Current Pharmaceutical Design, 2012, 18, 000-000 1

1381-6128/12 $58.00+.00 © 2012 Bentham Science Publishers

Structure Conversion and Structure Separation of G-Quadruplexes Investigated by Carbazole Derivatives

Ta-Chau Chang1,2,

* , Jen-Fei Chu1,3

, Yu-Lin Tsai1,2

and Zi-Fu Wang1,2

1Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan, Republic of China;

2Department of Chemistry,

National Taiwan University, Taipei 10617, Taiwan, Republic of China; 3Department of Chemistry, National Taiwan Normal Univer-

sity, Taipei, 11677, Taiwan, Republic of China

Abstract: The challenge of G-quadruplexes is that the G-rich sequences can adopt various G4 structures and possibly interconvert among

them, particularly under the change of environmental conditions. Both NMR and circular dichroism (CD) show the spectral conversion of d[AG3(T2AG3)3] (HT22) from Na-form to K-form after Na+/K+ ion exchange. No appreciable change on the induced CD spectra of

BMVC molecule and the single molecule tethered particle motion of HT22 in Na+ solution upon K+ titration suggests that the spectral conversion is unlikely due to the structural conversion via fully unfolded intermediate. Although a number of mechanisms were proposed

for the spectral change induced by the Na+/K+ ion exchange, determining the precise structures of HT22 in K+ solution may be essential to unravel the mechanism of the structural conversion. Thus, development of a new method for separating different structures is of criti-

cal importance for further individual verification. In the second part of this review, we describe a new approach based on “micelle-enhanced ultrafiltration” method for DNA structural separation. The BMVC, a G-quadruplex ligand, is first modified and then forms a

large size of emulsion after ultrasonic emulsification, together with its different binding affinities to various DNA structures; for the first time, we are able to separate different DNA structures after membrane filtration. Verification of the possible structural conversion and

investigation of structural diversity among various G4 structures are essential for exploring their potential biological roles and for devel-oping new anticancer drugs.

Keywords: DNA structures, G-quadruplexes, structural conversion, structural separation, BMVC, emulsification, filtration.

1. INTRODUCTION

Telomeres, the ends of chromosomes, are essential for the in-tegrity of chromosomes by protecting them from degradation and from end-to-end fusion [1,2]. It is believed that the length of te-lomere is closely related to aging [3] and cancer [4] in our life. Telomeres progressively shorten with each cell cycle in somatic cells and eventually result in cellular senescence [5,6]. In contrast, the telomere length in tumor cells is maintained by telomerase and leads to cellular immortalization [7]. Note that telomerase activity appears in ~85% of tumor cells and is not found in most somatic cells [8]. The importance of telomeres and telomerase was recog-nized by Nobel Committee and the Nobel Prize in Physiology or Medicine was awarded to E.H. Blackburn, C.W. Greider, and J.W. Szostak for the discovery of “how chromosomes are protected by telomeres and the enzyme telomerase” 2009.

Telomeres generally consist of many tandem repeats of gua-nine-rich (G-rich) motifs, for example, the hexameric repeats of TTAGGG/CCCTAA in human telomeres [9]. Of interest is that a short 3’-overhang of G-rich single-stranded sequence with 100-200 bases long could adopt intramolecular G-quadruplex (G4) struc-tures. It is suggested that the formation of the G4 structure at the 3’ end of telomeric DNA could effectively hinder telomerase activity [10], since the G4 structure is not a template of telomerase [11]. Such a structure might be a potential target for cancer therapeutic intervention [4,12-14]. Small molecules that can induce G4 struc-ture and further stabilize G4 structure have the potential to serve as anticancer agents. Thus, verification of the telomeric G4 structure is not only for its own biological importance but also for its potential target for drug design.

However, the G-rich sequences can adopt various G4 structures under different environmental conditions. For example, NMR analysis showed that human telomeric sequence, d[AG3(T2AG3)3]

*Address correspondence to this author at the Institute of Atomic and Mo-

lecular Sciences, Academia Sinica, Taipei 10617, Taiwan, Republic of

China; Tel: 8862 2366 8231; Fax: 8862 2362 0200; E-mail: tcchang@po.iams.sinica.edu.tw

(HT22), forms antiparallel basket G4 structure (Scheme IA) in Na+

solution [15] while X-ray crystallography showed that HT22 forms a parallel propeller G4 structure (Scheme IB) in the presence of K

+

[16]. In addition, NMR analyses showed the major conformations of d[TAG3(T2AG3)3] (TA-Tel21) and d[TAG3(T2AG3)3TT] (TA-Tel21-TT) sequences to be hybrid-I (Scheme IC) and hybrid-II (Scheme ID) types in K

+ solution, respectively [17-19]. On the

other hand, the d[G3(T2AG3)3T] (Tel21-T) in K+ solution predomi-

nantly forms a basket antiparallel G4 structure with only two G-quartet layers (Scheme IE) [20]. It appears that slight sequence variations of human telomeres can form different types of G4 struc-tures. Although NMR is a powerful method in determining G4 structures, the presence of two major conformations of HT22 in K

+

solution, which is also suggested by NMR spectra, poses a problem for structural analysis [21]. Thus, development of a new method for separating the various species present in solution, for further indi-vidual study, is of critical importance to the G4 research commu-nity.

Structural diversity is not the only factor to the G-quadruplex complexity. Recent results based on both NMR and circular dichro-ism (CD) measurements showed a rapid spectral conversion from the Na-form to the K-form of HT22 upon K

+ titration [21,22]. A

number of possible mechanisms for structural changes between different types of structures were proposed for this spectral conver-sion [21-27]. Considering the possible biological roles of intercon-version of G-quadruplexes and the structure-based rational drug design, we put our attention on the structure conversion and struc-ture separation of G-quadruplexes in this work.

We have designed and synthesized a 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) for recognizing the G4 structures of d(T2AG3)4 (HT24) [28]. BMVC is not only a G4 stabilizer but also a fluorescence probe. BMVC can increase the melting temperature of G-quadruplex and inhibit telomerase activ-ity [29]. More importance is that BMVC induces accelerated te-lomere shortening and inhibits tumorigenic potential in H1299 lung cancer cells [30]. On the other hand, significant increase of fluores-cence yield and distinctive fluorescence properties of BMVC upon

2 Current Pharmaceutical Design, 2012, Vol. 18, No. 00 Chang et al.

binding to various DNA structures have allowed us for the first time to verify the presence of G4 structure in human metaphase chromo-somes [31]. Here the CD spectra of HT24 and the induced CD spec-tra of BMVC are used to monitor the spectral change upon K

+ titra-

tion. No appreciable difference on the induced CD spectra of BMVC during the change of the CD patterns of HT24 attracts our attention in examining the structural conversion of the G4 struc-tures. Since BMVC has higher binding affinity to quadruplex than to duplex DNA [28], we then adapt the idea of “micelle-enhanced ultrafiltration” [32] to synthesize a BMVC derivative by substitut-ing a bromododecyl chain at 9-position of BMVC, named BMVC-12C-Br, for the separation of the linear duplexes from the G4 struc-tures and the non-parallel structures from the parallel G4 structures [33]. Scheme II shows the chemical structures of BMVC and BMVC-12C-Br. In addition, Table 1 lists the DNA sequences stud-ied in this work.

2. INTERCONVERSION OF G-QUADRUPLEXES AND BMVC

Spectral Conversion Upon Na+/K

+ Ion Exchange

The CD spectra have been extensively applied to study the G4 structures [34-40]. It is well known that linear parallel G4 struc-tures, such as propeller form, give a positive band ~265 nm and a negative band ~240 nm, while antiparallel G4 structures, such as basket and chair forms, show two positive bands ~295 and ~240 nm and a negative band ~265 nm. These spectral features are mainly attributed to the specific guanine stacking in various structures. The CD spectrum of HT24 in 150 mM Na

+ solution is quite different

from that in 150 mM K+ solution, as shown in Fig. (1A). Of interest

is that a spectral conversion from the Na-form to the K-form was observed upon K

+ titration, as shown in Fig. (1B). This spectral

conversion was further confirmed by imino proton spectra upon addition of 150 mM K

+, as shown in Fig. (1C). Yang et al. [21]

proposed a structural conversion via a strand-reorientation mecha-nism for the spectral conversion. This model involves a triplex in-termediate followed by a strand rearrangement to convert the di-

agonal loop in the basket type G4 structure to a lateral loop in the hybrid-I type G4 structure. Note that the basket type dominated for HT22 in Na

+ solution [15] and the hybrid types favored by several

telomeric sequences in K+ solution [17-19,21]. In addition, single

molecule FRET studies by Lee et al. [27] indicated that intercon-version between the two G4 folded states at 2 mM K

+ requires un-

folded intermediate.

Structural Conversion Monitored by a Binding Ligand: BMVC

If the strand-reorientation mechanism is responsible for the structural conversion of HT24 quadruplexes, one would expect that the end G-quartet of G4 structure is disturbed during the conver-sion. Thus, this model may be verified by monitoring the signal from an end-stacked G4 binding ligand. We took the advantage of BMVC ligand which is externally stacked to the end G-quartet of the G4 structure [41] to test the hypothesis of the strand-reorientation model for structural conversion from a basket type to a hybrid-I type G4 structures [22]. Figs. (2A and 2B) show the CD spectra of 10 μM HT24 and the induced CD spectra of 1.5 eq. BMVC in 150 mM Na

+ solution upon K

+ titration, respectively. We

found no appreciable difference on the induced CD pattern of BMVC during the change of the CD patterns of HT24 upon K

+

titration. It implies that the spectral conversion of HT24 in Na+

solution upon K+ titration is unlikely due to the proposed structural

conversion via strand-reorientation mechanism.

It is expected that the structural conversion is more complicated for HT78 than HT24. We have examined the spectral conversion of HT78 upon K

+ titration. Fig. (2C) shows the induced CD spectra of

BMVC/HT78 at a molar ratio around 12 in Na+ solution upon K

+

titration. Again, we have detected spectral change on the CD pat-terns of HT78. However, the induced CD spectra show signal en-hancement right after the first 10 mM K

+ titration and no apprecia-

ble difference for the further K+ titration. Considering that the G4

structure of HT78 surrounded by local environments is more com-plex and its melting temperature (Tm) can be increased by 17

oC

using 12 eq. BMVC, the rapid spectral change upon K+ titration is

A (basket) B (propeller) C (hybrid-I) D (hybrid-II) E (basket).

Scheme I.

BMVC BMVC-12C-Br

Scheme II.

N

Br10

N+N+I- I-

NH

NNI I

Structure Conversion and Structure Separation Current Pharmaceutical Design, 2012, Vol. 18, No. 00 3

unlikely due to the switch between different types of the G4 struc-tures [22]. Fig. (2D) shows the plots of the CD intensity at 265 nm of HT24, HT54, and HT78 in 150 mM Na

+ solution after adding

110 mM K+ as a function of time. It is found that the spectral

change of HT24 is very fast and almost negligible after adding K+

for 10 min. On the other hand, a drastically spectral change fol-lowed by a slow change is observed for both HT54 and HT78. This difference may be due to local environment of the G4 structure of HT78 surrounded by the residues of various single-stranded T2AG3 repeats.

Fluorescence resonance energy transfer (FRET) is a powerful method to monitor the structural conversion of G-quadruplexes due to the distance-dependent energy transfer from a donor to an accep-tor attached to the 5’ and 3’ ends of the oligonucleotide [42,43]. Since the fluorescence yield of BMVC increases about two orders of magnitude upon interaction with DNA, it is particularly useful to monitor structural conversion using BMVC as a donor for FRET to an acceptor of Cy5 labeled HT22. Fig. (3A) shows the time trace of fluorescence emission of the FRET pair BMVC/Cy5-HT22 in Na

+

solution at 675 nm after addition of 75 mM K+, 200 mM Na

+, and

excess 5’-(C3TA2)2 (c12) antisense DNA. Our results show no de-crease for the Cy5 emission upon adding K

+, suggesting that the

end G-quartet is not appreciably disturbed during the structural conversion from Na-form to K-form.

In addition, a stopped-flow FRET measurement with 100 ms time resolution and 200 ms dead time was used to test whether the unfolding of G4 structure could occur on the subsecond timescale under the same Na/K conversion experiments. Fig. (3B) shows the real time stopped-flow FRET signals of BMVC/Cy5-HT22 in Na

+

solution at 675 nm after addition of 75 mM K+ and 200 mM Na

+.

We did not observe any decrease in the FRET signals on a millisec-ond timescale, indicating that the unfolded intermediate state is not detected by the addition of K

+ on the subsecond timescale.

Folding and Unfolding Monitored by Single Molecule Tethered

Particle Motion

In contrast to conventional ensemble-averaged measurements, single-molecule methods can be used to measure the time course of individual molecules during a biochemical process and provide novel information about the reaction mechanisms. The tethered particle motion (TPM) method measures the change of DNA length

by monitoring the Brownian motion (BM) of beads attached to individual DNA molecules tethered at the coverglass surface, as shown in Fig. (4A). The details of single molecule TPM method can be found elsewhere [26]. It is known that both Na

+ and K

+ can

stabilize G4 structures. Furthermore, the concentrations of them are known to modulate the folding equilibrium constant. Figs. (4B,C) shows the distribution of the folded and unfolded states of HT22 in the presence of 15 mM and 150 mM Na

+, respectively. The bead

BM displays a distribution that can be fitted by two Gaussians at 15 mM Na

+: one centered around 10-12 nm, and the other around 24

nm. The first distribution coincides with the folded state of HT22 studied at 150 mM Na

+, and the latter coincides with the unfolded

state studied with a mixture of HT22 and antisense c12 [26]. In addition, the area under each Gaussian represents the population of each conformation under a specific salt concentration. Therefore, the folding equilibrium constant is measured to be 3.40±0.11 (N=74) at 15 mM Na

+. Note that the unfolded state is not observed

at 150 mM Na+. In fact, the unfolded state is hardly detected at 30

mM Na+.

Since the structural conversion between the folded and unfolded states of human telomeric sequence HT22 leads to a DNA tether length change, it is feasible to use TPM experiments to directly monitor the structural conversion process in real time. Fig. (4D) shows the typical time trace for monitoring the unfolding process of the folded HT22 in the presence of a 10,000-fold excess c12 an-tisense DNA. It is found that the conversion efficiency is highly dependent of the concentration of antisense. Under the 10,000-fold excess of c12, the percent of conversion efficiency is ~25% (27 out of 107 tethers). Fig. (4E) shows a typical time trace of HT22 in 150 mM Na

+ solution upon buffer change with 100 mM K

+ solution.

The time course of Na/K ion exchange does not show any totally unfolded state (61 out of 63 tethers). The rest of two tethers showed only larger BM fluctuations. Our results show no distinct interme-diate state, implying that a triplex intermediate followed by a strand rearrangement is unlikely a major pathway during the conversion from a basket type to a mixed type G4 structures.

Temperature Effect on Structural Conversion

It is known that the potassium cation can not only induce G4 structures but also stabilize G4 structures [44,45]. Fig. (5A) shows the CD signals at 295 nm for HT24 as a function of temperature under different [K

+]. Fig. (5B) shows the plot of Tm of HT24 as a

Table 1. The DNA Sequences used in this Work

Sequence Abbreviation Structure (K+) Reference

(T2AG3)4 HT24 2 coexist 17,21

(T2AG3)9 HT54

(T2AG3)13 HT78

AG3(T2AG3)3 HT22 2 coexist 17,21

TAG3T2AG3T HT12 2 coexist 58

(C3TA2)2 C12

ATGCGCAATTGCGCAT LD16 duplex 28

T2G3(T2AG3)3A Tel24-M hybrid 52

(TAG3)2TG3TAG3 Tel19-M parallel 57

G2T2G2TGTG2T2G2 TBA15 chair 61

TGAG3TG4AG3TG4A2G2 Pu24 parallel 62

4 Current Pharmaceutical Design, 2012, Vol. 18, No. 00 Chang et al.

function of ln[K+] with the linear regression fits. The Tm increases

as the [K+] increases are mainly due to the electrostatic interaction

between the cations and the DNA scaffold; this can reduce electro-static repulsion of the DNA phosphate groups and result in a more stable structure [46]. This finding is consistent with the observation of the interconversion detected at low salt concentrations (such as 2 mM K

+ or 5 mM Na

+), but hardly observed at high salt concentra-

tions (such as 150 mM K+ or Na

+) at room temperature.

In addition, Green et al. [43] have measured the activation en-ergy for the unfolding of d[G3(T2AG3)3] (Tel21) in 100 mM Na

+

solution to be 23.3 kcal/mol. This high unfolding activation barrier is different from our observation of the fast converted spectra via Na

+/ K

+ ion exchange. Fig. (5C) shows CD spectra of HT22 in 150

mM Na+ solution at 25

oC (black), 37

oC (red), and 55

oC (blue),

before (dash line) and 20 min after (solid line) the addition of 75 mM K

+. Comparing the CD spectra before the K

+ addition (dash

lines) at 25 oC and 55

oC (~ Tm), the spectral difference is mainly

due to the decrease of the antiparallel G4 structure at 55 oC. After

the addition of K+, these different Na-form spectra at different tem-

peratures rapidly convert to the same K-form spectrum within 20 min. These converted K-form spectra (solid lines) are similar to the spectrum taken after thermal annealing at 95

oC. The fast converted

K-form spectra are independent of temperature, implying that the energy barrier for structural conversion from Na-form to K-form is quite low or even negligible. Mashimo et al. [47] showed that the G-triplet is more stable than the hairpin conformation and equally stable when compared to the G-tetrad, implying that the G-triplex is

Fig. (1). (A) CD spectra of 10 μM HT24 in the solutions containing 150 mM of Na+ or K+ cation. (B) CD spectra of 20 μM HT24 in 150 mM Na+ solution

upon K+ titration. Each CD spectrum was recorded right after the K+ titration. (C) The imino proton 1H-NMR spectra of HT22 in 150 mM Na+ solution before

and 20 min after addition of 150 mM K+ at 25 oC, after further annealing, and HT22 in 150 mM K+ solution after annealing.

Structure Conversion and Structure Separation Current Pharmaceutical Design, 2012, Vol. 18, No. 00 5

a possible intermediate. However, the triplet intermediate has not been detected yet.

Kinetic Studies for Structural Conversion

Recently, Gray et al. [23] have monitored real-time CD signals at 291 and 261 nm of HT22 in 30 mM Na

+ solution after adding 50

mM K+. They found a rapid change in CD signal occurred (Na I1)

during the first ~5 s mixing dead time followed by two relaxation times of 40-50 s for an intermediate state and 600-800 s to a final structure at 25

oC. Particularly, the decay in 291 nm can be referred

to the partially unfolded intermediate state (I1 I2), and the rising in 291 nm corresponds to the refolding to the final state (I2 K). Again, the G-triplet was adopted as an intermediate state (I2) for the transition from basket type to hybrid type. Their results indicate that multiphasic kinetic processes are involved in the transition from Na-form to K-form on a timescale of minutes. Thus, the TPM ex-periment is possible to detect an appreciable percentage of an in-termediate state (I2) during the transition. The fact that we saw no change in induced CD, FRET, and BM suggests that such a G-

Fig. (2). (A) CD spectra of 10 μM HT24 and (B) the corresponding induced CD spectra of BMVC at molar ratio 1:1.5 in Na+ solution upon K+ titration. (C)

The induced CD spectra of BMVC/HT78 at a molar ratio 12 in Na+ solution upon K+ titration. (D) The plots of the CD intensity change at 265 nm of HT24,

HT54 [d(T2AG3)9], and HT78 [d(T2AG3)13] in 150 mM Na+ solution as a function of time after adding 110 mM K+.

Fig. (3). (A) The time trace of fluorescence emission of the FRET pair BMVC/Cy5-HT22 at 675 nm after addition of 75 mM K+, 200 mM Na+, and excess 5’-

(C3TA2)2 (c12) antisense DNA. (B) The real time stopped-flow FRET signals of BMVC/Cy5-HT22 at 675 nm after addition of 75 mM K+ and 200 mM Na+.

6 Current Pharmaceutical Design, 2012, Vol. 18, No. 00 Chang et al.

triplex is unlikely a major intermediate during the transition from basket to hybrid-I folded states.

Considering a two-step consecutive reaction, A I with k1 rate and I B with k2 rate, the detection of the intermediate state (I) depends on two factors. First, the intermediate needs to have dis-tinct signal differing from the initial reactant and the final product. Second, the accumulation of the intermediate is sufficient for detec-tion. It is known that unfolding of G4 structure is rather slow but folding is almost instant. The steady-state approximation in the reaction kinetics suggests that it is hardly to accumulate intermedi-ates when k2 > 100k1 [48]. This might explain why we are not able to observe the decrease in the FRET and the change in the induced CD. However, this is not in agreement with the finding of two re-laxation times, ~50 s and ~800 s, measured for relatively slow structural rearrangements during the transition from initial basket type to final hybrid-I type G4 structures. The question is what is the structure of this intermediate.

G-quadruplex Structure

Renciuk et al. [25] have used CD spectra to show that K+-

induced folded states can adopt several different structures, depend-ing on the salt and DNA concentration used. They found that both Na

+- and K

+-induced folded states have the same antiparallel struc-

tures under the low DNA concentration as used in our single-molecule studies. Since the reactants and the products have the same structures, it is likely that the G4 structure stays the same throughout the Na/K exchange, with only a minor difference in

structural compactness due to the larger-sized potassium ions. This is consistent with our experimental observation of no appreciable change in the FRET, induced CD, and BM upon Na/K exchange.

Lim et al. [20] found that the major G4 structure of Tel-T in K+

solution is a basket form with only two G-quartet layers, with even higher stability. Is it possible that the three G-quartet layers, anti-parallel Na

+ basket structure could be converted into the two G-

quartet layers, antiparallel K+ basket structure [20]? Recently, Yang

et al. [24] reported that the two G-quartet conformation is likely to be an intermediate form of the interconversion of different te-lomeric G4 conformations, rather than a stable form of the human telomeric sequence. Nevertheless, it implies that the structural con-version may involve local unfolding and base rearrangement for the spectral conversion upon Na

+/K

+ ion exchange. Considering the

underlying complexity and diversity of these G4 structures, further evidences are necessary to determine the transition from Na-form to K-form. In addition to determine the structure of intermediate, it is also important to determine the precise structures of HT22 in K

+

solution, which may play a key role in unraveling the mechanism of the structural conversion.

3. STRUCTURAL SEPARATION OF G-QUADRUPLEXES BY BMVC-12C-BR EMULSION

A number of groups [17,21,22,34,35,49-51] have attempted to determine the folding topology of four-repeated human telomeric sequences. The platinum cross-linking studies of HT22 and HT24 suggested that the basket type structure coexists with other G4

Fig. (4). (A) Schematic diagram of single molecule tethered particle motion. (B) BM histogram of HT22 at 15 mM Na+. (C) BM histogram of HT22 at 150

mM Na+. (D) The real time single-molecule TPM for monitoring G4 structures disrupted by c12 antisense oligonucleotide. The HT22 substrates were prepared

at 150 mM Na+ (folded state, green bar) and subjected to buffer change (gray area) with 10,000 excess of c12. The dead time of the experiment (~80 s) was

due to the buffer change and restabilization of the stage for imaging. The recordings were then taken for another ~8 min (red bar) to monitor the unfolding

process. (E) The real time single-molecule TPM for monitoring G4 structures induced by Na/K ion exchange.

Structure Conversion and Structure Separation Current Pharmaceutical Design, 2012, Vol. 18, No. 00 7

structures in both Na+ and K

+ solutions [49]. In addition, the

125I-

radioprobing studies suggested that a chair type structure is a major structure in K

+ solution [50]. Yang et al. modified telomere se-

quence by flanking sequence [21] and strongly suggested that the hybrid type intramolecular G4 structures are the major conforma-tions formed in human telomeric sequences in K

+ solution [24].

Patel et al. [52] studied both the wide type TA-Tel21 and the modi-fied d[TTGGG(T2AG)3A] (Tel24-M) sequences by NMR analysis and concluded that the major G4 structure is the hybrid type chair form. Sugiyama et al. [53] used 8-bromoguanine to lock individual units of the human telomere and found that the HT22 exists as a mixture of hybrid type form and antiparallel form. Although the hybrid type form is likely a major structure for the HT22 in K

+

solution, the precise G4 structure of HT22 in K+ solution still re-

mains undetermined. Particularly, it is evident that slight difference or minor modifications of human telomeric sequences could result in very different structures in recent NMR studies [54]. More cau-tion is required in experimental interpretation.

NMR analysis showed that two types of G4 structures of HT22 coexisted in K

+ solution, which poses a problem for structural

analysis [21,24]. Sheardy et al [55] have confirmed the presence of two types of G4 structures of HT24 in 150 mM K

+ solution by find-

ing two melting temperatures of 51 oC and 66

oC in DSC. In addi-

tion, Miyoshi et al. [56] found that the G-rich sequences favor the parallel G-quadruplex formation in K

+ solution using 40% polyeth-

ylene glycol (PEG) as a co-solvent. The high complexity of various G4 structures, under different solution conditions, makes structural analysis even more difficult. Development of a new method for separating the various species present in solution, for further indi-vidual study, is of critical importance to the G4 research commu-nity.

Methods and Molecule Design

In order to separate different DNA structures, we first take the advantages of BMVC, which has binding preference to quadruplex HT24 over linear duplex (ATGCGCAATTGCGCAT) (LD16) by ~5 times. We then adapt the idea from micelle-enhanced ultrafiltra-tion [32] to design a new molecule of 3,6-bis(1-methyl-4-vinyl-pyridinium iodide)-9-bromododecyl carbazole (BMVC-C12-Br) by substituting a bromododecyl chain at 9-position of BMVC. Due to the hydrophobic moiety of the alkyl chain, this derivative acts

Fig. (5). (A) The CD signals at 295 nm for HT24 as a function of temperature at different [K+]. (B) The plots of Tm of 20 μM HT24 measured from CD as a

function of ln[salt] with linear regression fits. (C) The CD spectra of HT22 in 150 mM Na+ solution at 25 oC (black), 37 oC (red), and 55 oC (blue), before (dash

line) and after (solid line) addition of 75 mM K+ for 20 min.

8 Current Pharmaceutical Design, 2012, Vol. 18, No. 00 Chang et al.

as a surfactant between oil and water. After ultrasonic emulsifica-tion, an oil-in-water (o/w) emulsion is formed. The larger size of the ~2 μm of BMVC-12C-Br emulsion than the 0.22 μm pore size of mixed cellulose esters (MCE) membrane, together with different binding affinities of BMVC-12C-Br emulsion to various DNA structures, allows us to demonstrate, for the first time, that the emulsified BMVC-12C-Br can be used to separate different DNA structures after filtration [33].

Separation of Linear Duplex from G-quadruplex DNA

Since the binding affinities of BMVC-12C-Br to HT24 and LD16 are ~1.42 10

7 and ~4.01 10

6, respectively, we have first

examined whether this BMVC-12C-Br emulsion could separate HT24 from LD16. Fig. (6A) shows the CD spectra before and after six sequences of emulsion induced filtration (EIF). A discernible decrease in the 295 nm CD signal was detected after each EIF. This change was not observed in the filtrate from the HT24/LD16 mix-ture without emulsion. Fig. (6B) shows the spectral difference be-tween the CD spectra before filtration and after the final EIF. The CD pattern for the spectral difference is quite similar to the CD pattern for HT24, implying the loss of the HT24 component after EIF. In addition, the CD pattern observed after the final EIF is very similar to the CD pattern for LD16, as shown in Fig. (6B). We fur-ther performed imino proton NMR to examine whether the NMR spectra of these DNA structures can be separated after EIF. Fig.

(6C) shows the imino proton NMR spectra of 100 μM LD16, 100 μM HT24, and the mixture of them before and after EIF in 150 mM Na

+ solution. It is found that the imino protons are located at 10.5-

12.5 ppm for G4 structure and at 12.5-14 ppm for Watson-Crick duplex [54]. The spectrum for the mixture after EIF is almost iden-tical to the spectrum of LD16, indicating that BMVC-12C-Br EIF is able to separate LD16 linear duplex from HT24 quadruplex struc-tures.

Separation of Non-Parallel from parallel G4 structures of Hu-

man Telomeres

We now examine the separation of parallel from non-parallel G-quadruplexes. Although the precise structures of HT24 in K

+

solution remain undetermined, the structures of two modified te-lomeric sequences, Tel24-M [52] and Tel19-M [57] were analyzed by NMR. Patel et al. [52] have shown that the hybrid type G4 struc-ture is the major conformation of Tel24-M in K

+ solution, while

Phan et al. [57] have illustrated that the parallel propeller type G4 structure is a predominant conformation of Tel19-M in K

+ solution.

Fig. (7A) shows the NMR spectra for 100 μM Tel24-M, 100 μM Tel19-M and the mixture of them before and after EIF in 150 mM K

+ solution together with the spectral difference before and after

EIF. It is found that the spectrum for the mixture after EIF is almost identical to the spectrum of Tel24-M and the spectral difference for the mixture before and after EIF is the same as the spectrum of

Fig. (6). (A) CD spectra of 20 μM HT24 and 20 μM LD16 mixture in 150 mM Na+ solution before and after 6 sequences of EIF. (B) Comparison of the spec-

tral difference between the initial and the final spectra (black) with the CD spectrum of HT24 in 150 mM Na+ solution (green). Also comparison of the final

filtrate (red) and LD16 (blue) in 150 mM Na+. (C) NMR spectra of DNA sequences of LD16 and HT24 before and after EIF

Structure Conversion and Structure Separation Current Pharmaceutical Design, 2012, Vol. 18, No. 00 9

Tel19-M. The gel assays Fig. (7B) further confirm this finding, i.e. a large decrease in the Tel19-M component, but a slight decrease in the Tel24-M component after EIF. It is suggested that BMVC-12C-Br EIF is able to separate Tel24-M hybrid type from Tel19-M par-allel type G4 structures.

In addition, NMR analysis shows that the two repeated human telomeric sequence d(TAG3TTAG3T) (HT12) can form both paral-

lel and antiparallel G4 structures in K+ solution [58]. Fig. (7C)

shows the CD spectra of HT12 in 150 mM K+ solution before and

after two sequences of EIF. The large decrease in the 265 nm CD band after the first filtration suggests that parallel G4 structures can be separated after EIF, as shown in the inset. Moreover, Fig. (7D) shows the imino proton NMR of HT12 in 150 mM K

+ solution

before and after EIF together with the spectral difference between them. There are a set of minor peaks and a set of major peaks in the

Fig. (7). (A) NMR spectra of Tel24-M and Tel19-M in 150 mM K+ solution before and after EIF. (B) Gel assays of Tel19-M (Lane 1), Tel24-M (Lane 2), and

the mixture of them before (Lane 3) and after EIF (Lane 4). (C) CD and (D) NMR spectra of HT12 in 150 mM K+ solution before and after EIF. (E) CD spec-

tra of HT22 in 150 mM K+ solution upon addition of 40% (w/v) PEG 200 before and after two sequences of EIF. The inset shows spectral difference between

each spectrum and the final spectrum. (F) NMR spectra of HT22 and upon addition of 50% (v/v) EtOH for overnight before and after EIF together with their

spectral difference.

10 Current Pharmaceutical Design, 2012, Vol. 18, No. 00 Chang et al.

spectrum before EIF. After EIF, the minor peaks remained in the filtrate are due to antiparallel G4 structure and the major peaks obtained from the spectral difference are attributed to parallel G4 structure. Again, BMVC-12C-Br EIF can separate the antiparallel type from the parallel type G4 structures formed by the same se-quence.

This method is further applied to verify whether the parallel G4 structure is a major conformation of HT22 in 150 mM K

+ solution.

The absence of a large change in the 265 nm CD band and no ap-preciable difference in the imino proton NMR spectra after EIF (data not shown) suggest that the parallel G4 structure is not a ma-jor conformation of HT22 in K

+ solution.

Recently, Xue et al. [59] found conformational change from nonparallel G4 structures to parallel G4 structures of human te-lomeres in K

+ solution under adding 40% (w/v) PEG 200. It is of

interest to investigate whether BMVC-12C-Br EIF can separate the parallel form after structural conversion from the non-parallel form of HT22 under PEG condition. Fig. (7E) shows the CD spectra of HT22 in 150 mM K

+ solution upon addition of 40 % (w/v) PEG

200 before and after two sequences of EIF. Again, the significant decrease in the 265 nm CD band after each EIF is consistent with the previous results, i.e. the parallel G4 structure after conversion is selected by the BMVC-12C-Br emulsion. It appears that the struc-tural change from the non-parallel type G4 structure to the parallel G4 structure of HT22 in K

+ solution indeed occurs under PEG con-

dition.

Since PEG is inconvenient for NMR spectrum and has a high viscosity and a low dielectric constant, we use ethanol as suggested by Renciuk et al. [25]. Although the stability of emulsion can be perturbed by ethanol, it takes about 30 min to turn the turbid BMVC-12C-Br emulsion into clear solution after adding 50% (v/v) ethanol. Here we conducted each EIF process within 5 min to as-sure its function. Fig. (7F) shows the imino proton NMR spectra of HT22 in 150 mM K

+ solution and upon addition of 50% (v/v) etha-

nol for overnight before and after EIF together with the spectral difference between them. Considering that BMVC-12C-Br emul-sion has better selectivity to the parallel structure, we believe that

the spectral difference characterized by the major signals between 11.1-11.3 ppm is due to the parallel type G4 structure induced by ethanol.

Separation of Non-parallel Form From Parallel Form of Non-telomeric G4 Structures

We have further examined whether this EIF method is also valid for the separation of non-telomeric G4 structures. NMR analyses showed that a chair type G4 structure is the major confor-mation for a thrombin-binding aptamer sequence d(G2T2G2TGTG2

T2G2) (TBA15) [60,61] and a parallel type G4 structure is predomi-nated for a c-myc promoter region sequence d(TGAG3TG4AG3TG4

AAGG) (Pu24) [62] in K+ solution. Fig. (8A) shows the NMR spec-

tra for 100 μM TBA15, 100 μM Pu24 and the mixture of them be-fore and after EIF in 150 mM K

+ solution together with the spectral

difference before and after EIF. The gel assays Fig. (8B) also show a large decrease in the Pu24 component, but only a slight change in the TBA15 component after EIF. This EIF method was also applied to separate the bcl2mid monomer from the bcl2mid dimer formed in 150 mM K

+ solution [33]. These results clearly indicate that this

EIF method can well separate the non-parallel G4 structures from the parallel G4 structures.

The BMVC-12C-Br EIF process has been proven to separate linear duplexes from G4 structures and non-parallel G4 structures from parallel G4 structures. This study provides “proof-of-concept” evidence for the selective targeting of emulsified agents to specific DNA structure for structural separation. Currently, we are searching for a new G4 ligand that has differential binding affinities to anti-parallel and hybrid type G4 structures.

4. SUMMARY

We have used BMVC as a probe to monitor the spectral con-version of HT24 upon Na

+/K

+ ion exchange. The induced CD and

FRET results showed no discernible difference after Na+/K

+ ion

exchange, implying that the end quartets of G-quadruplex are not appreciably perturbed during the structural conversion from the basket type of Na-form to the hybrid type of K-form. In addition, it is found that the energy barrier for structural conversion from Na-

Fig. (8). (A) NMR spectra of TBA15 and Pu24 in 150 mM K+ solution before and after EIF. (B) Gel assays of Pu24 (Lane 1), TBA15 (Lane 2), the mixture of

them before (Lane 3) and after EIF (Lane 4).

Structure Conversion and Structure Separation Current Pharmaceutical Design, 2012, Vol. 18, No. 00 11

form to K-form of human telomeres is quite low or even negligible. This low energy barrier may be important for biological signifi-cance, since the kinetic process is dominated in the physiological condition. Thus, we consider that the spectral change might be due to Na

+/K

+ ion exchange resulting in local unfolding and base rear-

rangement. Further studies are necessary to verify the possible mechanism of conversion from Na-form to K-form. Understanding the folding and unfolding of G-quadruplex and the conformational flexibility is essential not only for exploring the biological role of G-quadruplexes, but also for designing new anticancer agents.

In order to unravel the mechanism of structural conversion, we consider that the best way is to determine the initial and final struc-tures and monitor the intermediate states. Thus, we have developed a novel method to separate linear duplexes from G4 structures and non-parallel G4 structures from parallel G4 structures. This method combines a methodology for membrane emulsion/filtration and a lipophilic analogue of BMVC that can form an oil-in-water emul-sion with high binding affinity to specific DNA structure. This method allows us to show that the parallel structure is a major con-formation in HT12, but is negligible in HT22 in K

+ solution. Al-

though we are not able to separate the antiparallel and mixed G4 structures at present, the design principle based on a combination of a distinct methodology and a novel molecule opens a possible di-rection for structure separation. The study of telomeric structure is critical to drug design for telomerase inhibitor and telomere length shortening because G-quadruplexes may be sites of action for can-cer drugs.

ACKNOWLEDGEMENT

This work was supported by Academia Sinica (AS-98-TP-A04) and the National Science Council of the Republic of China (Grant NSC-98-2113-M001-025). The authors thank Cheng-Chung Chang, Chi-Chih Kang, Chih-Wei Chien, Wei-Chun Huang, Ting-Yuan Tseng, Ching-Yuan Tseng for their participation in the study of G-quadruplexes.

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Received: October 19, 2011 Accepted: November 28, 2011