. The role of CbulletC 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 CbulletC base pairing. The fact that poly(dC) was shown to form a self-complex via CbulletC base pair formation with a pK(a) 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 CbulletC 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 CbulletC base pairing would not be inconsistent with our observed aggregation phenomenon in d(CGG)(4).

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 CbulletC 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)(4) 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)(4), similar results have also been found with d(CGG)(2), d(CGG)(3), 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] leq 0.75 M. This, however, does not alter our interpretation on the observed aggregation phenomena induced by [K] leq 1 M.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant CA-42682, Army Medical Research Grant DAMD17-94-J-4474, and a subproject of Minority Biomedical Research Support Grant S06GM0892. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: HEPPS, N`-2-hydroxyethylpiperazine-N`-propanesulfonic acid.


ACKNOWLEDGEMENTS

I thank C. Liu for running the gel and D. Dunson, T. Krugh, and M. Stone for helpful comments on the manuscript.


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