How are thermodynamically stable G-quadruplex–duplexhybrids?

Iolanda Fotticchia • Jussara Amato • Bruno Pagano •

Ettore Novellino • Luigi Petraccone • Concetta Giancola

Received: 20 November 2014 / Accepted: 17 February 2015

� Akademiai Kiado, Budapest, Hungary 2015

Abstract In the last decade, DNA duplex and G-quadru-

plex motifs have been investigated for their potential appli-

cations in nanotechnology. Recently, G-quadruplex–duplex

hybrids, generated from the juxtaposition of these two

structural elements, have been considered as DNA nanos-

tructures for nanotechnological applications to take advan-

tage of both duplex and G-quadruplex peculiarities. The

junction between the two structural motifs can play an im-

portant role both for the structure and stability of these hy-

brids. Here, we analyze the thermodynamics of a number of

G-quadruplex–duplex hybrids differing in the bases com-

position in proximity of the junction. Differential scanning

calorimetry, circular dichroism, and gel electrophoresis

methodologies were employed to highlight differences in

their stability.

Keywords DNA nanostructures � G-quadruplex–duplex

hybrids � Thermodynamic stability � DSC � Circular

dichroism

Introduction

Nowadays, nucleic acids are considered interesting mate-

rials for the design and development of nanostructures,

nanomaterials, mechanical devices, and biosensors [1, 2].

Their ability to be used as building blocks for the creation

of nanoarchitectures makes them particularly attractive for

these applications [3]. The advantages of using nucleic

acids in nanotechnology lie in the ability of complementary

strands to hybridize in a controllable fashion. The steerable

hybridization allows to predict the structure obtained. At

first, only DNA duplexes were exploited for the design of

DNA nanoarchitectures. However, one of the major

limitations of nanostructures based exclusively on Watson–

Crick base pairing lies in the difficulty in making func-

tional modifications and in the lack of a certain sensitivity

to chemical stimuli. On the contrary, G-quadruplex-based

nanostructures appear to be more useful because of their

greater conformational flexibility in response to a chemical

stimulus [4, 5]. Recently, the attention has been focused on

the design of DNA G-quadruplex–duplex hybrids gener-

ated by the juxtaposition of these two structural elements.

These hybrids take advantage of the high specificity of the

duplex and the remarkable polymorphism and sensitivity

of the G-quadruplexes [6–8]. In particular, Phan et al.

investigated several G-quadruplex–duplex hybrids with

different topologies, demonstrating that a good juxtaposi-

tion of these two structural elements is possible and that the

base composition proximal to the junction can play an

important role both for the structure and the stability of

these hybrids [9].

In this work, we studied the thermodynamic stability of

G-quadruplex–duplex hybrids looking at the enthalpic and

entropic contributions to the overall stability. For this

purpose, we selected a pool of hybrids starting from a

structure, called Q–D, which presents a parallel-stranded

G-quadruplex with three G-tetrads and a duplex hairpin,

with six base pairs, in perpendicular orientation to the axis

of the ‘‘core’’ of the G-quadruplex (Fig. 1) [8]. In this

I. Fotticchia � J. Amato � B. Pagano � E. Novellino �C. Giancola (&)

Department of Pharmacy, University of Naples Federico II,

Via D. Montesano 49, 80131 Naples, Italy

e-mail: giancola@unina.it

L. Petraccone

Department of Chemical Sciences, University of Naples

Federico II, Via Cintia, 80126 Naples, Italy

123

J Therm Anal Calorim

DOI 10.1007/s10973-015-4588-y

structure, two base pair breaking thymines situated in the

G-quadruplex–duplex junction at positions 10 and 26 of the

sequence (T10 and T26) seem to assure the orthogonal

orientation of the two structural elements [8]. The other

hybrids investigated have similar sequences, but are char-

acterized by specific modifications at the junction or in

proximity of the junction (Table 1). In the first sequence,

T10 was substituted with an adenine (Q–D[A10]); in the

second one, the two thymines were substituted with a cy-

tosine and a guanine (Q–D[C10/G26]); in the third one,

two bases in close proximity of the two thymines were

substituted with an adenine and a thymine (Q–D[A11/

T25]); in the fourth one, an additional thymine was added

(Q–D[1T11]).

The thermodynamic study of the modified hybrids was

performed also comparing their stability with those of the

hybrid Q–D and the single G-quadruplex. The study was

carried out using differential scanning calorimetry (DSC)

and circular dichroism (CD). DSC is a good tool to study

G-quadruplex stability, being the calorimetric profiles very

sensitive to variations in G-tetrad number, cation type, and

loops length [10–12]. Moreover, polyacrylamide gel elec-

trophoresis experiments were carried out to verify the

molecularity of the studied species.

Experimental

Sample preparation

All the oligonucleotide sequences used for this study were

synthesized on an ABI 394 DNA/RNA synthesizer using

products from Glen Research. The oligonucleotides were de-

protected following the manufacturer’s protocols and purified

using Poly-Pak cartridges. Samples were dialyzed successively

against water, 10 mM KCl solution, and water again. They

were subsequently frozen, lyophilized, and suspended in a

buffer containing 10 mM lithium phosphate and 10 mM KCl

(pH 7.0). The lithium cation was used for the stabilization of the

duplex and the potassium cation to allow the formation of the

G-quadruplex. The resulting solutions were annealed by heat-

ing at 368 K for 5 min and faster cooled to room temperature

before each experiment. The concentration of the oligonu-

cleotides was evaluated by UV measurement at 260 nm, at a

temperature of 368 K, using molar extinction coefficient values

calculated by the nearest-neighbor model [13].

Circular dichroism

CD spectra were recorded using a Jasco J-715 spectropo-

larimeter equipped with a Peltier-type temperature control

system (PTC-423). Oligonucleotides concentration of

1 lM was used to record CD spectra between 220 and

320 nm employing cells with 1.0 cm path length. A time

constant of 4 s, a 2-nm bandwidth, and a scan rate of

20 nm min-1 were used to acquire the data. The spectra

were signal-averaged over at least three scans and baseline

corrected by subtracting a buffer spectrum.

Melting curves were recorded over the range

293–373 K, in 10 mM lithium phosphate and 10 mM KCl,

pH 7.0, by following the change of the CD signal at

262 nm, with a scanning rate of 1.0 K min-1. The CD

melting curves were modelled by a two-state transition

according to the van’t Hoff analysis implemented in Origin

7.0 software [14]. The melting temperature (Tm) and the

enthalpy change (DH) values provide the best fit of the

experimental melting data.

26

10

3'

5'

Fig. 1 Schematic illustration of the hybrid Q–D

Table 1 Investigated sequences

Q TTGGGTGGG T GCA T GGGTGGGT

D CGCGAAGCATTCGCG

Q–D TTGGGTGGG T CGCGAAGCATTCGCG T GGGTGGGT

Q–D[A10] TTGGGTGGG A CGCGAAGCATTCGCG T GGGTGGGT

Q–D[C10/G26] TTGGGTGGG C CGCGAAGCATTCGCG G GGGTGGGT

Q–D[T11/A25] TTGGGTGGG T TGCGAAGCATTCGCA T GGGTGGGT

Q–D[1T11] TTGGGTGGG TT CGCGAAGCATTCGCG T GGGTGGGT

I. Fotticchia et al.

123

Differential scanning calorimetry

Differential scanning calorimetry measurements were per-

formed on the last generation nano-DSC (TA Instruments).

The experiments were performed at a DNA single-strand

concentration of 40 lM. Scans were performed at

1 K min-1 in the 278–373 K temperature range. Rev-

ersibility was established for each sample by 3–4 scans

after cooling. A buffer–buffer scan was subtracted from the

buffer–sample scans, and sigmoidal baselines were drawn

for each scan. Baseline-corrected curves were then nor-

malized for the single-strand molar concentration to obtain

the corresponding molar heat capacity curves.

The process calorimetric enthalpies, DHTm, were ob-

tained by integrating the area under the heat capacity

versus the temperature curves. Tm is the temperature cor-

responding to the maximum of each peak. Entropy values

were obtained by integrating the curve DCp/T versus T

(where DCp is the molar heat capacity and T is the tem-

perature in Kelvin), and the free-energy values were

computed by the equation:

DG ¼ DH � TDS ð1Þ

The DSC curves were modelled by a two-state transition

according to the van’t Hoff analysis to determine the DHvH.

The reported errors on the thermodynamic parameters

(Table 2) are the standard deviations of the mean from

three determinations.

Nondenaturing polyacrylamide gel electrophoresis

(PAGE)

Nondenaturing gel electrophoresis was performed at room

temperature using 20 % polyacrylamide gel (acrylamide/

bisacrylamide 29:1), in which 1 9 TBE (89 mM of Tris

and boric acid and 2 mM EDTA, pH 7.5) was used as

running buffer. Buffer was supplemented with 10 mM

KCl. Bands in the gels were visualized by UV shadowing.

A concentration of 35 lM was used for each sample, and

the total volume was 10 lL. All samples were subjected to

the ‘‘fast annealing’’ procedure (as described above) in

10 mM lithium phosphate (pH 7.0) and 10 mM KCl. Prior

to loading the samples onto the gel, 1 lL of glycerol so-

lution (60 % glycerol v/v) was added to each one.

Results

CD measurements

In this work, the CD studies were addressed not only to

acquire the melting temperatures, but also to obtain the

melting enthalpies. The hybrids taken in consideration for

this study show, in some cases, a tendency to aggregate.

Taking in mind that the aim of our study is a thermody-

namic characterization of the stability of monomolecular

species in solution, we utilized an ad hoc ‘‘fast annealing’’

procedure to avoid aggregate formation. Each sample was

heated at 368 K, acquiring spectra overtime until two

consecutive spectra became superimposable, thus indicat-

ing a complete denaturation. The sample was then fast

cooled at 293 K to block in a kinetic trap the unimolecular

species, a spectrum was acquired, and CD melting/an-

nealing curves were recorded in the temperature range of

293–373 K. Finally, a spectrum at 293 K was collected.

Figure 2 shows the spectra of the investigated sequences at

363 K and at 293 K before and after the acquisition of CD

melting/annealing curves. For each sequence, the two

spectra recorded at 293 K result to be superimposable. The

spectra of hybrids show similar profiles with a minimum at

245 and a maximum at 262 nm. In Fig. 3, the spectra of

G-quadruplex, duplex, and of the unmodified hybrid Q–

D are shown. The spectrum resulting from the arithmetic

sum of G-quadruplex and duplex spectra is also shown in

Fig. 3 as dashed line. Although not completely superim-

posable, the calculated and experimental spectra are quite

Table 2 Thermodynamic parameters from DSC and CD measurements

DSC CD

Tm/K DHTm/kJ mol-1 DHvH/kJ mol-1 DSTm/kJ mol-1K-1 DG298K/kJ mol-1 Tm/K DHvH/kJ mol-1

Q 337.0 ± 0.5 191 ± 10 181 ± 15 0.56 ± 0.05 24 ± 4 335 ± 1 183 ± 15

D – – – – – 345 ± 1 97 ± 10

Q–D 338.0 ± 0.5 237 ± 12 222 ± 19 0.70 ± 0.07 28 ± 4 337 ± 1 238 ± 14

Q–D[T11/A25] 337.0 ± 0.5 238 ± 16 – – – 335 ± 1 –

Q–D[A10] 342.0 ± 0.5 283 ± 14 276 ± 20 0.83 ± 0.08 36 ± 5 339 ± 1 280 ± 22

Q–D[C10/G26] 341.0 ± 0.5 189 ± 10 180 ± 14 0.55 ± 0.05 25 ± 4 338 ± 1 185 ± 15

Q–D[1T11] 341.0 ± 0.5 263 ± 13 254 ± 15 0.74 ± 0.07 43 ± 6 339 ± 1 266 ± 21

G-quadruplex–duplex hybrids

123

similar, thus indicating that both the G-quadruplex and

duplex structures are retained, as already found in the

previous work [9]. The melting profiles at 262 nm are

shown in Fig. 4. All the hybrids show sigmoidal profiles

with melting temperatures in the range of 335–339 K. The

enthalpy change, DHvH, was calculated applying the van’t

Hoff equation assuming that the melting is a two-state

N¢D process [14]. The values are shown in Table 2. For

the hybrid Q–D[T11/A25], the DHvH was not calculated

since the gel electrophoresis analysis indicated the pres-

ence of two species in solution (see below). For the hybrids

Q–D[A10] and Q–D[1T11], the DHvH values were

slightly higher than that of Q–D, whereas the DHvH value

of Q–D[C10/G26] was appreciably higher.

Differential scanning calorimetry (DSC) measurements

DSC experiments were performed for a deeper thermody-

namic characterization of the stability of the hybrids. The

DSC curves for the investigated DNA structures are shown

in Fig. 5, and the corresponding thermodynamic pa-

rameters are listed in Table 2. First, the stability of

Q–D was compared to that of Q. The DH value of Q is in

line with the formation of three tetrads. In fact, it has been

220–5

0

5

10

–8

–4

0

4

8

12

–4

0

4

8 6

3

0

–3

–10

–4

0

4

8

–5

0

5

10

240

CD

sig

nal

CD

sig

nal

CD

sig

nal

CD

sig

nal

CD

sig

nal

CD

sig

nal

260 280 300 320 220 240 260 280 300 320

220 240 260 280 300 320 220 240 260 280 300 320

220 240 260 280 300 320

293 K363 K293 K after melting

220 240 260

λ/nm λ/nm

λ/nm λ/nm

λ/nm λ/nm

280 300 320

293 K363 K293 K after melting

293 K363 K293 K after melting

293 K363 K293 K after melting

293 K363 K293 K after melting

293 K363 K293 K after melting

(a) (b)

(c) (d)

(e) (f)

Fig. 2 CD spectra of Q (a), Q–D (b), Q–D[A10] (c), Q–D[C10/G26] (d), Q–D[T11/A25] (e), Q–D[1T11] (f). Each panel shows spectra before

melting at 293 K, at 363 K and at 293 K after melting and annealing

220 240 260 280

λ/nm300 320

–6

–4

–2

0

2

4

6

8

10

[ θ] ×

10–5

deg

dm

ol–1

cm

–1

Q

DQ – D

Q + D

Fig. 3 CD spectra of Q (—), D (m), Q–D (d) and the arithmetic sum

of CD spectra of Q and D (– –)

I. Fotticchia et al.

123

found an average contribution of 60–80 kJ mol-1 for each

G-tetrad, depending on factors such as cations, loops,

strand orientation, or stacking of additional bases on the

tetrad [10, 15]. The DH value of Q–D is clearly higher than

that of Q due to the contribution of the duplex. The melting

of the duplex cannot be followed by DSC, due to the low

heat contribution of a 12-mer hairpin dissociation. How-

ever, a DH of 97 kJ mol-1 was calculated from CD profile,

that, added to DH of Q, gives a total value of

280 kJ mol-1, higher than the experimental DH of

238 kJ mol-1 obtained for Q–D. This implies that some

interactions are lost in the formation of Q–D hybrid with

respect to the two isolated DNA structures.

For all the investigated hybrids, the Tm and DH values

from DSC are in good agreement with those obtained by

van’t Hoff analysis from both CD and DSC measurements.

This is a good indication that the melting of the studied

hybrids is a N¢D one-step process [16]. For the hybrid

Q–D[T11/A25], the Gibbs energy was not calculated since,

300

0

5

10

15

20

25

310 320 330Temperature/K

<ΔC

p> k

J m

ol–1

K–1

0

5

10

15

20

25

<ΔC

p> k

J m

ol–1

K–1

0

5

10

15

20

25

<ΔC

p> k

J m

ol–1

K–1

0

5

10

15

20

25

<ΔC

p> k

J m

ol–1

K–1

0

5

10

15

20

25

<ΔC

p> k

J m

ol–1

K–1

0

5

10

15

20

25

<ΔC

p> k

J m

ol–1

K–1

340 350 360 370 300 310 320 330Temperature/K

340 350 360 370

300 310 320 330Temperature/K

340 350 360 370 300 310 320 330Temperature/K

340 350 360 370

300 310 320 330Temperature/K

340 350 360 370 300 310 320 330Temperature/K

340 350 360 370

(a) (b)

(c) (d)

(e) (f)

Fig. 5 DSC profiles of Q (a), Q–D (b), Q–D[A10] (c), Q–D[C10/G26] (d), Q–D[T11/A25] (e), Q–D[1T11] (f) in 10 mM lithium phosphate

(pH 7.0) and 10 mM KCl

3000.0

0.2

0.4

0.6

0.8

1.0

312 324 336

Temperature/K

Frac

tion

fold

ed

348 360

QQ – DQ – D[A10]Q – D[C10/G26]Q – D[T11/A25]Q – D[+T11]

372

Fig. 4 CD melting curves of Q, Q–D, Q–D[A10], Q–D[C10/G26],Q–D[T11/A25], Q–D[1T11] in 10 mM lithium phosphate (pH 7.0)

and 10 mM KCl. The curves were obtained by recording the changes

in the CD signal at 262 nm as function of the temperature

G-quadruplex–duplex hybrids

123

as previously mentioned, the gel electrophoresis analysis

indicated the presence of two species in solution (see the

next section).

The Gibbs energy values do not show dramatic differ-

ence in stability with respect to that of Q–D hybrid,

although a higher stabilization was found for Q–D[A10]

and Q–D[1T11]. The melting temperatures for all the

hybrids fall in the range of 338–342 K, but the melting

temperatures alone cannot be used as criterion of stability.

Actually, Q–D[C10/G26] shows a melting temperature

higher than that of most of studied hybrids but, displaying

the smallest DH and DS values, exhibits a slightly lower

thermodynamic stability.

Gel electrophoresis

Gel electrophoresis analysis was performed in order to

assess the molecularity of the studied systems, employing a

polyacrylamide gel at 20 % in nondenaturing conditions

(Fig. 6). Gel mobility of the hybrids was monitored in

comparison with Q and with a human telomeric sequence

d[(TTAGGG)8TT], which folds into two consecutive

G-quadruplex structure (Q–Q50mer) [17, 18]. As expected,

the G-quadruplex Q and Q–Q50mer showed the fastest and

slowest migration, respectively. The hybrids exhibit an

intermediate mobility, as awaited for monomolecular spe-

cies being their migrations faster than that of the dimeric

Q–Q50mer structure. The hybrids display a similar migra-

tion pattern except for Q–D[T11/A25] that shows two

bands, probably due to two different conformations in so-

lution. The Q–D[A10] migration is slightly faster, it could

be due to a greater compactness of the structure.

Discussion

In this work, we studied the influence of the base compo-

sition in the G-quadruplex–duplex junction on the ther-

modynamic stability of G-quadruplex–duplex hybrids.

First, the stability of the starting structure Q–D was com-

pared to those of the G-quadruplex (Q) and duplex

(D) alone. The melting enthalpy was compared to the sum

of the melting enthalpies of duplex and G-quadruplex. It

resulted slightly lower, indicating the loss of few interac-

tions in the hybrid formation, according to a slightly lower

intensity in the CD spectrum compared to the arithmetic

sum of Q and D spectra.

The overall thermodynamic data show that the modified

hybrids have comparable stability in the range of the ex-

perimental errors, suggesting that the considered modifi-

cations have no dramatic effects on the stability. However,

a rationalization of the results can be made. The hybrid

Q–D[A10], in which the thymine 10 in the junction is

replaced by an adenine, shows a stabilization in terms of

enthalpy and Gibbs energy compared to Q–D. This could

be attributed to an additional A-T base pairing that stabi-

lizes and compacts the structure, as also confirmed by the

gel electrophoresis analysis. Conversely, the replacement

of the two thymines at the junction by G-C base pair in

Q–D[C10/G26] involves a slight destabilization of the

hybrid, probably due to a more stable base pairing that

confers rigidity and probable structural distortions closest

to the junction. It suggests that a greater conformational

flexibility is preferred at the junction. This is confirmed for

the hybrid Q–D[1T11] containing an additional thymine,

which further decreases the steric tension at the junction

points, allowing a stabilization of the overall structure.

In conclusion, the physicochemical characterization of

the investigated hybrids could aid to define the finest

conditions for future design and development of new DNA-

based nanostructures for potential applications in

nanotechnology.

Acknowledgements We thank Anh Tuan Phan and Kah Wai Lim

(Nanyang Technological University, Singapore) for generously pro-

viding the investigated oligonucleotide sequences. Financial support

from ‘‘Future in Research’’ (FIR) 2013 Grant is gratefully acknowl-

edged (Project code RBFR13XFXR).

Fig. 6 Gel electrophoresis analysis of Q, Q–D, Q–D[A10],Q–D[C10/G26], Q–D[T11/A25], Q–D[1T11] and Q–Q50mer on

20 % polyacrylamide gel; running buffer 1 9 TBE (pH 7.5) supple-

mented with 10 mM KCl

I. Fotticchia et al.

123

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