Stability of human telomere quadruplexes at high DNA concentrations

Stability of human telomere quadruplexes at high DNA concentrations

Iva Kejnovská1, Michaela Vorlíčková

1,2, Marie Brázdová

1, and Janos Sagi

3

1Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135,

CZ-612 65 Brno, Czech Republic

2CEITEC- Central European Institute of Technology, Masaryk University, Kamenice 5,

CZ-625 00 Brno, Czech Republic

3Rimstone Laboratory, RLI, 29 Lancaster Way, Cheshire, CT 06410, USA

Correspondence to: Michaela Vorlíčková, e-mail: [email protected]

Janos Sagi, e-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an‘Accepted Article’, doi: 10.1002/bip.22400© 2013 Wiley Periodicals, Inc.

2

ABSTRACT

For mimicking macromolecular crowding of DNA quadruplexes, various crowding agents have

been used, typically PEG, with quadruplexes of micromolar strand concentrations. Thermal and

thermodynamic stabilities of these quadruplexes increased with the concentration of the agents,

the rise depended on the crowder used. A different phenomenon was observed, and is presented

in this paper, when the crowder was the quadruplex itself. With DNA strand concentrations

ranging from 3 µM to 9 mM, the thermostability did not change up to ~2 mM, above which it

increased, indicating that the unfolding quadruplex units were not monomolecular above ~2 mM.

The results are explained by self-association of the G-quadruplexes above this concentration.

The ∆Go37 values, evaluated only below 2 mM, did not become more negative, as with the non-

DNA crowders, instead, slightly increased. Folding topology changed from antiparallel to hybrid

above 2 mM, and then to parallel quadruplexes at high, 6-9 mM strand concentrations. In this

range, the concentration of the DNA phosphate anions approached the concentration of the K+

counterions used. Volume exclusion is assumed to promote the topological changes of

quadruplexes toward the parallel, and the decreased screening of anions could affect their

stability.

INTRODUCTION

G-quadruplexes can adopt a large variety of folding topologies.1 For example, the human

telomere repeat d[AG3(TTAG3)3], htel-22, was found to fold into a 3-tetrad, 3-loop antiparallel

basket-type quadruplex in Na+ solution,

2 and into two types of hybrid (3+1)

3-5 and chair

6

quadruplexes in K+

solution, furthermore, parallel architectures were observed both in

solution 7-11

and in K+-crystals.

12 All these K

+-stabilized scaffolds have also been studied in

cosolvents-mimicked molecular crowding conditions.7,8,11

Minton and Wilf 13

coined the phrase

of macromolecular crowding based on the recognition that biological macromolecules occupy

30% (±10%) of the cellular volume, thus reaching 300 g/L concentrations. Therefore, the

experiments carried out in crowded conditions have become important. The crowding in K+

solutions has been imitated by various agents or cosolvents, such as EtOH (up to 60%),8,11,17

PEG 200 (0-60%), 9,11,14-21

PEG 400, 22

PEG 1000, 23

PEG 8000, 11

PEG 35000, 11

glycerol,

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ethylene glycol (0-15%), 24

histone peptide (20%) in Na+ solution,

17 acetonitrile (50%),

10,11

Ficoll 400, 11,14

and DMSO. 11

The common feature found in K+ solutions was that on the

increase of cosolvent concentration, at low, micromolar DNA strand concentrations, the

quadruplex folding changed from the K+-stabilized antiparallel or hybrid, respectively, to the

parallel fold, and the thermal and thermodynamic stabilities increased concurrently from the

beginning. 14,20,24

A few studies have also been published in which the quadruplex forming DNA

was the crowding agent instead of the cosolvents. The folding changes from micromolar to

millimolar strand concentrations of the d[G3(TTAG3)3] (htel-21) were the same as those found in

the presence of various cosolvents, as determined by CD. 8

Recent CD and Raman-based studies

discussed the formation of parallel aggregates by high concentration of the htel-22

d[AG3(TTAG3)3] quadruplexes in NaCl 25

and KCl 26

solutions. In the present study we show

how the folding topology and the association of quadruplexes are connected with their

concentration, and how the associations influence the parameters characterizing the thermal

stability of the quadruplex of htel-21.

RESULTS AND DISCUSSION

Circular Dichroism Spectra

Figure 1 shows the CD spectra of the K+-stabilized d[G3(TTAG3)3] quadruplexes measured at 3.6

µM, 6.4 mM and 8.8 mM DNA strand concentrations. The CD spectrum of the lowest DNA

concentration is typical for the K+-stabilized antiparallel quadruplex.

2,8,26 The spectrum arises

non-cooperatively upon addition of K+ ions to the Na

+-stabilized antiparallel basket-type

quadruplex; therefore, the resulting structure remains in the same topological family, i.e. in the

antiparallel form. 8 Various methods have confirmed this (references in

8), a Raman study has

just recently. 26

The K+-stabilized quadruplex of htel-22 d[AG3(TTAG3)], measured at

micromolar strand concentrations shows the same CD spectrum as does the htel-21, and it has

been designated by other laboratories as one of the two hybrid (3+1) forms. The designations

were based on the NMR structures; however, these structures were determined at high,

millimolar strand concentrations meeting the NMR requirement for sample concentrations. 3-5

Measured at the NMR-required millimolar strand concentrations the hybrid (3+1) structure

provides a CD spectrum, which is characterized 27

by two positive bands of approximately

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similar heights near 260 nm and 290 nm (Figure S1A), similar to the middle spectrum in Figure

1. On the basis of this CD spectrum the existence of the (3+1) quadruplex structure was

predicted 28

one year before the structure was determined by NMR 3-5

. At still higher DNA

concentrations the positive CD band at 260 nm further increases, the 295 nm band decreases, and

the CD spectrum characteristic of the parallel quadruplexes 28

is formed (Figures 1 and S1A).

The characteristic ∆ε signal values at 265 and 290 nm of the DNA concentrations studied are

collected in Table 1.

PAGE

Nondenaturing polyacrylamide gel electrophoresis shown in Figure 2 indicated that up to 1.72

mM strand concentrations the d[G3(TTAG3)3] adopted monomolecular structures. Starting from

3.7 mM the presence of minor bands corresponding to bi- and higher-molecular species were

noticeable. Simultaneously, a smear appeared in the electrophoretic lanes, the intensity and

extension of which increased with increasing DNA concentration. The presence of the smear was

not affected by temperature; it looked identical at 23oC, 5

oC, and 35

oC (not shown), and was also

present after denaturation and annealing of the samples and also in the sample, that did not form

parallel quadruplex at high DNA concentration (see below). Thus the smear is apparently not

related to quadruplex associations, it may be the result of the higher DNA concentrations. The

majority of substance was detectable in the fastest running bands also above 1.72 mM. This

indicates that the higher-order quadruplex structures dissociate in the electric field applied in the

PAGE runs. The bands of quadruplex dimers, trimers and still higher associates were better

perceptible at low temperature and lower voltage of the electric field (Figure S1B).

Thermal stability

At the lowest strand concentration of d[G3(TTAG3)3] used in this study, 3.57 µM, the thermal

stability (Tm) of the quadruplex was 75.9oC in 60 mM potassium phosphate, 150 mM KCl, pH

6.7. Stability barely changed from 3.57 µM to 1.72 mM. The Tm values started to increase above

1.72 mM, and continued to increase up to the highest DNA concentration measured, 8.79 mM, at

which it was 83.5oC (Table 1, Figure 3A).

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Formation of quadruplex associations

The Tm of monomolecular DNA structures does not change with the DNA concentration. In this

way, the melting unit of d[G3(TTAG3)3] was intramolecular up to 1.72 mM (Table 1), ln(c) ≈

0.6) (Figure 3A), i.e., in a concentration range of 500 times above the initial 3.57 µM (Table 1)

(The micromolar range is generally used in UV and CD absorption measurements.) The

increasing Tm values above 1.72 mM mean that the unfolding units were not monomolecular any

more, thus associates of quadruplexes must have melted. This is in line with minor bands

corresponding to bimolecular, trimolecular and multimolecular associates visible on the non-

denaturing PAGE images at DNA concentrations higher than 1.72 mM (Figure 2).

The hybrid (3+1) quadruplexes that form with strand concentrations higher than 1.7 mM

probably only weakly associate due to the steric hindrance of the lateral loops. The parallel folds

that according to the CD spectra formed here above 6 mM strand could more efficiently

associate probably through the terminal G-tetrads, as the propeller-type side loops formed do not

cause steric obstacles.

To visualize the associates we have undertaken an AFM study (Figure 4). We have used

the highest, 8.74 mM DNA strand concentration from a new batch of htel-21 oligonucleotides

(Figure S1A). We have compared the structures and the AFM images of the sample before heat-

denaturation and after denaturation and the annealing as described in Material and Methods

section. It is to be noted that no parallel quadruplexes are formed in the freshly dissolved

oligonucleotide, these are formed only after denaturation and annealing. In line with this, the CD

spectrum of the concentrated DNA solution before denaturation is similar to the CD spectrum of

the low DNA concentration solution (Figure 1 and S1). Only after denaturation and annealing

does the CD spectrum correspond to parallel quadruplexes (Figure 4, insert A). AFM shows that

the parallel quadruplexes provide smaller number of much larger images than the antiparallel

quadruplexes do before denaturation. The average size of the single antiparallel quadruplexes is

in line with that in an AFM image of the htel-24 in 100 mM K+.

29 Figure 4 is also

complemented by the electrophoretic results of the two samples. It can be seen that only the lane

with the parallel quadruplexes provides bands of quadruplex dimers, and higher associates

(Figures 4, insert B, and S1), which are not present in the lane with the non-heated quadruplex

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sample. The smear is however present in both lanes, and thus the smears are apparently not

related to parallel quadruplex associations. The main electrophoretic band, however, corresponds

to monomers (Figures 2 and S1) so that the associates apparently dissociate in the electric field in

the course of the electrophoresis. The quadruplex monomers probably are antiparallels as

indicated by the same position of the main bands of both samples, while parallel quadruplexes

generally run more slowly than antiparallel ones. 28 Dissociation of the quadruplex associates

proceeds more slowly at low temperatures. Two minor bands corresponding to quadruplex

dimers and higher associates are recognizable at low temperature and slightly lower electric

voltage (Figure S1B).

In addition to the Tm values, the hypochromicity of melting, recorded at 296 nm, has also

changed with the strand concentration (Table 1, Figure 3B). The average value of 43% at low

concentrations abruptly altered above ln(c) of 0.6, and diminished to 33%. The decrease is

probably in correlation with the structural changes, such as the association of quadruplexes. (The

decrease of the molar absorption of the hybrid and the parallel associates cannot be excluded, as

well.) In the thermal profiles no bi- or multiphase melting transitions could be seen (Figure 5).

Thus, apparently, above 1.72 mM strand concentration the associated hybrid and parallel

quadruplex folds can form in one step from random coil during slow cooling, and also, unfold in

one step during melting. To investigate this point, CD melting experiments have also been

performed.

CD melting studies

Figure 6 shows the temperature dependencies of CD spectra with a 0.265 mM (Figure 6A) and of

the 10-times diluted sample of a 6.86 mM (Figure 6B) strand concentration solutions of

d[G3(TTAG3)3]. The low concentration sample folds into individual K+-stabilized, defined as K

+-

antiparallel 8,26

architecture. Its CD spectra go through isodichroic points at 227 and 236 nm in

the course of the melting that indicates a two-state transition. At high concentrations, 6-9 mM,

the d[G3(TTAG3)3] molecules form associates of parallel folds (the association starts at lower

concentrations corresponding to associated hybrid quadruplexes). CD melting could not be

determined at these high concentrations as the small solution volume evaporates in the narrow

0.001 cm path-length demountable cells. Interestingly however, neither the PAGE profile (Figure

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6, insert, top right) nor the CD spectrum (Figure 6B) changed upon a 10-times dilution. That is,

the parallel quadruplex associates once formed continue to stay together on dilution, contrary to

their dissociation in the electric filed used in PAGE. CD spectra of the diluted, 0.686 mM sample

(Figure 6B) went through isodichroic points at 227 and 243 nm within 56-80oC, communicating

two-state melting. After denaturation the spectrum of the reannealed solution showed an

antiparallel quadruplex (Figure 6B), same as in Figure 6A. The 0.686 mM sample of

d[G3(TTAG3)3] actually folded into an antiparallel architecture.

Enthalpy, entropy and free energy

Thermal unfolding of G-quadruplexes of micromolar strand concentration has been generally

considered as a two-state process, and the UV absorbance-based data analyzed accordingly by

various model-dependent methods, as reviewed thoroughly by Mergny and Lacroix. 30

A recent

study by Gray et al. 31

described that the unfolding of the htel-22 d[AG3(TTAG3)3] and that of

the 2-aminopurine-containing htel-22 quadruplexes went through two and one sequential

intermediates, respectively, in 25 mM KCl, 10 mM tetrabutylammonium phosphate buffer.

(Repeating the experiments in 20 mM potassium phosphate buffer did not reproduce these

results, P. Mojzes, personal communication.) Based on their model of melting the authors

predicted in an earlier paper 32

that elevated K+ concentrations would stabilize a single topology.

The presence of melting intermediates would then depend on modifications, such as the 2-

aminopurine, and on cation concentration. Indeed, Sacca et al. 33

have proved that the melting of

htel-22 was a two-state process without intermediates at a higher, 100 mM KCl concentration.

The proof was based on the well matching enthalpies obtained from the van’t Hoff analysis of

the UV melting profiles and from the calorimetric results.

Throughout the present experiments with htel-21, we have used even higher K+

concentrations than the 100 mM of Sacca et al. 33

: 60 mM K+-phosphate, 150 mM KCl (pH 6.7),

equaling approximately 240 mM for K+ ions. Presumably, there was only a small probability for

melting intermediates at 240 mM K+. Actually, the melting (quadruplex unfolding) and

reannealing (folding) UV absorption profiles at 296 nm at each concentration of d[G3(TTAG3)3]

studied here were reversible and superimposable, referring to thermodynamic equilibria.

Examples for these order-disorder-order transitions are illustrated in Figure 5 with the profiles of

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the 3.57 µM and the 6.35 mM strand concentrations. The reversibility of melting, the

superimposable unfold and refold profiles, and the seemingly monophasic thermal curves

referred to, and the CD thermal profiles actually proved (Figure 6) that there were only two types

of structures in significant amounts in 240 mM K+ solutions during the thermal transitions from

50oC to 80

o for the low, and from 60

oC to 85

oC for the high DNA concentrations, and back: the

folded and unfolded structures. Based on the two-states, we have calculated the thermodynamic

data by shape analysis of the absorption thermal profiles using the MeltWin software, a non-

linear curve-fitting algorithm. 31

However, only the data for the concentration range of 3.57 µM

to 1.72 mM were used, in which range the Tm values did not change (Figure 3A). The

thermodynamic data are presented in Table 1. Above 1.72 mM concentrations the “folded” may

mean the associated hybrid or parallel quadruplexes, and the preconception is that the high-

concentration folding-unfolding is probably a multi-state process. Therefore, the calculated

thermodynamic data above 1.72 mM may be average values of multiphase transitions, which

could result in misinterpretation of the data. These values are in parentheses in Table 1.

The formation of quadruplexes above 3.57 µM of strands became slightly less exothermic

with the increase of concentration up to 1.72 mM, the ∆Go37 changed from -6.51 to -6.06

kcal/mol. These were enthalpy-based changes (Table 1). (The thermodynamic parameters began

to be less negative above 1.72 mM, indicating decreasing thermodynamic stabilities.) The 1.72

mM concentration corresponded to the breaks in the graphs of the Tm and the hypochromic

values in Figure 3 (at/above ln(c) = 0.6, which is about 1.8 mM).

Mimicking macromolecular crowding by the addition of cosolvents to micromolar strand

concentrations of quadruplexes the stability (Tm, ∆Go) of quadruplexes have been described to

increase with the increasing cosolvent concentrations. 14,20,24

The change of ∆Go37 values from

-3.5 to -5.5 kcal/mol of the TBA quadruplex d[G2T2G2TGTG2T2G2] of 5 µM strand

concentration has been found to be linear with 0-40% PEG 200, and both the 0% as well as the

40% PEG quadruplex folds were reported to be the two-tetrad, chair-type K+-antiparallels. As no

conformational change has occurred, both the Tm and the free energy change must have reflected

the effect of increasing concentration of PEG. 14

With 0-15% glycerol the free energy of

formation of d[G3(TTAG3)3] quadruplex also changed in a quasi-linear fashion from -2.6 to -6.6

kcal/mol. No conformation was rendered to the various stabilities. 24

The stability of 20 µM of

d[AG3(TTAG3)3] in Na+ solution, in which it formed an intramolecular antiparallel quadruplex,

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increased from 58.1oC to 66.9

oC in 40% PEG 200, and the enthalpy, entropy and free energy of

formation concomitantly became more negative, the latter from -3.1 to -5.5 kcal/mol, but no

conformational change was reported. 20

In contrast, in K+ solution the 40% PEG 200 induced

parallel conformation in the htel-23 d[TAG3(TTAG3)3] of about 40 µM strand concentration with

a corresponding increase of Tm from 63oC to 91

oC.

21 Similarly to these results, a stabilizing

effect of the increasing concentration of quadruplexes was anticipated in the present study. Our

results however deviated from those of the cosolvent-induced crowding: the thermal stability

started to increase only with the formation of quadruplex stacks, above 1.72 mM, and the

enthalpy, entropy, and free energy change values did not show increased stability even in the

3.57 µM to 1.72 mM range. They began to be slightly less negative, referring to somewhat

decreased thermodynamic stability as the concentration was increased. Stability may also depend

on the crowder, 34

which is the DNA quadruplex itself here, not like the generally studied

cosolvents. 14,20,24

We cannot accept and do not interpret the thermodynamic parameters obtained from the

melting curves of quadruplexes of strand concentrations above 1.72 mM (Table 1, in

parentheses), as these parameters may originate from non-two-state thermal transitions. Still, a

decreasing trend in free energy changes can be predicted, even if there are two known stabilizing

effects that can be taken into account. First, the folding topology from 1.72 mM up to 8.79 mM

changed from the antiparallel towards the parallel (Figure 1), and the parallel has been described

to be more stable than the antiparallel, expressed by both the Tm and the ∆Go25 values.

35 The

other effect is the entropy-based stabilization of the crowded macromolecules due to volume

exclusion. Although this view has been challenged by both experimentally and computation

(studies showed that the macromolecules can be thermodynamically disfavored in crowding

conditions), 34,36-40

there could be an entropic stabilization of the quadruplexes by crowding in

addition to the stabilization effect due to the topological changes from antiparallel towards the

parallel. However, another effect, the repulsion between the polyanionic structures may

overcome these stabilizations. With the increasing number of DNA strands, the concentration of

the phosphate anions increases, whereas the K+ cation concentration of about 240 mM remains

unchanged. At the lowest strand concentration studied (Table 1) the ratio of [K+]/[DNA

phosphate-] was about 3360. This ratio decreased with the increasing strand concentrations to

364, 28, and then to 7 at 1.72 mM strand, then to 3.2, 2.8, 1.9 and finally to 1.4 at 8.79 mM DNA

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strand concentration. The less effective screening of the phosphate anions by K+ ions could be a

reason for the decreasing thermodynamic stabilities. That is, the increasing repulsion between the

stacked hybrid and parallel folds (above 1.72 mM) could be manifested in less negative enthalpy,

entropy and free energy change values. At the DNA strand concentration of 8.79 mM the

concentration of the DNA phosphate anions reached 8.79 x 21 = 184.6 mM, which was about

70% of the cation concentration present, the [K+]/[DNA phosphate

-] was only ~1.3. The greatest

repulsion forces could form at this strand concentration between the associated anionic parallel

quadruplexes in the packed macromolecular solution. Interestingly, the effect of this repulsion

apparently was not reflected in the Tm values that started to increase right at the ion ratio of 3.2.

However, the increasing Tm values could also be average values, containing also the negative

effect of repulsion. As a conclusion we can say that as the DNA concentration increases, the

counterion cation - DNA phosphate anion balance changes in a way that the screening efficiency

of the phosphate anions by cations largely decreases. This may lead to less negative entropy,

which creates more flexible structures easing any conformational transition forced by the

environment, that is by the cramming of quadruplexes. As the parallel quadruplexes can more

easily associate (through the terminal tetrads) than the antiparallel or hybrid structures, thus

occupying less volume, the crowding of quadruplexes may induce the conversion of non-parallel

forms toward the parallel topology.

Higher-order quadruplex structures

Based on their recent NMR results, Heddi and Phan 11

suggested the formation of higher-order

and higher-symmetry structures under crowding conditions induced by 40-60% PEG 200 for five

htel-22-25 sequences. These higher-order structures were supposed to be stacked bimolecular,

propeller-type parallel quadruplexes. In another recent work of Phan‘s group 41

the authors

described that the 17-mer quadruplex of d[GTG2TG3TG3TG3T] in K+ also formed dimeric

propeller-type parallel quadruplexes, which were stacked at their 5’-end tetrads. In an NMR

study Trajkovski et al. 42

also described the formation of dimeric quadruplexes from 1.3 mM to

6.6 mM strand concentrations of a 19-mer sequence from the first intron of the N-myc gene,

d[TAG3CG3AG3AG3A2], in 120 mM K+. The monomers were three-tetrad parallel quadruplexes

with three flexible propeller-type single nucleotide loops. The threshold was 1.3 mM for the

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formation of dimers of the 19-mers. Abu-Ghazalah et al. 25

have found that the htel-22

quadruplex formed higher-order, multimolecular parallel structures, called also aggregated

complexes, from above 2 mM of strand concentrations in 0.1 M and 1 M NaCl (not KCl). The

19-mer’s 1.3 mM 42

and the 22-mer’s 2 mM 25

threshold strand concentrations for forming

higher-order structures are in good agreement with the 21-mer’s 1.72 mM threshold of the

present work. The conclusion drawn from the present study and those of Phan, 11,41

Trajkovski 42

and Abu-Ghazalah 25

is that stacks of quadruplexes form above a certain strand concentration.

The change of thermal stability is a good indicator for the formation of these stacks.

Differences between the effects of cosolvents and the high DNA concentrations

Effect of the cosolvents on quadruplex structure has been explained by dehydration

exerted either by small molecules, such as EtOH, 7,8,11

glycerol, 24

ethylene glycol, 24

DMSO, 11

and acetonitrile, 10,11

or by macromolecules, like various lengths of PEG, 9,11,14-21

and Ficoll. 11,14

Dehydration has been proved to stabilize quadruplexes, as demonstrated either by Tm and/or ∆Go

values, 14,20,21,24

and the high concentrations of cosolutes could induce formation of bimolecular

quadruplex stacks of the parallel types. 11

Folding topology of the quadruplexes at elevated

cosolute or at high DNA concentrations is apparently similar: Parallel. The two ways leading to

the parallel quadruplexes comprise different mechanisms, but both ways result in reduced water

activity that may induce the parallel fold. 21

Calculating with the molecular mass of 6653 g/L for

the htel-21 d[G3(TTAG3)3], the 8.79 mM strand concentration at which the parallel stacks

formed contains 5.8% DNA (5.8 g/100 mL). The average 30% found in cells is for all

macromolecules, bonded structural and functional, 13

so the amount of all DNA in a cell must be

much less than 30%. The 5.8% can be less or close to the average cellular DNA content, still, the

5.8% concentration of d[G3(TTAG3)3] proved to be sufficient to induce the formation of

associated parallel quadruplex folds. On the other hand, oligodeoxynucleotides of 3-20 µM

concentrations formed parallel stacks with as high as 20-60% of cosolvents. 19,21,25,28

The parallel

topology is thought by some 15

to be the in vivo relevant conformation of quadruplexes, whereas

others disagreed. 43

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CONCLUSIONS

The experiments presented here demonstrated that both the stability and the folding motives of a

htel-21 quadruplex is strongly connected to its concentrations in the micromolar to millimolar

range. Two parameters describing a thermal transition, the Tm and the hyperchromic change at

296 nm are strong indicators of the quadruplex concentrations at which the macromolecules start

to associate with each other. This strand concentration is ~2mM with the human telomere repeat

d[G3(TTAG3)3]. At concentrations less than 2 mM the Tm did not change, contrary to the

cosolvent-induced changes of quadruplex stability. With the increase of the DNA concentration

the ratio of cation/phosphate anion continuously decreases, which can increase the repulsion

between the polyanionic quadruplexes. The increased repulsion was theorized here to affect the

thermodynamic stability. The folding-unfolding phenomenon found with concentrated

quadruplex solutions may also foretell that the conformational properties of other

macromolecules may also differ in concentrated, cell-like conditions from those observed in

simulated conditions.

MATERIALS AND METHODS

The htel-21 deoxyoligonucleotide d[G3(TTAG3)3] was synthesized and HPLC-purified by VBC

Genomics (Vienna, Austria). The lyophilized oligonucleotide was dissolved in 1 mM Na-

phosphate buffer with 0.3 mM EDTA, pH 7 to yield a concentration of about 100 OD260/ml. The

sample was then further purified on Amicon Ultra 0.5 mL centrifugal filters 3K (Millipore),

concentrated to 1/10-1/20th

volumes and transformed into 60 mM K-phosphate and 0.15 M KCl,

pH 6.7. From this stock solution each DNA concentration sample was prepared separately by

dilution with 60 mM K-phosphate and 0.15 M KCl, pH 6.7 to the expected concentration and

was heated for 5 minutes at 95°C, then annealed by slow cooling over 3 hours from 95°C to

room temperature.

CD measurements were carried out in a Jobin-Yvon CD6 dichrograph (Longjumeau,

France) and Jasco 815 (Tokyo, Japan) in 1-cm to 0.001-cm path-length quartz Hellma cells

placed in a thermostated cell holder at 23°C. Scan rate was of 0.5 nm per second. A set of three

scans was averaged for each sample. Jasco 815 was used for the determination of the CD thermal

melting profiles measured in 0.01-cm path-length rectangular quartz Hellma cell. Spectra were

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acquired at a rate of 100 nm/min with averaging of four measurements. CD signal was expressed

as the difference in the molar absorption, ∆ε of the right- and left-handed circularly polarized

light. DNA concentrations in K+ solution were determined on the basis of the absorption at 260

nm at high, denaturing temperature using the molar absorption coefficient of 223,46 [M-1

cm-1

].

In the case of the highly concentrated samples whose absorption exceeded 3.5 at 260 nm (only

0.01 cm stopper cells could be used for temperature dependences) the concentrations were

calculated from their absorbance at 296 nm using the A296/A260 ratio determined at lower DNA

concentrations.

Native polyacrylamide gel electrophoresis (PAGE) was run in a temperature-controlled

electrophoretic apparatus (SE-600, Hoefer Scientific, San Francisco, CA). Gel concentration was

16 % (29:1 monomer to bis ratio, Applichem, Darmstadt, Germany). About two micrograms of

DNA was loaded on the 14 x 16 x 0.1 cm gel. Samples were electrophoresed at 23°C for 19

hours at 30 V, and also at 5°C for 18 h at 60 V, and at 35°C for 16 h and 25 V. Gel was stained

with Stains All (Sigma, St. Louis, MO) after electrophoresis and scanned using a Personal

Densitometer SI, model 375-A (Molecular Dynamics, Sunnyvale, CA).

The UV absorption melting curves were measured in a Varian Cary 4000 UV-Vis

spectrometer (Australia) in 1-cm to 0.01-cm path-length rectangular quartz Hellma cells from

20oC to 95 or 99

oC. Temperature was increased/decreased by 1°C intervals and the samples were

equilibrated for 3 min before each measurement. The thermal stability (Tm) and the

thermodynamic data, where applicable, have been calculated from the shape of the thermal

profiles, by using sloping baselines and adopting the finding of Olsen et al. 44

that during the

temperature-induced unfolding the heat capacity changes were negligible, e.g. in the case of htel-

22, among other quadruplexes. The monomolecular association option of the MeltWin v.3.0

software,45

a non-linear curve-fitting algorithm was applied for the calculations, as described

earlier by others and us. 46-49

Data of triplicate experiments are collected in Table 1.

For the AFM imaging 0.25 µl of DNA samples was diluted by 2.25 µl of buffer

containing 5 mM potassium-HEPES (pH 7.6) and 2 mM MgCl2 and placed onto the surface of

freshly cleaved mica V4 (SPI Supplies, USA) for 10 minutes, and then rinsed with 0.75 ml of

water. The remaining water was blown away by flow of compressed air. Samples were scanned

with Nanoscope V MultiMode VIII (Veeco) operating in Scan Assist mode in Air. Scananalysis-

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Air Cantilever with Silicon Tip (Bruker, USA) was used to image the samples.

ACKNOWLEDGEMENTS

Professor Peter Mojzes and Dr. Jan Palacky are thanked for valuable discussions. This work was

supported by Grants P205/12/0466 and 13-36108S provided by the Grant Agency of the Czech

Repubic and by the project ‘‘CEITEC – Central European Institute of Technology’’

(CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund.

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15

Text to Figures

Figure 1.

Selected CD spectra of different concentrations of d[G3(TTAG3)3] in 60 mM potassium

phosphate and 0.15 M KCl, pH 6.7. The strand concentrations were: 3.57 µM (green), 6.35 mM

(red), and 8.79 mM (blue). The spectra were measured after heat denaturation and slowly

annealing, as described in Material and Methods. Insert: ∆ε values measured at 265 nm (blue)

and 290 nm (green) as a functions of the logarithm of the d[G3(TTAG3)3] strand concentration.

Figure 2.

Non-denaturing PAGE (top) and its densitometric record (bottom) of d[G3(TTAG3)3] of

indicated concentrations running at 23°C. The samples were taken directly from the CD

experiments. The DNA ladder served as the electrophoretic marker.

Figure 3.

Thermal stability (A), and the temperature-induced hypochromic changes at 296 nm (B) as the

functions of the logarithm of the strand concentration of the d[G3(TTAG3)3] quadruplexes in 60

mM K-phosphate and 0.15 M KCl, pH 6.7. (The logarithmic scale of concentration was used to

better show the position of the breaks in the curves.)

Figure 4.

Atomic force microscopy of 8.7 mM strand concentration of d[G3(TTAG3)3] before (left) and

after (right) heating for 5 minutes at 95°C and slowly annealing. The scale represents 2,5 µm.

Upper figures are in planar projection; the lower are in 3D projection. Inserts: CD spectra (A)

and a native electrophoresis (B) of the same samples: Antiparallel quadruplex (blue, 1) and

parallel quadruplex (green, 2). The band of monomers dominates in the electrophoresis so that

the associates dissociate during its run. All the measurements were done at room temperature.

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Figure 5.

Examples of the thermal unfolding and reformation of d[G3(TTAG3)3] quadruplexes at 3.57 µM

and 6.35 mM strand concentrations, as monitored at 296 nm.

Figure 6.

The CD and UV absorption spectra of a low (0.265 mM, panel A, and insert A, top left) and of

the 10-times diluted sample of a high (6.86 mM, panel B, and insert B, top center) concentrations

of d[G3(TTAG3)3] at various temperatures from 20oC (blue) to 77

oC (green in A) or 82

oC (red in

B), and a single CD spectrum back at 20oC (black). Insert top right: non-denaturing PAGE at

23°C of d[G3(TTAG3)3], lane 1, concentrated sample (6.86 mM), lane 2, 10-times diluted sample

(0.686 mM) before melting, and lane 3, after melting, cooled down.

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Thermodynamic parameters for the formation of dG3(TTAG3)3 quadruplexes in 60 mM potassium

phosphate, 150 mM KCl, pH 6.7.

*Hypo. stands for the temperature-induced hypochromic changes; Standard deviation of the

∆Ho values was ±5.9%, and ±6.1% of the ∆S

o values.

#

strand

conc.

(mM)

Tm (oC)

∆∆∆∆Tm (oC)

Hypo.

at 296

nm*

(%)

∆∆∆∆Ho

(kcal/

mol)*

T∆∆∆∆So(k

cal/mol)

*

∆∆∆∆Go37

(kcal/mol)

∆∆∆∆∆∆∆∆Go3

7

(kcal/

mol)

∆∆∆∆εεεε at

265

nm

∆∆∆∆εεεε at

290

nm

1 0.00357 75.9±0.1 0 42.1 -58.4 -51.9 -6.51±0.06 0 65.9 118.2

2 0.033 76.6±0.1 0.7 43.6 -57.2 -50.7 -6.48±0.25 0.03 69.1 117.6

3 0.424 76.0±0.1 0.1 42.5 -54.2 -48.1 -6.05±0.05 0.46 66.4 117.7

4 1.72 76.1±0.4 0.2 42.7 -54.1 -48.1 -6.06±0.28 0.45 78.0 99.5

5 3.73 77.0±0.1 1.1 41.8 (-50.8) (-45.0) (-5.80±0.01) 84.1 107.7

6 4.25 79.2±0.1 3.3 39.8 (-45.6) (-40.1) (-5.54±0.3) 102.9 104.1

7 6.35 81.1±0.3 5.2 35.6 (-41.2) (-35.9) (-5.22±0.5) 105.1 92.4

8 8.79 83.5±0.6 7.6 32.6 (-36.6) (-31.6) (-5.03±0.6) 155.1 68.8

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144x164mm (300 x 300 DPI)

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195x300mm (300 x 300 DPI)

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90x53mm (300 x 300 DPI)

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143x101mm (300 x 300 DPI)

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111x69mm (300 x 300 DPI)

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181x215mm (300 x 300 DPI)

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