Hindawi Publishing CorporationISRN BiochemistryVolume 2013 Article ID 631875 7 pageshttpdxdoiorg1011552013631875

Research ArticleVariable-Temperature Size Exclusion Chromatography forthe Study of the Structural Changes in G-Quadruplex

Sanae Benabou1 Ramon Eritja2 and Raimundo Gargallo1

1 Solution Equilibria and Chemometrics Group (Associate Unit UB-CSIC) Department of Analytical ChemistryUniversity of Barcelona Diagonal 647 08028 Barcelona Spain

2 Institute of Advanced Chemistry of Catalonia (IQAC-CSIC) CIBER-BBN Networking Research Centre on BioengineeringBiomaterials and Nanomedicine Jordi Girona 18-26 08034 Barcelona Spain

Correspondence should be addressed to Raimundo Gargallo raimon gargalloubedu

Received 22 July 2013 Accepted 26 September 2013

Academic Editors E Kandimalla and Y Mely

Copyright copy 2013 Sanae Benabou et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The conformational equilibria of a guanine-rich sequence found at the promoter region of the human c-kit oncogene are studied bymeans of circular dichroism spectroscopy (CD) and variable-temperature size exclusion chromatography (SEC) It is shown that thewild sequence ckit21 exists as a mixture of monomeric and multimeric G-quadruplexes Appropriate mutation of several bases inthe wild sequence produces the shift from parallel to antiparallel G-quadruplex as well as the disappearance of multimeric speciesThe shift from the antiparallel to the parallel conformation induced by temperature is reflected in both CD and SEC profiles

1 Introduction

Guanine-rich regions can fold into complex structuresknown asG-quadruplexes whose building block is the tetradan almost planar arrangement of four guanine bases bondedby hydrogen bonds [1] Cations such as Na+ or K+ areknown to strongly stabilize such structures by intercalationin the middle or among the tetrads Depending on therelative orientation of the guanine tracts antiparallel parallelor mixed topologies can be found The formation of G-quadruplexes in sequences corresponding to the telomeresand to the promoter regions of several oncogenes has beendeeply studied in vitro by means of disparate instrumentaltechniques [2 3] Recently the presence in vivo of thesestructures has been observed [4]

In their pioneer work Fernando et al identified a G-quadruplex forming sequence upstreamof the c-kit transcrip-tion initiation site [5] The wild sequence (herein identifiedas ckit21 Figure 1) contains three tracts of three guaninesand a fourth tract comprising four guanines Based on CDNMR and UV-monitored melting experiments the mutatedsequence ckit21T18 was shown to be less heterogeneous thanckit21 whereas a homogeneous parallel G-quadruplex was

proposed for themutated sequence ckit21T21 In a later workHsu et al studied the conformational space of ckit21 andckit21T21 by means of NMR [6]Their results confirmed thatin presence of a concentration of K+ around 100mM ckit21existed as an ensemble of monomeric structures that sharea parallel-stranded propeller-type conformation Schemati-cally these structures produced two similar folding patternsnamed dangling-end and blunt-end (Figure 1) Hence thesequence ckit21T18 should form the blunt-end structureincluding a two-base third loop whereas the sequenceckit21T21 should form the dangling-end structure includinga single-base reversal-loopAlmost simultaneously to the pre-vious paper the existence of a monomer-dimer equilibriumfor the ckit21T21 sequence was also proposed [7] Based onNMR measurements it was suggested that concentrations ofK+ sim100mM shifted the equilibrium to the dimer speciesVery recently the polymorphism of the ckit21T21 sequencehas been studied by means of size exclusion chromatography(SEC) [8] At sim150mMK+ and 200mMDNA concentrationthe existence of monomeric dimeric and tetrameric speciesin equilibrium was proposed However none of the recordedNMR spectra for the purified species matched the previouslyreported NMR data for that sequence It seems that the

2 ISRN Biochemistry

Sequence 5998400 L1 L2 L3 3998400

ckit21T21

ckit21

ckit21T18

ckit21T

C GA

A

C

C T

C

C

C CGCGA

CGCGA

CGCGA GGG AT

T GGG T GGG TGTGT GGG T GGG G

GGG

GGG

GGG

GGG

GGG

GGG

GGG

GGG

GGG

GGG

GGG

(a)

C1 C5

G4

A17G18

G19

G20

G21

G16

G14 C1C5

G4A17

G18G19

G20

G21

G16

G14

(b)

Figure 1 (a) Sequences studied in this work (b) Dangling- to blunt-end equilibrium in ckit21

formation of a particular quadruplex is dependent on manyfactors not only on the sequence [9 10]

In the present work the effect of several mutations onthe ckit21 sequence by means of CD and SEC measurementsdone at different temperatures was studied The DNA con-centration used for both measurements was around 2ndash5120583M(the order of magnitude used in most of the spectroscopicmeasurements) and the K+ concentration was 150mM Inaddition to the three sequences already mentioned (ckit21ckit21T18 and ckit21T21) a fourth sequence (ckit21T) whereall cytosine and adenine bases were mutated to thymine wasstudied The results showed that the mutation in appropri-ate locations was clearly reflected into the chromatogramsThe complete mutation in ckit21T produced a dramaticreduction of the conformational polymorphism shown bythe wild sequence Besides variable-temperature HPLC hasbeen already used to study the conformational equilibria ofoligonucleotides [11] to our knowledge this is the first reporton the use of variable-temperature SEC to complement spec-troscopic information related to the G-quadruplex structure

2 Material and Methods

21 Reagents DNA stock solutions were prepared in MilliQwater stored atminus20∘C and diluted to working concentrationsin MilliQ water immediately before use The compositionof quadruplex stabilizing buffer (QSB) was 7mM KH

2PO4

10mM Na2HPO4 and 147mM KCl pH 72 Oligonucleotide

51015840-d[CG3CG3CGCGAG

3AG4]-31015840 designated as ckit21 was

a G-quadruplex forming element in the promoter regionof the human c-kit protooncogene Oligonucleotide 51015840-d[TG3TG3TGTGTG

3TG4]-31015840 designated as ckit21T was

a mutated sequence in which adenine and cytosine basesof the wild type ckit21 were systematically replaced withthymine Two additional sequences ckit21T18 and ckit21T21

were designed to freeze the dangling-end to blunt-end equi-librium These oligonucleotides were prepared as describedelsewhere [13] Oligonucleotide concentration was deter-mined by UV absorbance measurements at 260 nm usingcalculated extinction coefficients and the nearest-neighbormethod as implemented in OligoCalc webpage [14] Theintegrity of DNA samples was checked by means of MSmeasurements Oligonucleotides were annealed by raisingthe temperature to 90∘C for 10min and then cooling to roomtemperature overnight prior to melting or SEC measure-ments

22 Instrumentation and Procedures Absorbance and CDspectra were measured using an Agilent HP8453 photodiodearray spectrophotometer and a Jasco J-810 spectropolarime-ter respectively Both of them are equipped with stirrer andPeltier units mounted in the spindle of the thermoelectriccuvette holders Melting curves were collected on the J-810instrument The concentration of DNA samples was around3 120583M Samples were heated at a linear temperature ramp of03∘Cmin with data collection beginning at 25∘C and endingat 95∘C A blank dataset taken from the buffer solution alonewas also recorded and used for blank subtraction Meltingtemperature (119879

119898) values are the average value of at least a pair

of 119879119898values recorded during repeated melting experiments

The chromatographic system consisted of an Agilent 1100Series HPLC instrument equipped with a G1311A quater-nary pump a G1379A degasser a G1392A autosampler aG1315B photodiode array detector furnished with a 13120583Lflow cell and an Agilent ChemStation for data acquisi-tion and analysis (Rev A 1002) all of them from AgilentTechnologies (Waldbronn Germany) The temperature wascontrolled using an HPLC column heater GECKO 2000(CIL France) A Phenomenex BioSep-SEC-S 2000 column(300 times 78mm particle size of 5120583m and pore of size 145 A)

ISRN Biochemistry 3

from Phenomenex (Torrance CA USA) was used for thechromatographic separation The mobile phase was a pH 71buffer (01M potassium phosphate) The flow rate was set to10mLmin A volume of 15120583L of the sample (5120583M in DNA)was injected and the temperature was set to 20 30 and 40∘CAbsorbance spectra were recorded between 200 and 500 nm

23 Data Analysis In this study the analysis of multivariatedata recorded along the melting experiments was done usinghouse-made software based on the implementation of thevanrsquot Hoff equation [15]

Spectra recorded along melting experiments werearranged in a table or data matrix whose size was 119898 rows(spectra recorded) and 119899 columns (wavelengths measured)

The goal of the data analysis was to calculate the distri-bution diagrams and pure (individual) spectra for all the 119899119888spectroscopically active species considered throughout theexperiment The distribution diagram provides informationabout the thermodynamics of the melting processes Inaddition the shape and intensity of the pure spectra mayprovide qualitative information about the structure of thespecies

With this goal in mind data matrix D was decomposedaccording to Beer-Lambert-Bouguerrsquos law in matrix form

D = CST + E (1)

where C is the matrix (119898 times 119899119888) containing the distributiondiagram ST is thematrix (119899119888times119899) containing the pure spectraand E is the matrix of data (119898 times 119899) not explained by theproposed decomposition

The mathematical decomposition of D into matrices CST and E may be done basically in two different waysdepending on whether a physicochemical model is initiallyproposed (hard-modeling approach) or not (soft-modelingapproach) In this work a hard-modeling approach hasbeen applied In this case the physicochemical model isrelated to the thermodynamics of DNA unfolding Hencefor the unfolding of intramolecular structures such as thosestudied here the chemical equation and the correspondingequilibrium constant may be written as

DNAfolded + heatlarrrarr DNAunfolded

Kunfolding =[DNAunfolded]

[DNAfolded]

(2)

The concentration of the folded and unfolded forms istemperature-dependent Accordingly the equilibrium con-stant depends on temperature according to the vanrsquot Hoffequation

ln119870unfolding = minusΔ119867V119867

119877119879

+

Δ119878V119867

119877

(3)

In this case it is assumed that Δ119867V119867 and Δ119878V119867 willnot change throughout the range of temperatures studiedhere Whenever a physicochemical model is applied thedistribution diagram inC complies with the proposedmodel

Accordingly the proposed values for the equilibrium con-stants and the shape of the pure spectra in ST are refinedto explain satisfactorily data in D whereas residuals in Eare minimized Hard modeling of melting experiments wasdone with a modified version of the MCR-ALS procedurethat includes the model proposed in (3) for the unfolding ofintramolecular structures

3 Results and Discussion

Figure 2 shows the SEC chromatograms and CD spectra(insets) recorded for all four sequences at 20 30 and 40∘CHigher temperatures could not be achieved due to thelimitations of the chromatographic column DNA sampleswere prepared by direct dilution from a stock solution heatedat 95∘C for 10 minutes and slowly cooled overnight beforemeasurements were done

The shape of the CD spectrum of a ckit21 samplemeasured at pH 7 and 20∘C showed the typical featuresrelated to parallel G-quadruplexes like a large positive bandaround 265 nm and a negative band around 240 nm Thesmall shoulder at 295 nm may reflect a certain contributionof antiparallel structure The CD spectrum of ckit21T on thecontrary did not show any contribution at 295 nm reflectinga clear parallel conformation The CD spectrum of ckit21T18at pH 7 and 25∘C was clearly different from that measuredfor ckit21 because it showed two large positive bands around265 and 295 nm which denoted a mixed antiparallelparalleltopology in the same molecule or the presence of a mixtureof structures in different molecules Finally the CD spectrumof ckit21T21 was similar to that measured for ckit21 as itcontained a large positive band centered at 265 nm whichdenoted a major parallel topology and a small shoulder at295 nm

Melting experiments simultaneously monitored with CDand molecular absorption spectroscopies were carried outto evaluate the relative stability of the structures formedby the ckit21-based sequences Analysis of the whole set ofspectra recorded along the melting experiments was doneby means of a multivariate data analysis method [15] Thenumber of species present along the melting process aswell as the associated thermodynamic parameters (119879

119898and

changes in enthalpy and entropy) was determined in this wayUpon heating all four G-quadruplex structures unfoldedwith 119879

119898values equal to 74∘C (ckit21) 82∘C (ckit21T)

66∘C (ckit21T18) and 71∘C (ckit21T21) The unfolding ofthe ckit21T18 clearly took place in two steps (Figure 3)Upon heating the band at 295 nm was completely lost at55∘C whereas the intensity of the band at 265 nm increaseddramatically At this point the parallel structure was clearlythe major one Finally this structure was lost after heatingup to 90∘C The determined value of 119879

119898was 66 plusmn 1∘C

which was clearly lower than for ckit21 It seems that theexistence of a two-base third loop produced the shift ofthe parallel to antiparallel equilibrium proposed by Hsuet al to this last position [6] Thermodynamic parameterscalculated from melting experiments are reported in Table 1

4 ISRN Biochemistry

6 7 8 9 10 11 12012345678

Retention time (min)

Abso

rban

ce (m

AU)

220 240 260 280 300 320minus5

0

5

10

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

times106

(a)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus4

minus2

0

2

4

6

8times10

6

Retention time (min)

Abso

rban

ce (m

AU)

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(b)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus3minus2minus1

012345times10

6

Wavelength (nm)

Retention time (min)

Abso

rban

ce (m

AU)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(c)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus4minus2

02

46

810

Retention time (min)

Abso

rban

ce (m

AU)

times106

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(d)

Figure 2 SEC profiles and CD spectra recorded for ckit21 (a) ckit21T (b) ckit21T18 (c) and ckit21T21 (d) at 21 (blue) 30 (green) and 40∘C(red) and pH 71

Table 1Thermodynamic parameters related to the unfolding of the three sequences studied in this work Experiments have been performedat least three times Parameters are done in terms of mean value and standard deviation

Sequence Δ119867

0 (kcalsdotmolminus1) Δ119878

0 (calsdotKminus1 sdotmolminus1) Δ119866

0 (kcalsdotmolminus1) at 37∘C 119879

119898(∘C) Reference

ckit21 51 plusmn 3 145 plusmn 9 54 plusmn 06 74 plusmn 1 This workckit21T 46 130 58 82 [12]ckit21T18 44 plusmn 2 131 plusmn 3 38 plusmn 04 66 plusmn 2 This workckit21T21 46 plusmn 4 135 plusmn 12 45 plusmn 04 71 plusmn 1 This work

The native sequence showed the highest values for the ther-modynamic parameters which reflected the higher stabilityof this sequence in front of the mutated ones The standardchange in free energy comes from the high values of standardchanges both in enthalpy and in entropy On the contrarythe ckit21T18 sequence showed the lowest values whereasckit21T21 showed intermediate values From the data shownin this table it was clear that the native sequence was themost stable at 37∘C Also it seems that the G18T mutationreduced dramatically the stability of the structure It is knownthat the formation of a tetrad implies the release of about15ndash25 kcalmol depending on factors such as a stacking ofadditional bases on the tetrad In our case unfolding of theckit21 sequence required a change in enthalpy per tetradof 17 kcalmol Overall unfolding of this sequence requiredaround 6 kcalmol more than those of the mutated sequencesckit21T18 and ckit21T21 This difference could be due to the

additional stacking of the terminal guanine at the 31015840 endin the wild sequence In the case of ckit21T21 the limitedcontribution of the terminal thymine bases to the overallstability of quadruplex structures had been already pointedout [1]

CD spectroscopy is a low-resolution technique whichdoes not allow discerning completely the presence of mix-tures or mixed conformations On the other hand therecorded SEC chromatograms may reveal clear differencesamong the conformational space spanned by the four dif-ferent sequences Overall SEC results allowed a qualitativecorrelation with the results obtained from spectroscopy

The chromatogram of the ckitG4 was the simplest amongthe four considered because the recorded peak at 85minutesis almost Gaussian-shaped and it lacks minor peaks at lowerand higher elution times than the principal peak Only ashoulder may be glimpsed at around 8 minutes According

ISRN Biochemistry 5

220 230 240 250 260 270 280 290 300 310 320

minus5

0

5

10

15

20

Wavelength (nm)

Ellip

ticity

(mde

g)

(a)

220 230 240 250 260 270 280 290 300 310 3200

0102030405060708

Wavelength (nm)

Abso

rban

ce (m

AU)

(b)

20 30 40 50 60 70 80 900

051

152

253

Con

cent

ratio

n (M

)

Temperature (∘C)

times10minus6

(c)

220 230 240 250 260 270 280 290 300 310 320minus2

0246

220 230 240 250 260 270 280 290 300 310 3200

1

2

Wavelength (nm)

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

olminus1)

Mol

ar ab

sorp

tivity

(cm

2m

olminus1)

times106

times105

(d)

Figure 3 Melting experiment of ckit21T18 monitored simultaneously by CD and molecular absorption spectroscopies (a) ExperimentalCD spectra (b) Experimental molecular absorption spectra (c) Calculated distribution diagram (d) Calculated pure CD and molecularabsorption spectra Concentration of DNA equal to 33 microM pH 71 and 150mM ionic strength

45

44

43

42

41

40

39

38

37

36

3564 66 68 70 72 74 76 78

Retention time (min)

log

(MW

)

y = minus05717x + 80943

R2 = 09856

Figure 4 Calibration of chromatographic system The oligonucleotides injected are 51015840-T21-31015840 (MW 63262 gsdotmolminus1) 51015840-T

24-31015840 (MW

72387 gsdotmolminus1) 51015840-ACC CCC TGC ATC TGC ATG CCC CCT CCC ACC CCC T-31015840 (MW 100575 gsdotmolminus1) 51015840-ACC CCC TGC ATC TTTTTG CCC CCT CCC ACC CCC T-31015840 (MW 100765 gsdotmolminus1) and the Watson-Crick duplex formed by 51015840-ACC CCC TGC ATC TGC ATGCCC CCT CCC ACC CCC T-31015840 and 51015840-AGG GGG TGG GAG GGG GCA TGC AGA TGC AGG GGG T-31015840 (MW 208727 gsdotmolminus1) All ofthese sequences form lineal structures at pH 71 and 20∘C

to the SEC calibration with lineal structures (Figure 4) thelinear structure of ckit21T should be eluted at around 75minutes Accordingly the main peak at 85 minutes wasrelated to a folded structure whose hydrodynamic volumewas smaller than that corresponding to the linear structureAccording to the CD spectrum the folded structure wasrelated to a parallel G-quadruplex The presence of a single

almost-Gaussian SEC band was correlated with the clearparallel G-quadruplex conformation detected by CD In thetemperature range tested no clear changes were observable

In contrast to ckit21T the wild ckit21 sequence showeda more complex conformational space reflected in thepresence of two main peaks at 72 and 88 minutes Asbefore the peak at 86 minutes was related to the parallel

6 ISRN Biochemistry

G-quadruplex On the other hand the peak at 72 minutesmay be associated with some kind of multimeric specieslike dimers [16] The mutation of cytosine and adenine bythymine bases in ckit21T seems to prevent the formation ofsuch aggregates As reflected in the CD spectra the shape ofthe SEC chromatograms hardly changes upon heating

Concomitant with the features observed in the CDspectrum at 20∘C the chromatogram of ckit21T18 was theone that showed the most striking differences in relationto the other sequences The chromatogram showed a largepeak at 85 minutes and two small peaks at 71 and 105minutes According to the previous results these peakswere related to the parallel G-quadruplex aggregates andthe antiparallel conformation respectively Upon heatingthe peak at 105 minutes slowly disappeared to merge withthat corresponding to the G-quadruplex This behavior wasconcomitant with that observed in CD spectroscopy and wasrelated to the shift of the conformational equilibrium to theparallel conformation Based on the SEC results it seems thatthe CD spectrum at 20∘C is related to a mixture of speciesshowing parallel and antiparallel conformations rather thanwith a unique species showing a mixed conformation

Finally the chromatogram of ckit21T21 showed threemain peaks at 72 81 and 89 minutesThis profile was quali-tatively similar to that of the wild sequence which again wasin accordance with both CD and melting experiments Onlythe presence of a peak at 81 minutes makes the differencebetween both sequences These results agree qualitativelywith those obtained at higher concentration of DNA [8]

4 Conclusions

Several conclusions may be drawn from the results shownhere Firstly the utility of variable-temperature SEC chro-matography as a useful tool to study the conformationalequilibria of G-quadruplexes has been shownThe chromato-graphic profilesmay be qualitatively related to those obtainedbymeans of other techniques such asCD Secondlymutationof bases at the loops produces dramatic changes in the confor-mational equilibria The assumption that four guanine tractsof equal length produce conformational homogeneity maynot be true andmust be checked bymeans of complementarytechniques such as SEC

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors acknowledge funding from the Spanish Gov-ernment (CTQ2012-38616-C02-02 andCTQ2010-20541-C03-01)

References

[1] J L Mergny J Gros A D Cian et al ldquoEnergetics kinetics anddynamics of quadruplex foldingrdquo in Quadruplex Nucleic AcidsS Neidle and S Balasubramanian Eds pp 31ndash72 The RoyalSociety of Chemistry Cambridge UK 2006

[2] J Jaumot and R Gargallo ldquoExperimental methods for studyingthe interactions between G-quadruplex structures and ligandsrdquoCurrent Pharmaceutical Design vol 18 no 14 pp 1900ndash19162012

[3] D Miyoshi A Nakao and N Sugimoto ldquoStructural transi-tion from antiparallel to parallel G-quadruplex of d(G

4T4G4)

induced by Ca2+rdquoNucleic Acids Research vol 31 no 4 pp 1156ndash1163 2003

[4] G Biffi D Tannahill J McCafferty and S BalasubramanianldquoQuantitative visualization of DNAG-quadruplex structures inhuman cellsrdquo Nature Chemistry vol 5 pp 182ndash186 2013

[5] H Fernando A P Reszka J Huppert et al ldquoA conservedquadruplexmotif located in a transcription activation site of thehuman c-kit oncogenerdquo Biochemistry vol 45 no 25 pp 7854ndash7860 2006

[6] S-T D Hsu P Varnai A Bugaut A P Reszka S Neidleand S Balasubramanian ldquoA G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex havingasymmetric G-tetrad dynamicsrdquo Journal of the American Chem-ical Society vol 131 no 37 pp 13399ndash13409 2009

[7] V Kuryavyi A T Phan and D J Patel ldquoSolution structuresof all parallel-stranded monomeric and dimeric G-quadruplexscaffolds of the human c-kit2 promoterrdquoNucleic Acids Researchvol 38 no 19 pp 6757ndash6773 2010

[8] M C Miller H T Le W L Dean P A Holt J B Chairesand J O Trent ldquoPolymorphism and resolution of oncogene pro-moter quadruplex-forming sequencesrdquo Organic and Biomolec-ular Chemistry vol 9 no 22 pp 7633ndash7637 2011

[9] H T Le M C Miller R Buscaglia et al ldquoNot all G-quadruplexes are created equally an investigation of thestructural polymorphism of the c-Myc G-quadruplex-formingsequence and its interaction with the porphyrin TMPyP4rdquoOrganic ampBiomolecular Chemistry vol 10 pp 9393ndash9404 2012

[10] L Spindler M Rigler I Drevensek-Olenik N Marsquoani Hessariand M Webba da Silva ldquoEffect of base sequence on G-wireformation in solutionrdquo Journal of Nucleic Acids vol 2010Article ID 431651 8 pages 2010

[11] W H Braunlin I Giri L Beadling and K J BreslauerldquoConformational screening of oligonucleotides by variable-temperature high performance liquid chromatographydissecting the duplex-hairpin-coil equilibria ofd(CGCGAATTCGCG)rdquo Biopolymers vol 74 no 3 pp221ndash231 2004

[12] P Bucek J Jaumot A Avino R Eritja and R GargalloldquoPH-modulatedWatson-Crick duplex-quadruplex equilibria ofguanine-rich and cytosine-rich DNA sequences 140 base pairsupstream of the c-kit transcription initiation siterdquo Chemistryvol 15 no 46 pp 12663ndash12671 2009

[13] N Khan A Avino R Tauler C Gonzalez R Eritja and RGargallo ldquoSolution equilibria of the i-motif-forming regionupstream of the B-cell lymphoma-2 P1 promoterrdquo Biochimievol 89 no 12 pp 1562ndash1572 2007

[14] W A Kibbe ldquoOligoCalc an online oligonucleotide propertiescalculatorrdquoNucleic Acids Research vol 35 ppW43ndashW46 2007

[15] S Fernandez R Eritja A Avino J Jaumot and R GargalloldquoInfluence of pH temperature and the cationic porphyrin

ISRN Biochemistry 7

TMPyP4 on the stability of the i-motif formed by the 51015840-(C3TA2)4-31015840 sequence of the human telomererdquo InternationalJournal of Biological Macromolecules vol 49 no 4 pp 729ndash7362011

[16] N Smargiasso F Rosu W Hsia et al ldquoG-quadruplex DNAassemblies loop length cation identity and multimer forma-tionrdquo Journal of the American Chemical Society vol 130 no 31pp 10208ndash10216 2008

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2 ISRN Biochemistry

Sequence 5998400 L1 L2 L3 3998400

ckit21T21

ckit21

ckit21T18

ckit21T

C GA

A

C

C T

C

C

C CGCGA

CGCGA

CGCGA GGG AT

T GGG T GGG TGTGT GGG T GGG G

GGG

GGG

GGG

GGG

GGG

GGG

GGG

GGG

GGG

GGG

GGG

(a)

C1 C5

G4

A17G18

G19

G20

G21

G16

G14 C1C5

G4A17

G18G19

G20

G21

G16

G14

(b)

Figure 1 (a) Sequences studied in this work (b) Dangling- to blunt-end equilibrium in ckit21

formation of a particular quadruplex is dependent on manyfactors not only on the sequence [9 10]

In the present work the effect of several mutations onthe ckit21 sequence by means of CD and SEC measurementsdone at different temperatures was studied The DNA con-centration used for both measurements was around 2ndash5120583M(the order of magnitude used in most of the spectroscopicmeasurements) and the K+ concentration was 150mM Inaddition to the three sequences already mentioned (ckit21ckit21T18 and ckit21T21) a fourth sequence (ckit21T) whereall cytosine and adenine bases were mutated to thymine wasstudied The results showed that the mutation in appropri-ate locations was clearly reflected into the chromatogramsThe complete mutation in ckit21T produced a dramaticreduction of the conformational polymorphism shown bythe wild sequence Besides variable-temperature HPLC hasbeen already used to study the conformational equilibria ofoligonucleotides [11] to our knowledge this is the first reporton the use of variable-temperature SEC to complement spec-troscopic information related to the G-quadruplex structure

2 Material and Methods

21 Reagents DNA stock solutions were prepared in MilliQwater stored atminus20∘C and diluted to working concentrationsin MilliQ water immediately before use The compositionof quadruplex stabilizing buffer (QSB) was 7mM KH

2PO4

10mM Na2HPO4 and 147mM KCl pH 72 Oligonucleotide

51015840-d[CG3CG3CGCGAG

3AG4]-31015840 designated as ckit21 was

a G-quadruplex forming element in the promoter regionof the human c-kit protooncogene Oligonucleotide 51015840-d[TG3TG3TGTGTG

3TG4]-31015840 designated as ckit21T was

a mutated sequence in which adenine and cytosine basesof the wild type ckit21 were systematically replaced withthymine Two additional sequences ckit21T18 and ckit21T21

were designed to freeze the dangling-end to blunt-end equi-librium These oligonucleotides were prepared as describedelsewhere [13] Oligonucleotide concentration was deter-mined by UV absorbance measurements at 260 nm usingcalculated extinction coefficients and the nearest-neighbormethod as implemented in OligoCalc webpage [14] Theintegrity of DNA samples was checked by means of MSmeasurements Oligonucleotides were annealed by raisingthe temperature to 90∘C for 10min and then cooling to roomtemperature overnight prior to melting or SEC measure-ments

22 Instrumentation and Procedures Absorbance and CDspectra were measured using an Agilent HP8453 photodiodearray spectrophotometer and a Jasco J-810 spectropolarime-ter respectively Both of them are equipped with stirrer andPeltier units mounted in the spindle of the thermoelectriccuvette holders Melting curves were collected on the J-810instrument The concentration of DNA samples was around3 120583M Samples were heated at a linear temperature ramp of03∘Cmin with data collection beginning at 25∘C and endingat 95∘C A blank dataset taken from the buffer solution alonewas also recorded and used for blank subtraction Meltingtemperature (119879

119898) values are the average value of at least a pair

of 119879119898values recorded during repeated melting experiments

The chromatographic system consisted of an Agilent 1100Series HPLC instrument equipped with a G1311A quater-nary pump a G1379A degasser a G1392A autosampler aG1315B photodiode array detector furnished with a 13120583Lflow cell and an Agilent ChemStation for data acquisi-tion and analysis (Rev A 1002) all of them from AgilentTechnologies (Waldbronn Germany) The temperature wascontrolled using an HPLC column heater GECKO 2000(CIL France) A Phenomenex BioSep-SEC-S 2000 column(300 times 78mm particle size of 5120583m and pore of size 145 A)

ISRN Biochemistry 3

from Phenomenex (Torrance CA USA) was used for thechromatographic separation The mobile phase was a pH 71buffer (01M potassium phosphate) The flow rate was set to10mLmin A volume of 15120583L of the sample (5120583M in DNA)was injected and the temperature was set to 20 30 and 40∘CAbsorbance spectra were recorded between 200 and 500 nm

23 Data Analysis In this study the analysis of multivariatedata recorded along the melting experiments was done usinghouse-made software based on the implementation of thevanrsquot Hoff equation [15]

Spectra recorded along melting experiments werearranged in a table or data matrix whose size was 119898 rows(spectra recorded) and 119899 columns (wavelengths measured)

The goal of the data analysis was to calculate the distri-bution diagrams and pure (individual) spectra for all the 119899119888spectroscopically active species considered throughout theexperiment The distribution diagram provides informationabout the thermodynamics of the melting processes Inaddition the shape and intensity of the pure spectra mayprovide qualitative information about the structure of thespecies

With this goal in mind data matrix D was decomposedaccording to Beer-Lambert-Bouguerrsquos law in matrix form

D = CST + E (1)

where C is the matrix (119898 times 119899119888) containing the distributiondiagram ST is thematrix (119899119888times119899) containing the pure spectraand E is the matrix of data (119898 times 119899) not explained by theproposed decomposition

The mathematical decomposition of D into matrices CST and E may be done basically in two different waysdepending on whether a physicochemical model is initiallyproposed (hard-modeling approach) or not (soft-modelingapproach) In this work a hard-modeling approach hasbeen applied In this case the physicochemical model isrelated to the thermodynamics of DNA unfolding Hencefor the unfolding of intramolecular structures such as thosestudied here the chemical equation and the correspondingequilibrium constant may be written as

DNAfolded + heatlarrrarr DNAunfolded

Kunfolding =[DNAunfolded]

[DNAfolded]

(2)

The concentration of the folded and unfolded forms istemperature-dependent Accordingly the equilibrium con-stant depends on temperature according to the vanrsquot Hoffequation

ln119870unfolding = minusΔ119867V119867

119877119879

+

Δ119878V119867

119877

(3)

In this case it is assumed that Δ119867V119867 and Δ119878V119867 willnot change throughout the range of temperatures studiedhere Whenever a physicochemical model is applied thedistribution diagram inC complies with the proposedmodel

Accordingly the proposed values for the equilibrium con-stants and the shape of the pure spectra in ST are refinedto explain satisfactorily data in D whereas residuals in Eare minimized Hard modeling of melting experiments wasdone with a modified version of the MCR-ALS procedurethat includes the model proposed in (3) for the unfolding ofintramolecular structures

3 Results and Discussion

Figure 2 shows the SEC chromatograms and CD spectra(insets) recorded for all four sequences at 20 30 and 40∘CHigher temperatures could not be achieved due to thelimitations of the chromatographic column DNA sampleswere prepared by direct dilution from a stock solution heatedat 95∘C for 10 minutes and slowly cooled overnight beforemeasurements were done

The shape of the CD spectrum of a ckit21 samplemeasured at pH 7 and 20∘C showed the typical featuresrelated to parallel G-quadruplexes like a large positive bandaround 265 nm and a negative band around 240 nm Thesmall shoulder at 295 nm may reflect a certain contributionof antiparallel structure The CD spectrum of ckit21T on thecontrary did not show any contribution at 295 nm reflectinga clear parallel conformation The CD spectrum of ckit21T18at pH 7 and 25∘C was clearly different from that measuredfor ckit21 because it showed two large positive bands around265 and 295 nm which denoted a mixed antiparallelparalleltopology in the same molecule or the presence of a mixtureof structures in different molecules Finally the CD spectrumof ckit21T21 was similar to that measured for ckit21 as itcontained a large positive band centered at 265 nm whichdenoted a major parallel topology and a small shoulder at295 nm

Melting experiments simultaneously monitored with CDand molecular absorption spectroscopies were carried outto evaluate the relative stability of the structures formedby the ckit21-based sequences Analysis of the whole set ofspectra recorded along the melting experiments was doneby means of a multivariate data analysis method [15] Thenumber of species present along the melting process aswell as the associated thermodynamic parameters (119879

119898and

changes in enthalpy and entropy) was determined in this wayUpon heating all four G-quadruplex structures unfoldedwith 119879

119898values equal to 74∘C (ckit21) 82∘C (ckit21T)

66∘C (ckit21T18) and 71∘C (ckit21T21) The unfolding ofthe ckit21T18 clearly took place in two steps (Figure 3)Upon heating the band at 295 nm was completely lost at55∘C whereas the intensity of the band at 265 nm increaseddramatically At this point the parallel structure was clearlythe major one Finally this structure was lost after heatingup to 90∘C The determined value of 119879

119898was 66 plusmn 1∘C

which was clearly lower than for ckit21 It seems that theexistence of a two-base third loop produced the shift ofthe parallel to antiparallel equilibrium proposed by Hsuet al to this last position [6] Thermodynamic parameterscalculated from melting experiments are reported in Table 1

4 ISRN Biochemistry

6 7 8 9 10 11 12012345678

Retention time (min)

Abso

rban

ce (m

AU)

220 240 260 280 300 320minus5

0

5

10

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

times106

(a)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus4

minus2

0

2

4

6

8times10

6

Retention time (min)

Abso

rban

ce (m

AU)

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(b)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus3minus2minus1

012345times10

6

Wavelength (nm)

Retention time (min)

Abso

rban

ce (m

AU)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(c)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus4minus2

02

46

810

Retention time (min)

Abso

rban

ce (m

AU)

times106

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(d)

Figure 2 SEC profiles and CD spectra recorded for ckit21 (a) ckit21T (b) ckit21T18 (c) and ckit21T21 (d) at 21 (blue) 30 (green) and 40∘C(red) and pH 71

Table 1Thermodynamic parameters related to the unfolding of the three sequences studied in this work Experiments have been performedat least three times Parameters are done in terms of mean value and standard deviation

Sequence Δ119867

0 (kcalsdotmolminus1) Δ119878

0 (calsdotKminus1 sdotmolminus1) Δ119866

0 (kcalsdotmolminus1) at 37∘C 119879

119898(∘C) Reference

ckit21 51 plusmn 3 145 plusmn 9 54 plusmn 06 74 plusmn 1 This workckit21T 46 130 58 82 [12]ckit21T18 44 plusmn 2 131 plusmn 3 38 plusmn 04 66 plusmn 2 This workckit21T21 46 plusmn 4 135 plusmn 12 45 plusmn 04 71 plusmn 1 This work

The native sequence showed the highest values for the ther-modynamic parameters which reflected the higher stabilityof this sequence in front of the mutated ones The standardchange in free energy comes from the high values of standardchanges both in enthalpy and in entropy On the contrarythe ckit21T18 sequence showed the lowest values whereasckit21T21 showed intermediate values From the data shownin this table it was clear that the native sequence was themost stable at 37∘C Also it seems that the G18T mutationreduced dramatically the stability of the structure It is knownthat the formation of a tetrad implies the release of about15ndash25 kcalmol depending on factors such as a stacking ofadditional bases on the tetrad In our case unfolding of theckit21 sequence required a change in enthalpy per tetradof 17 kcalmol Overall unfolding of this sequence requiredaround 6 kcalmol more than those of the mutated sequencesckit21T18 and ckit21T21 This difference could be due to the

additional stacking of the terminal guanine at the 31015840 endin the wild sequence In the case of ckit21T21 the limitedcontribution of the terminal thymine bases to the overallstability of quadruplex structures had been already pointedout [1]

CD spectroscopy is a low-resolution technique whichdoes not allow discerning completely the presence of mix-tures or mixed conformations On the other hand therecorded SEC chromatograms may reveal clear differencesamong the conformational space spanned by the four dif-ferent sequences Overall SEC results allowed a qualitativecorrelation with the results obtained from spectroscopy

The chromatogram of the ckitG4 was the simplest amongthe four considered because the recorded peak at 85minutesis almost Gaussian-shaped and it lacks minor peaks at lowerand higher elution times than the principal peak Only ashoulder may be glimpsed at around 8 minutes According

ISRN Biochemistry 5

220 230 240 250 260 270 280 290 300 310 320

minus5

0

5

10

15

20

Wavelength (nm)

Ellip

ticity

(mde

g)

(a)

220 230 240 250 260 270 280 290 300 310 3200

0102030405060708

Wavelength (nm)

Abso

rban

ce (m

AU)

(b)

20 30 40 50 60 70 80 900

051

152

253

Con

cent

ratio

n (M

)

Temperature (∘C)

times10minus6

(c)

220 230 240 250 260 270 280 290 300 310 320minus2

0246

220 230 240 250 260 270 280 290 300 310 3200

1

2

Wavelength (nm)

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

olminus1)

Mol

ar ab

sorp

tivity

(cm

2m

olminus1)

times106

times105

(d)

Figure 3 Melting experiment of ckit21T18 monitored simultaneously by CD and molecular absorption spectroscopies (a) ExperimentalCD spectra (b) Experimental molecular absorption spectra (c) Calculated distribution diagram (d) Calculated pure CD and molecularabsorption spectra Concentration of DNA equal to 33 microM pH 71 and 150mM ionic strength

45

44

43

42

41

40

39

38

37

36

3564 66 68 70 72 74 76 78

Retention time (min)

log

(MW

)

y = minus05717x + 80943

R2 = 09856

Figure 4 Calibration of chromatographic system The oligonucleotides injected are 51015840-T21-31015840 (MW 63262 gsdotmolminus1) 51015840-T

24-31015840 (MW

72387 gsdotmolminus1) 51015840-ACC CCC TGC ATC TGC ATG CCC CCT CCC ACC CCC T-31015840 (MW 100575 gsdotmolminus1) 51015840-ACC CCC TGC ATC TTTTTG CCC CCT CCC ACC CCC T-31015840 (MW 100765 gsdotmolminus1) and the Watson-Crick duplex formed by 51015840-ACC CCC TGC ATC TGC ATGCCC CCT CCC ACC CCC T-31015840 and 51015840-AGG GGG TGG GAG GGG GCA TGC AGA TGC AGG GGG T-31015840 (MW 208727 gsdotmolminus1) All ofthese sequences form lineal structures at pH 71 and 20∘C

to the SEC calibration with lineal structures (Figure 4) thelinear structure of ckit21T should be eluted at around 75minutes Accordingly the main peak at 85 minutes wasrelated to a folded structure whose hydrodynamic volumewas smaller than that corresponding to the linear structureAccording to the CD spectrum the folded structure wasrelated to a parallel G-quadruplex The presence of a single

almost-Gaussian SEC band was correlated with the clearparallel G-quadruplex conformation detected by CD In thetemperature range tested no clear changes were observable

In contrast to ckit21T the wild ckit21 sequence showeda more complex conformational space reflected in thepresence of two main peaks at 72 and 88 minutes Asbefore the peak at 86 minutes was related to the parallel

6 ISRN Biochemistry

G-quadruplex On the other hand the peak at 72 minutesmay be associated with some kind of multimeric specieslike dimers [16] The mutation of cytosine and adenine bythymine bases in ckit21T seems to prevent the formation ofsuch aggregates As reflected in the CD spectra the shape ofthe SEC chromatograms hardly changes upon heating

Concomitant with the features observed in the CDspectrum at 20∘C the chromatogram of ckit21T18 was theone that showed the most striking differences in relationto the other sequences The chromatogram showed a largepeak at 85 minutes and two small peaks at 71 and 105minutes According to the previous results these peakswere related to the parallel G-quadruplex aggregates andthe antiparallel conformation respectively Upon heatingthe peak at 105 minutes slowly disappeared to merge withthat corresponding to the G-quadruplex This behavior wasconcomitant with that observed in CD spectroscopy and wasrelated to the shift of the conformational equilibrium to theparallel conformation Based on the SEC results it seems thatthe CD spectrum at 20∘C is related to a mixture of speciesshowing parallel and antiparallel conformations rather thanwith a unique species showing a mixed conformation

Finally the chromatogram of ckit21T21 showed threemain peaks at 72 81 and 89 minutesThis profile was quali-tatively similar to that of the wild sequence which again wasin accordance with both CD and melting experiments Onlythe presence of a peak at 81 minutes makes the differencebetween both sequences These results agree qualitativelywith those obtained at higher concentration of DNA [8]

4 Conclusions

Several conclusions may be drawn from the results shownhere Firstly the utility of variable-temperature SEC chro-matography as a useful tool to study the conformationalequilibria of G-quadruplexes has been shownThe chromato-graphic profilesmay be qualitatively related to those obtainedbymeans of other techniques such asCD Secondlymutationof bases at the loops produces dramatic changes in the confor-mational equilibria The assumption that four guanine tractsof equal length produce conformational homogeneity maynot be true andmust be checked bymeans of complementarytechniques such as SEC

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors acknowledge funding from the Spanish Gov-ernment (CTQ2012-38616-C02-02 andCTQ2010-20541-C03-01)

References

[1] J L Mergny J Gros A D Cian et al ldquoEnergetics kinetics anddynamics of quadruplex foldingrdquo in Quadruplex Nucleic AcidsS Neidle and S Balasubramanian Eds pp 31ndash72 The RoyalSociety of Chemistry Cambridge UK 2006

[2] J Jaumot and R Gargallo ldquoExperimental methods for studyingthe interactions between G-quadruplex structures and ligandsrdquoCurrent Pharmaceutical Design vol 18 no 14 pp 1900ndash19162012

[3] D Miyoshi A Nakao and N Sugimoto ldquoStructural transi-tion from antiparallel to parallel G-quadruplex of d(G

4T4G4)

induced by Ca2+rdquoNucleic Acids Research vol 31 no 4 pp 1156ndash1163 2003

[4] G Biffi D Tannahill J McCafferty and S BalasubramanianldquoQuantitative visualization of DNAG-quadruplex structures inhuman cellsrdquo Nature Chemistry vol 5 pp 182ndash186 2013

[5] H Fernando A P Reszka J Huppert et al ldquoA conservedquadruplexmotif located in a transcription activation site of thehuman c-kit oncogenerdquo Biochemistry vol 45 no 25 pp 7854ndash7860 2006

[6] S-T D Hsu P Varnai A Bugaut A P Reszka S Neidleand S Balasubramanian ldquoA G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex havingasymmetric G-tetrad dynamicsrdquo Journal of the American Chem-ical Society vol 131 no 37 pp 13399ndash13409 2009

[7] V Kuryavyi A T Phan and D J Patel ldquoSolution structuresof all parallel-stranded monomeric and dimeric G-quadruplexscaffolds of the human c-kit2 promoterrdquoNucleic Acids Researchvol 38 no 19 pp 6757ndash6773 2010

[8] M C Miller H T Le W L Dean P A Holt J B Chairesand J O Trent ldquoPolymorphism and resolution of oncogene pro-moter quadruplex-forming sequencesrdquo Organic and Biomolec-ular Chemistry vol 9 no 22 pp 7633ndash7637 2011

[9] H T Le M C Miller R Buscaglia et al ldquoNot all G-quadruplexes are created equally an investigation of thestructural polymorphism of the c-Myc G-quadruplex-formingsequence and its interaction with the porphyrin TMPyP4rdquoOrganic ampBiomolecular Chemistry vol 10 pp 9393ndash9404 2012

[10] L Spindler M Rigler I Drevensek-Olenik N Marsquoani Hessariand M Webba da Silva ldquoEffect of base sequence on G-wireformation in solutionrdquo Journal of Nucleic Acids vol 2010Article ID 431651 8 pages 2010

[11] W H Braunlin I Giri L Beadling and K J BreslauerldquoConformational screening of oligonucleotides by variable-temperature high performance liquid chromatographydissecting the duplex-hairpin-coil equilibria ofd(CGCGAATTCGCG)rdquo Biopolymers vol 74 no 3 pp221ndash231 2004

[12] P Bucek J Jaumot A Avino R Eritja and R GargalloldquoPH-modulatedWatson-Crick duplex-quadruplex equilibria ofguanine-rich and cytosine-rich DNA sequences 140 base pairsupstream of the c-kit transcription initiation siterdquo Chemistryvol 15 no 46 pp 12663ndash12671 2009

[13] N Khan A Avino R Tauler C Gonzalez R Eritja and RGargallo ldquoSolution equilibria of the i-motif-forming regionupstream of the B-cell lymphoma-2 P1 promoterrdquo Biochimievol 89 no 12 pp 1562ndash1572 2007

[14] W A Kibbe ldquoOligoCalc an online oligonucleotide propertiescalculatorrdquoNucleic Acids Research vol 35 ppW43ndashW46 2007

[15] S Fernandez R Eritja A Avino J Jaumot and R GargalloldquoInfluence of pH temperature and the cationic porphyrin

ISRN Biochemistry 7

TMPyP4 on the stability of the i-motif formed by the 51015840-(C3TA2)4-31015840 sequence of the human telomererdquo InternationalJournal of Biological Macromolecules vol 49 no 4 pp 729ndash7362011

[16] N Smargiasso F Rosu W Hsia et al ldquoG-quadruplex DNAassemblies loop length cation identity and multimer forma-tionrdquo Journal of the American Chemical Society vol 130 no 31pp 10208ndash10216 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Biochemistry 3

from Phenomenex (Torrance CA USA) was used for thechromatographic separation The mobile phase was a pH 71buffer (01M potassium phosphate) The flow rate was set to10mLmin A volume of 15120583L of the sample (5120583M in DNA)was injected and the temperature was set to 20 30 and 40∘CAbsorbance spectra were recorded between 200 and 500 nm

23 Data Analysis In this study the analysis of multivariatedata recorded along the melting experiments was done usinghouse-made software based on the implementation of thevanrsquot Hoff equation [15]

Spectra recorded along melting experiments werearranged in a table or data matrix whose size was 119898 rows(spectra recorded) and 119899 columns (wavelengths measured)

The goal of the data analysis was to calculate the distri-bution diagrams and pure (individual) spectra for all the 119899119888spectroscopically active species considered throughout theexperiment The distribution diagram provides informationabout the thermodynamics of the melting processes Inaddition the shape and intensity of the pure spectra mayprovide qualitative information about the structure of thespecies

With this goal in mind data matrix D was decomposedaccording to Beer-Lambert-Bouguerrsquos law in matrix form

D = CST + E (1)

where C is the matrix (119898 times 119899119888) containing the distributiondiagram ST is thematrix (119899119888times119899) containing the pure spectraand E is the matrix of data (119898 times 119899) not explained by theproposed decomposition

The mathematical decomposition of D into matrices CST and E may be done basically in two different waysdepending on whether a physicochemical model is initiallyproposed (hard-modeling approach) or not (soft-modelingapproach) In this work a hard-modeling approach hasbeen applied In this case the physicochemical model isrelated to the thermodynamics of DNA unfolding Hencefor the unfolding of intramolecular structures such as thosestudied here the chemical equation and the correspondingequilibrium constant may be written as

DNAfolded + heatlarrrarr DNAunfolded

Kunfolding =[DNAunfolded]

[DNAfolded]

(2)

The concentration of the folded and unfolded forms istemperature-dependent Accordingly the equilibrium con-stant depends on temperature according to the vanrsquot Hoffequation

ln119870unfolding = minusΔ119867V119867

119877119879

+

Δ119878V119867

119877

(3)

In this case it is assumed that Δ119867V119867 and Δ119878V119867 willnot change throughout the range of temperatures studiedhere Whenever a physicochemical model is applied thedistribution diagram inC complies with the proposedmodel

Accordingly the proposed values for the equilibrium con-stants and the shape of the pure spectra in ST are refinedto explain satisfactorily data in D whereas residuals in Eare minimized Hard modeling of melting experiments wasdone with a modified version of the MCR-ALS procedurethat includes the model proposed in (3) for the unfolding ofintramolecular structures

3 Results and Discussion

Figure 2 shows the SEC chromatograms and CD spectra(insets) recorded for all four sequences at 20 30 and 40∘CHigher temperatures could not be achieved due to thelimitations of the chromatographic column DNA sampleswere prepared by direct dilution from a stock solution heatedat 95∘C for 10 minutes and slowly cooled overnight beforemeasurements were done

The shape of the CD spectrum of a ckit21 samplemeasured at pH 7 and 20∘C showed the typical featuresrelated to parallel G-quadruplexes like a large positive bandaround 265 nm and a negative band around 240 nm Thesmall shoulder at 295 nm may reflect a certain contributionof antiparallel structure The CD spectrum of ckit21T on thecontrary did not show any contribution at 295 nm reflectinga clear parallel conformation The CD spectrum of ckit21T18at pH 7 and 25∘C was clearly different from that measuredfor ckit21 because it showed two large positive bands around265 and 295 nm which denoted a mixed antiparallelparalleltopology in the same molecule or the presence of a mixtureof structures in different molecules Finally the CD spectrumof ckit21T21 was similar to that measured for ckit21 as itcontained a large positive band centered at 265 nm whichdenoted a major parallel topology and a small shoulder at295 nm

Melting experiments simultaneously monitored with CDand molecular absorption spectroscopies were carried outto evaluate the relative stability of the structures formedby the ckit21-based sequences Analysis of the whole set ofspectra recorded along the melting experiments was doneby means of a multivariate data analysis method [15] Thenumber of species present along the melting process aswell as the associated thermodynamic parameters (119879

119898and

changes in enthalpy and entropy) was determined in this wayUpon heating all four G-quadruplex structures unfoldedwith 119879

119898values equal to 74∘C (ckit21) 82∘C (ckit21T)

66∘C (ckit21T18) and 71∘C (ckit21T21) The unfolding ofthe ckit21T18 clearly took place in two steps (Figure 3)Upon heating the band at 295 nm was completely lost at55∘C whereas the intensity of the band at 265 nm increaseddramatically At this point the parallel structure was clearlythe major one Finally this structure was lost after heatingup to 90∘C The determined value of 119879

119898was 66 plusmn 1∘C

which was clearly lower than for ckit21 It seems that theexistence of a two-base third loop produced the shift ofthe parallel to antiparallel equilibrium proposed by Hsuet al to this last position [6] Thermodynamic parameterscalculated from melting experiments are reported in Table 1

4 ISRN Biochemistry

6 7 8 9 10 11 12012345678

Retention time (min)

Abso

rban

ce (m

AU)

220 240 260 280 300 320minus5

0

5

10

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

times106

(a)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus4

minus2

0

2

4

6

8times10

6

Retention time (min)

Abso

rban

ce (m

AU)

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(b)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus3minus2minus1

012345times10

6

Wavelength (nm)

Retention time (min)

Abso

rban

ce (m

AU)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(c)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus4minus2

02

46

810

Retention time (min)

Abso

rban

ce (m

AU)

times106

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(d)

Figure 2 SEC profiles and CD spectra recorded for ckit21 (a) ckit21T (b) ckit21T18 (c) and ckit21T21 (d) at 21 (blue) 30 (green) and 40∘C(red) and pH 71

Table 1Thermodynamic parameters related to the unfolding of the three sequences studied in this work Experiments have been performedat least three times Parameters are done in terms of mean value and standard deviation

Sequence Δ119867

0 (kcalsdotmolminus1) Δ119878

0 (calsdotKminus1 sdotmolminus1) Δ119866

0 (kcalsdotmolminus1) at 37∘C 119879

119898(∘C) Reference

ckit21 51 plusmn 3 145 plusmn 9 54 plusmn 06 74 plusmn 1 This workckit21T 46 130 58 82 [12]ckit21T18 44 plusmn 2 131 plusmn 3 38 plusmn 04 66 plusmn 2 This workckit21T21 46 plusmn 4 135 plusmn 12 45 plusmn 04 71 plusmn 1 This work

The native sequence showed the highest values for the ther-modynamic parameters which reflected the higher stabilityof this sequence in front of the mutated ones The standardchange in free energy comes from the high values of standardchanges both in enthalpy and in entropy On the contrarythe ckit21T18 sequence showed the lowest values whereasckit21T21 showed intermediate values From the data shownin this table it was clear that the native sequence was themost stable at 37∘C Also it seems that the G18T mutationreduced dramatically the stability of the structure It is knownthat the formation of a tetrad implies the release of about15ndash25 kcalmol depending on factors such as a stacking ofadditional bases on the tetrad In our case unfolding of theckit21 sequence required a change in enthalpy per tetradof 17 kcalmol Overall unfolding of this sequence requiredaround 6 kcalmol more than those of the mutated sequencesckit21T18 and ckit21T21 This difference could be due to the

additional stacking of the terminal guanine at the 31015840 endin the wild sequence In the case of ckit21T21 the limitedcontribution of the terminal thymine bases to the overallstability of quadruplex structures had been already pointedout [1]

CD spectroscopy is a low-resolution technique whichdoes not allow discerning completely the presence of mix-tures or mixed conformations On the other hand therecorded SEC chromatograms may reveal clear differencesamong the conformational space spanned by the four dif-ferent sequences Overall SEC results allowed a qualitativecorrelation with the results obtained from spectroscopy

The chromatogram of the ckitG4 was the simplest amongthe four considered because the recorded peak at 85minutesis almost Gaussian-shaped and it lacks minor peaks at lowerand higher elution times than the principal peak Only ashoulder may be glimpsed at around 8 minutes According

ISRN Biochemistry 5

220 230 240 250 260 270 280 290 300 310 320

minus5

0

5

10

15

20

Wavelength (nm)

Ellip

ticity

(mde

g)

(a)

220 230 240 250 260 270 280 290 300 310 3200

0102030405060708

Wavelength (nm)

Abso

rban

ce (m

AU)

(b)

20 30 40 50 60 70 80 900

051

152

253

Con

cent

ratio

n (M

)

Temperature (∘C)

times10minus6

(c)

220 230 240 250 260 270 280 290 300 310 320minus2

0246

220 230 240 250 260 270 280 290 300 310 3200

1

2

Wavelength (nm)

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

olminus1)

Mol

ar ab

sorp

tivity

(cm

2m

olminus1)

times106

times105

(d)

Figure 3 Melting experiment of ckit21T18 monitored simultaneously by CD and molecular absorption spectroscopies (a) ExperimentalCD spectra (b) Experimental molecular absorption spectra (c) Calculated distribution diagram (d) Calculated pure CD and molecularabsorption spectra Concentration of DNA equal to 33 microM pH 71 and 150mM ionic strength

45

44

43

42

41

40

39

38

37

36

3564 66 68 70 72 74 76 78

Retention time (min)

log

(MW

)

y = minus05717x + 80943

R2 = 09856

Figure 4 Calibration of chromatographic system The oligonucleotides injected are 51015840-T21-31015840 (MW 63262 gsdotmolminus1) 51015840-T

24-31015840 (MW

72387 gsdotmolminus1) 51015840-ACC CCC TGC ATC TGC ATG CCC CCT CCC ACC CCC T-31015840 (MW 100575 gsdotmolminus1) 51015840-ACC CCC TGC ATC TTTTTG CCC CCT CCC ACC CCC T-31015840 (MW 100765 gsdotmolminus1) and the Watson-Crick duplex formed by 51015840-ACC CCC TGC ATC TGC ATGCCC CCT CCC ACC CCC T-31015840 and 51015840-AGG GGG TGG GAG GGG GCA TGC AGA TGC AGG GGG T-31015840 (MW 208727 gsdotmolminus1) All ofthese sequences form lineal structures at pH 71 and 20∘C

to the SEC calibration with lineal structures (Figure 4) thelinear structure of ckit21T should be eluted at around 75minutes Accordingly the main peak at 85 minutes wasrelated to a folded structure whose hydrodynamic volumewas smaller than that corresponding to the linear structureAccording to the CD spectrum the folded structure wasrelated to a parallel G-quadruplex The presence of a single

almost-Gaussian SEC band was correlated with the clearparallel G-quadruplex conformation detected by CD In thetemperature range tested no clear changes were observable

In contrast to ckit21T the wild ckit21 sequence showeda more complex conformational space reflected in thepresence of two main peaks at 72 and 88 minutes Asbefore the peak at 86 minutes was related to the parallel

6 ISRN Biochemistry

G-quadruplex On the other hand the peak at 72 minutesmay be associated with some kind of multimeric specieslike dimers [16] The mutation of cytosine and adenine bythymine bases in ckit21T seems to prevent the formation ofsuch aggregates As reflected in the CD spectra the shape ofthe SEC chromatograms hardly changes upon heating

Concomitant with the features observed in the CDspectrum at 20∘C the chromatogram of ckit21T18 was theone that showed the most striking differences in relationto the other sequences The chromatogram showed a largepeak at 85 minutes and two small peaks at 71 and 105minutes According to the previous results these peakswere related to the parallel G-quadruplex aggregates andthe antiparallel conformation respectively Upon heatingthe peak at 105 minutes slowly disappeared to merge withthat corresponding to the G-quadruplex This behavior wasconcomitant with that observed in CD spectroscopy and wasrelated to the shift of the conformational equilibrium to theparallel conformation Based on the SEC results it seems thatthe CD spectrum at 20∘C is related to a mixture of speciesshowing parallel and antiparallel conformations rather thanwith a unique species showing a mixed conformation

Finally the chromatogram of ckit21T21 showed threemain peaks at 72 81 and 89 minutesThis profile was quali-tatively similar to that of the wild sequence which again wasin accordance with both CD and melting experiments Onlythe presence of a peak at 81 minutes makes the differencebetween both sequences These results agree qualitativelywith those obtained at higher concentration of DNA [8]

4 Conclusions

Several conclusions may be drawn from the results shownhere Firstly the utility of variable-temperature SEC chro-matography as a useful tool to study the conformationalequilibria of G-quadruplexes has been shownThe chromato-graphic profilesmay be qualitatively related to those obtainedbymeans of other techniques such asCD Secondlymutationof bases at the loops produces dramatic changes in the confor-mational equilibria The assumption that four guanine tractsof equal length produce conformational homogeneity maynot be true andmust be checked bymeans of complementarytechniques such as SEC

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors acknowledge funding from the Spanish Gov-ernment (CTQ2012-38616-C02-02 andCTQ2010-20541-C03-01)

References

[1] J L Mergny J Gros A D Cian et al ldquoEnergetics kinetics anddynamics of quadruplex foldingrdquo in Quadruplex Nucleic AcidsS Neidle and S Balasubramanian Eds pp 31ndash72 The RoyalSociety of Chemistry Cambridge UK 2006

[2] J Jaumot and R Gargallo ldquoExperimental methods for studyingthe interactions between G-quadruplex structures and ligandsrdquoCurrent Pharmaceutical Design vol 18 no 14 pp 1900ndash19162012

[3] D Miyoshi A Nakao and N Sugimoto ldquoStructural transi-tion from antiparallel to parallel G-quadruplex of d(G

4T4G4)

induced by Ca2+rdquoNucleic Acids Research vol 31 no 4 pp 1156ndash1163 2003

[4] G Biffi D Tannahill J McCafferty and S BalasubramanianldquoQuantitative visualization of DNAG-quadruplex structures inhuman cellsrdquo Nature Chemistry vol 5 pp 182ndash186 2013

[5] H Fernando A P Reszka J Huppert et al ldquoA conservedquadruplexmotif located in a transcription activation site of thehuman c-kit oncogenerdquo Biochemistry vol 45 no 25 pp 7854ndash7860 2006

[6] S-T D Hsu P Varnai A Bugaut A P Reszka S Neidleand S Balasubramanian ldquoA G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex havingasymmetric G-tetrad dynamicsrdquo Journal of the American Chem-ical Society vol 131 no 37 pp 13399ndash13409 2009

[7] V Kuryavyi A T Phan and D J Patel ldquoSolution structuresof all parallel-stranded monomeric and dimeric G-quadruplexscaffolds of the human c-kit2 promoterrdquoNucleic Acids Researchvol 38 no 19 pp 6757ndash6773 2010

[8] M C Miller H T Le W L Dean P A Holt J B Chairesand J O Trent ldquoPolymorphism and resolution of oncogene pro-moter quadruplex-forming sequencesrdquo Organic and Biomolec-ular Chemistry vol 9 no 22 pp 7633ndash7637 2011

[9] H T Le M C Miller R Buscaglia et al ldquoNot all G-quadruplexes are created equally an investigation of thestructural polymorphism of the c-Myc G-quadruplex-formingsequence and its interaction with the porphyrin TMPyP4rdquoOrganic ampBiomolecular Chemistry vol 10 pp 9393ndash9404 2012

[10] L Spindler M Rigler I Drevensek-Olenik N Marsquoani Hessariand M Webba da Silva ldquoEffect of base sequence on G-wireformation in solutionrdquo Journal of Nucleic Acids vol 2010Article ID 431651 8 pages 2010

[11] W H Braunlin I Giri L Beadling and K J BreslauerldquoConformational screening of oligonucleotides by variable-temperature high performance liquid chromatographydissecting the duplex-hairpin-coil equilibria ofd(CGCGAATTCGCG)rdquo Biopolymers vol 74 no 3 pp221ndash231 2004

[12] P Bucek J Jaumot A Avino R Eritja and R GargalloldquoPH-modulatedWatson-Crick duplex-quadruplex equilibria ofguanine-rich and cytosine-rich DNA sequences 140 base pairsupstream of the c-kit transcription initiation siterdquo Chemistryvol 15 no 46 pp 12663ndash12671 2009

[13] N Khan A Avino R Tauler C Gonzalez R Eritja and RGargallo ldquoSolution equilibria of the i-motif-forming regionupstream of the B-cell lymphoma-2 P1 promoterrdquo Biochimievol 89 no 12 pp 1562ndash1572 2007

[14] W A Kibbe ldquoOligoCalc an online oligonucleotide propertiescalculatorrdquoNucleic Acids Research vol 35 ppW43ndashW46 2007

[15] S Fernandez R Eritja A Avino J Jaumot and R GargalloldquoInfluence of pH temperature and the cationic porphyrin

ISRN Biochemistry 7

TMPyP4 on the stability of the i-motif formed by the 51015840-(C3TA2)4-31015840 sequence of the human telomererdquo InternationalJournal of Biological Macromolecules vol 49 no 4 pp 729ndash7362011

[16] N Smargiasso F Rosu W Hsia et al ldquoG-quadruplex DNAassemblies loop length cation identity and multimer forma-tionrdquo Journal of the American Chemical Society vol 130 no 31pp 10208ndash10216 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 ISRN Biochemistry

6 7 8 9 10 11 12012345678

Retention time (min)

Abso

rban

ce (m

AU)

220 240 260 280 300 320minus5

0

5

10

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

times106

(a)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus4

minus2

0

2

4

6

8times10

6

Retention time (min)

Abso

rban

ce (m

AU)

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(b)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus3minus2minus1

012345times10

6

Wavelength (nm)

Retention time (min)

Abso

rban

ce (m

AU)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(c)

6 7 8 9 10 11 120

2

4

6

8

10

220 240 260 280 300 320minus4minus2

02

46

810

Retention time (min)

Abso

rban

ce (m

AU)

times106

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

ol

)minus1

(d)

Figure 2 SEC profiles and CD spectra recorded for ckit21 (a) ckit21T (b) ckit21T18 (c) and ckit21T21 (d) at 21 (blue) 30 (green) and 40∘C(red) and pH 71

Table 1Thermodynamic parameters related to the unfolding of the three sequences studied in this work Experiments have been performedat least three times Parameters are done in terms of mean value and standard deviation

Sequence Δ119867

0 (kcalsdotmolminus1) Δ119878

0 (calsdotKminus1 sdotmolminus1) Δ119866

0 (kcalsdotmolminus1) at 37∘C 119879

119898(∘C) Reference

ckit21 51 plusmn 3 145 plusmn 9 54 plusmn 06 74 plusmn 1 This workckit21T 46 130 58 82 [12]ckit21T18 44 plusmn 2 131 plusmn 3 38 plusmn 04 66 plusmn 2 This workckit21T21 46 plusmn 4 135 plusmn 12 45 plusmn 04 71 plusmn 1 This work

The native sequence showed the highest values for the ther-modynamic parameters which reflected the higher stabilityof this sequence in front of the mutated ones The standardchange in free energy comes from the high values of standardchanges both in enthalpy and in entropy On the contrarythe ckit21T18 sequence showed the lowest values whereasckit21T21 showed intermediate values From the data shownin this table it was clear that the native sequence was themost stable at 37∘C Also it seems that the G18T mutationreduced dramatically the stability of the structure It is knownthat the formation of a tetrad implies the release of about15ndash25 kcalmol depending on factors such as a stacking ofadditional bases on the tetrad In our case unfolding of theckit21 sequence required a change in enthalpy per tetradof 17 kcalmol Overall unfolding of this sequence requiredaround 6 kcalmol more than those of the mutated sequencesckit21T18 and ckit21T21 This difference could be due to the

additional stacking of the terminal guanine at the 31015840 endin the wild sequence In the case of ckit21T21 the limitedcontribution of the terminal thymine bases to the overallstability of quadruplex structures had been already pointedout [1]

CD spectroscopy is a low-resolution technique whichdoes not allow discerning completely the presence of mix-tures or mixed conformations On the other hand therecorded SEC chromatograms may reveal clear differencesamong the conformational space spanned by the four dif-ferent sequences Overall SEC results allowed a qualitativecorrelation with the results obtained from spectroscopy

The chromatogram of the ckitG4 was the simplest amongthe four considered because the recorded peak at 85minutesis almost Gaussian-shaped and it lacks minor peaks at lowerand higher elution times than the principal peak Only ashoulder may be glimpsed at around 8 minutes According

ISRN Biochemistry 5

220 230 240 250 260 270 280 290 300 310 320

minus5

0

5

10

15

20

Wavelength (nm)

Ellip

ticity

(mde

g)

(a)

220 230 240 250 260 270 280 290 300 310 3200

0102030405060708

Wavelength (nm)

Abso

rban

ce (m

AU)

(b)

20 30 40 50 60 70 80 900

051

152

253

Con

cent

ratio

n (M

)

Temperature (∘C)

times10minus6

(c)

220 230 240 250 260 270 280 290 300 310 320minus2

0246

220 230 240 250 260 270 280 290 300 310 3200

1

2

Wavelength (nm)

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

olminus1)

Mol

ar ab

sorp

tivity

(cm

2m

olminus1)

times106

times105

(d)

Figure 3 Melting experiment of ckit21T18 monitored simultaneously by CD and molecular absorption spectroscopies (a) ExperimentalCD spectra (b) Experimental molecular absorption spectra (c) Calculated distribution diagram (d) Calculated pure CD and molecularabsorption spectra Concentration of DNA equal to 33 microM pH 71 and 150mM ionic strength

45

44

43

42

41

40

39

38

37

36

3564 66 68 70 72 74 76 78

Retention time (min)

log

(MW

)

y = minus05717x + 80943

R2 = 09856

Figure 4 Calibration of chromatographic system The oligonucleotides injected are 51015840-T21-31015840 (MW 63262 gsdotmolminus1) 51015840-T

24-31015840 (MW

72387 gsdotmolminus1) 51015840-ACC CCC TGC ATC TGC ATG CCC CCT CCC ACC CCC T-31015840 (MW 100575 gsdotmolminus1) 51015840-ACC CCC TGC ATC TTTTTG CCC CCT CCC ACC CCC T-31015840 (MW 100765 gsdotmolminus1) and the Watson-Crick duplex formed by 51015840-ACC CCC TGC ATC TGC ATGCCC CCT CCC ACC CCC T-31015840 and 51015840-AGG GGG TGG GAG GGG GCA TGC AGA TGC AGG GGG T-31015840 (MW 208727 gsdotmolminus1) All ofthese sequences form lineal structures at pH 71 and 20∘C

to the SEC calibration with lineal structures (Figure 4) thelinear structure of ckit21T should be eluted at around 75minutes Accordingly the main peak at 85 minutes wasrelated to a folded structure whose hydrodynamic volumewas smaller than that corresponding to the linear structureAccording to the CD spectrum the folded structure wasrelated to a parallel G-quadruplex The presence of a single

almost-Gaussian SEC band was correlated with the clearparallel G-quadruplex conformation detected by CD In thetemperature range tested no clear changes were observable

In contrast to ckit21T the wild ckit21 sequence showeda more complex conformational space reflected in thepresence of two main peaks at 72 and 88 minutes Asbefore the peak at 86 minutes was related to the parallel

6 ISRN Biochemistry

G-quadruplex On the other hand the peak at 72 minutesmay be associated with some kind of multimeric specieslike dimers [16] The mutation of cytosine and adenine bythymine bases in ckit21T seems to prevent the formation ofsuch aggregates As reflected in the CD spectra the shape ofthe SEC chromatograms hardly changes upon heating

Concomitant with the features observed in the CDspectrum at 20∘C the chromatogram of ckit21T18 was theone that showed the most striking differences in relationto the other sequences The chromatogram showed a largepeak at 85 minutes and two small peaks at 71 and 105minutes According to the previous results these peakswere related to the parallel G-quadruplex aggregates andthe antiparallel conformation respectively Upon heatingthe peak at 105 minutes slowly disappeared to merge withthat corresponding to the G-quadruplex This behavior wasconcomitant with that observed in CD spectroscopy and wasrelated to the shift of the conformational equilibrium to theparallel conformation Based on the SEC results it seems thatthe CD spectrum at 20∘C is related to a mixture of speciesshowing parallel and antiparallel conformations rather thanwith a unique species showing a mixed conformation

Finally the chromatogram of ckit21T21 showed threemain peaks at 72 81 and 89 minutesThis profile was quali-tatively similar to that of the wild sequence which again wasin accordance with both CD and melting experiments Onlythe presence of a peak at 81 minutes makes the differencebetween both sequences These results agree qualitativelywith those obtained at higher concentration of DNA [8]

4 Conclusions

Several conclusions may be drawn from the results shownhere Firstly the utility of variable-temperature SEC chro-matography as a useful tool to study the conformationalequilibria of G-quadruplexes has been shownThe chromato-graphic profilesmay be qualitatively related to those obtainedbymeans of other techniques such asCD Secondlymutationof bases at the loops produces dramatic changes in the confor-mational equilibria The assumption that four guanine tractsof equal length produce conformational homogeneity maynot be true andmust be checked bymeans of complementarytechniques such as SEC

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors acknowledge funding from the Spanish Gov-ernment (CTQ2012-38616-C02-02 andCTQ2010-20541-C03-01)

References

[1] J L Mergny J Gros A D Cian et al ldquoEnergetics kinetics anddynamics of quadruplex foldingrdquo in Quadruplex Nucleic AcidsS Neidle and S Balasubramanian Eds pp 31ndash72 The RoyalSociety of Chemistry Cambridge UK 2006

[2] J Jaumot and R Gargallo ldquoExperimental methods for studyingthe interactions between G-quadruplex structures and ligandsrdquoCurrent Pharmaceutical Design vol 18 no 14 pp 1900ndash19162012

[3] D Miyoshi A Nakao and N Sugimoto ldquoStructural transi-tion from antiparallel to parallel G-quadruplex of d(G

4T4G4)

induced by Ca2+rdquoNucleic Acids Research vol 31 no 4 pp 1156ndash1163 2003

[4] G Biffi D Tannahill J McCafferty and S BalasubramanianldquoQuantitative visualization of DNAG-quadruplex structures inhuman cellsrdquo Nature Chemistry vol 5 pp 182ndash186 2013

[5] H Fernando A P Reszka J Huppert et al ldquoA conservedquadruplexmotif located in a transcription activation site of thehuman c-kit oncogenerdquo Biochemistry vol 45 no 25 pp 7854ndash7860 2006

[6] S-T D Hsu P Varnai A Bugaut A P Reszka S Neidleand S Balasubramanian ldquoA G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex havingasymmetric G-tetrad dynamicsrdquo Journal of the American Chem-ical Society vol 131 no 37 pp 13399ndash13409 2009

[7] V Kuryavyi A T Phan and D J Patel ldquoSolution structuresof all parallel-stranded monomeric and dimeric G-quadruplexscaffolds of the human c-kit2 promoterrdquoNucleic Acids Researchvol 38 no 19 pp 6757ndash6773 2010

[8] M C Miller H T Le W L Dean P A Holt J B Chairesand J O Trent ldquoPolymorphism and resolution of oncogene pro-moter quadruplex-forming sequencesrdquo Organic and Biomolec-ular Chemistry vol 9 no 22 pp 7633ndash7637 2011

[9] H T Le M C Miller R Buscaglia et al ldquoNot all G-quadruplexes are created equally an investigation of thestructural polymorphism of the c-Myc G-quadruplex-formingsequence and its interaction with the porphyrin TMPyP4rdquoOrganic ampBiomolecular Chemistry vol 10 pp 9393ndash9404 2012

[10] L Spindler M Rigler I Drevensek-Olenik N Marsquoani Hessariand M Webba da Silva ldquoEffect of base sequence on G-wireformation in solutionrdquo Journal of Nucleic Acids vol 2010Article ID 431651 8 pages 2010

[11] W H Braunlin I Giri L Beadling and K J BreslauerldquoConformational screening of oligonucleotides by variable-temperature high performance liquid chromatographydissecting the duplex-hairpin-coil equilibria ofd(CGCGAATTCGCG)rdquo Biopolymers vol 74 no 3 pp221ndash231 2004

[12] P Bucek J Jaumot A Avino R Eritja and R GargalloldquoPH-modulatedWatson-Crick duplex-quadruplex equilibria ofguanine-rich and cytosine-rich DNA sequences 140 base pairsupstream of the c-kit transcription initiation siterdquo Chemistryvol 15 no 46 pp 12663ndash12671 2009

[13] N Khan A Avino R Tauler C Gonzalez R Eritja and RGargallo ldquoSolution equilibria of the i-motif-forming regionupstream of the B-cell lymphoma-2 P1 promoterrdquo Biochimievol 89 no 12 pp 1562ndash1572 2007

[14] W A Kibbe ldquoOligoCalc an online oligonucleotide propertiescalculatorrdquoNucleic Acids Research vol 35 ppW43ndashW46 2007

[15] S Fernandez R Eritja A Avino J Jaumot and R GargalloldquoInfluence of pH temperature and the cationic porphyrin

ISRN Biochemistry 7

TMPyP4 on the stability of the i-motif formed by the 51015840-(C3TA2)4-31015840 sequence of the human telomererdquo InternationalJournal of Biological Macromolecules vol 49 no 4 pp 729ndash7362011

[16] N Smargiasso F Rosu W Hsia et al ldquoG-quadruplex DNAassemblies loop length cation identity and multimer forma-tionrdquo Journal of the American Chemical Society vol 130 no 31pp 10208ndash10216 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Biochemistry 5

220 230 240 250 260 270 280 290 300 310 320

minus5

0

5

10

15

20

Wavelength (nm)

Ellip

ticity

(mde

g)

(a)

220 230 240 250 260 270 280 290 300 310 3200

0102030405060708

Wavelength (nm)

Abso

rban

ce (m

AU)

(b)

20 30 40 50 60 70 80 900

051

152

253

Con

cent

ratio

n (M

)

Temperature (∘C)

times10minus6

(c)

220 230 240 250 260 270 280 290 300 310 320minus2

0246

220 230 240 250 260 270 280 290 300 310 3200

1

2

Wavelength (nm)

Wavelength (nm)

Ellip

ticity

(mde

gmiddotcm

2m

olminus1)

Mol

ar ab

sorp

tivity

(cm

2m

olminus1)

times106

times105

(d)

Figure 3 Melting experiment of ckit21T18 monitored simultaneously by CD and molecular absorption spectroscopies (a) ExperimentalCD spectra (b) Experimental molecular absorption spectra (c) Calculated distribution diagram (d) Calculated pure CD and molecularabsorption spectra Concentration of DNA equal to 33 microM pH 71 and 150mM ionic strength

45

44

43

42

41

40

39

38

37

36

3564 66 68 70 72 74 76 78

Retention time (min)

log

(MW

)

y = minus05717x + 80943

R2 = 09856

Figure 4 Calibration of chromatographic system The oligonucleotides injected are 51015840-T21-31015840 (MW 63262 gsdotmolminus1) 51015840-T

24-31015840 (MW

72387 gsdotmolminus1) 51015840-ACC CCC TGC ATC TGC ATG CCC CCT CCC ACC CCC T-31015840 (MW 100575 gsdotmolminus1) 51015840-ACC CCC TGC ATC TTTTTG CCC CCT CCC ACC CCC T-31015840 (MW 100765 gsdotmolminus1) and the Watson-Crick duplex formed by 51015840-ACC CCC TGC ATC TGC ATGCCC CCT CCC ACC CCC T-31015840 and 51015840-AGG GGG TGG GAG GGG GCA TGC AGA TGC AGG GGG T-31015840 (MW 208727 gsdotmolminus1) All ofthese sequences form lineal structures at pH 71 and 20∘C

to the SEC calibration with lineal structures (Figure 4) thelinear structure of ckit21T should be eluted at around 75minutes Accordingly the main peak at 85 minutes wasrelated to a folded structure whose hydrodynamic volumewas smaller than that corresponding to the linear structureAccording to the CD spectrum the folded structure wasrelated to a parallel G-quadruplex The presence of a single

almost-Gaussian SEC band was correlated with the clearparallel G-quadruplex conformation detected by CD In thetemperature range tested no clear changes were observable

In contrast to ckit21T the wild ckit21 sequence showeda more complex conformational space reflected in thepresence of two main peaks at 72 and 88 minutes Asbefore the peak at 86 minutes was related to the parallel

6 ISRN Biochemistry

G-quadruplex On the other hand the peak at 72 minutesmay be associated with some kind of multimeric specieslike dimers [16] The mutation of cytosine and adenine bythymine bases in ckit21T seems to prevent the formation ofsuch aggregates As reflected in the CD spectra the shape ofthe SEC chromatograms hardly changes upon heating

Concomitant with the features observed in the CDspectrum at 20∘C the chromatogram of ckit21T18 was theone that showed the most striking differences in relationto the other sequences The chromatogram showed a largepeak at 85 minutes and two small peaks at 71 and 105minutes According to the previous results these peakswere related to the parallel G-quadruplex aggregates andthe antiparallel conformation respectively Upon heatingthe peak at 105 minutes slowly disappeared to merge withthat corresponding to the G-quadruplex This behavior wasconcomitant with that observed in CD spectroscopy and wasrelated to the shift of the conformational equilibrium to theparallel conformation Based on the SEC results it seems thatthe CD spectrum at 20∘C is related to a mixture of speciesshowing parallel and antiparallel conformations rather thanwith a unique species showing a mixed conformation

Finally the chromatogram of ckit21T21 showed threemain peaks at 72 81 and 89 minutesThis profile was quali-tatively similar to that of the wild sequence which again wasin accordance with both CD and melting experiments Onlythe presence of a peak at 81 minutes makes the differencebetween both sequences These results agree qualitativelywith those obtained at higher concentration of DNA [8]

4 Conclusions

Several conclusions may be drawn from the results shownhere Firstly the utility of variable-temperature SEC chro-matography as a useful tool to study the conformationalequilibria of G-quadruplexes has been shownThe chromato-graphic profilesmay be qualitatively related to those obtainedbymeans of other techniques such asCD Secondlymutationof bases at the loops produces dramatic changes in the confor-mational equilibria The assumption that four guanine tractsof equal length produce conformational homogeneity maynot be true andmust be checked bymeans of complementarytechniques such as SEC

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors acknowledge funding from the Spanish Gov-ernment (CTQ2012-38616-C02-02 andCTQ2010-20541-C03-01)

References

[1] J L Mergny J Gros A D Cian et al ldquoEnergetics kinetics anddynamics of quadruplex foldingrdquo in Quadruplex Nucleic AcidsS Neidle and S Balasubramanian Eds pp 31ndash72 The RoyalSociety of Chemistry Cambridge UK 2006

[2] J Jaumot and R Gargallo ldquoExperimental methods for studyingthe interactions between G-quadruplex structures and ligandsrdquoCurrent Pharmaceutical Design vol 18 no 14 pp 1900ndash19162012

[3] D Miyoshi A Nakao and N Sugimoto ldquoStructural transi-tion from antiparallel to parallel G-quadruplex of d(G

4T4G4)

induced by Ca2+rdquoNucleic Acids Research vol 31 no 4 pp 1156ndash1163 2003

[4] G Biffi D Tannahill J McCafferty and S BalasubramanianldquoQuantitative visualization of DNAG-quadruplex structures inhuman cellsrdquo Nature Chemistry vol 5 pp 182ndash186 2013

[5] H Fernando A P Reszka J Huppert et al ldquoA conservedquadruplexmotif located in a transcription activation site of thehuman c-kit oncogenerdquo Biochemistry vol 45 no 25 pp 7854ndash7860 2006

[6] S-T D Hsu P Varnai A Bugaut A P Reszka S Neidleand S Balasubramanian ldquoA G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex havingasymmetric G-tetrad dynamicsrdquo Journal of the American Chem-ical Society vol 131 no 37 pp 13399ndash13409 2009

[7] V Kuryavyi A T Phan and D J Patel ldquoSolution structuresof all parallel-stranded monomeric and dimeric G-quadruplexscaffolds of the human c-kit2 promoterrdquoNucleic Acids Researchvol 38 no 19 pp 6757ndash6773 2010

[8] M C Miller H T Le W L Dean P A Holt J B Chairesand J O Trent ldquoPolymorphism and resolution of oncogene pro-moter quadruplex-forming sequencesrdquo Organic and Biomolec-ular Chemistry vol 9 no 22 pp 7633ndash7637 2011

[9] H T Le M C Miller R Buscaglia et al ldquoNot all G-quadruplexes are created equally an investigation of thestructural polymorphism of the c-Myc G-quadruplex-formingsequence and its interaction with the porphyrin TMPyP4rdquoOrganic ampBiomolecular Chemistry vol 10 pp 9393ndash9404 2012

[10] L Spindler M Rigler I Drevensek-Olenik N Marsquoani Hessariand M Webba da Silva ldquoEffect of base sequence on G-wireformation in solutionrdquo Journal of Nucleic Acids vol 2010Article ID 431651 8 pages 2010

[11] W H Braunlin I Giri L Beadling and K J BreslauerldquoConformational screening of oligonucleotides by variable-temperature high performance liquid chromatographydissecting the duplex-hairpin-coil equilibria ofd(CGCGAATTCGCG)rdquo Biopolymers vol 74 no 3 pp221ndash231 2004

[12] P Bucek J Jaumot A Avino R Eritja and R GargalloldquoPH-modulatedWatson-Crick duplex-quadruplex equilibria ofguanine-rich and cytosine-rich DNA sequences 140 base pairsupstream of the c-kit transcription initiation siterdquo Chemistryvol 15 no 46 pp 12663ndash12671 2009

[13] N Khan A Avino R Tauler C Gonzalez R Eritja and RGargallo ldquoSolution equilibria of the i-motif-forming regionupstream of the B-cell lymphoma-2 P1 promoterrdquo Biochimievol 89 no 12 pp 1562ndash1572 2007

[14] W A Kibbe ldquoOligoCalc an online oligonucleotide propertiescalculatorrdquoNucleic Acids Research vol 35 ppW43ndashW46 2007

[15] S Fernandez R Eritja A Avino J Jaumot and R GargalloldquoInfluence of pH temperature and the cationic porphyrin

ISRN Biochemistry 7

TMPyP4 on the stability of the i-motif formed by the 51015840-(C3TA2)4-31015840 sequence of the human telomererdquo InternationalJournal of Biological Macromolecules vol 49 no 4 pp 729ndash7362011

[16] N Smargiasso F Rosu W Hsia et al ldquoG-quadruplex DNAassemblies loop length cation identity and multimer forma-tionrdquo Journal of the American Chemical Society vol 130 no 31pp 10208ndash10216 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 ISRN Biochemistry

G-quadruplex On the other hand the peak at 72 minutesmay be associated with some kind of multimeric specieslike dimers [16] The mutation of cytosine and adenine bythymine bases in ckit21T seems to prevent the formation ofsuch aggregates As reflected in the CD spectra the shape ofthe SEC chromatograms hardly changes upon heating

Concomitant with the features observed in the CDspectrum at 20∘C the chromatogram of ckit21T18 was theone that showed the most striking differences in relationto the other sequences The chromatogram showed a largepeak at 85 minutes and two small peaks at 71 and 105minutes According to the previous results these peakswere related to the parallel G-quadruplex aggregates andthe antiparallel conformation respectively Upon heatingthe peak at 105 minutes slowly disappeared to merge withthat corresponding to the G-quadruplex This behavior wasconcomitant with that observed in CD spectroscopy and wasrelated to the shift of the conformational equilibrium to theparallel conformation Based on the SEC results it seems thatthe CD spectrum at 20∘C is related to a mixture of speciesshowing parallel and antiparallel conformations rather thanwith a unique species showing a mixed conformation

Finally the chromatogram of ckit21T21 showed threemain peaks at 72 81 and 89 minutesThis profile was quali-tatively similar to that of the wild sequence which again wasin accordance with both CD and melting experiments Onlythe presence of a peak at 81 minutes makes the differencebetween both sequences These results agree qualitativelywith those obtained at higher concentration of DNA [8]

4 Conclusions

Several conclusions may be drawn from the results shownhere Firstly the utility of variable-temperature SEC chro-matography as a useful tool to study the conformationalequilibria of G-quadruplexes has been shownThe chromato-graphic profilesmay be qualitatively related to those obtainedbymeans of other techniques such asCD Secondlymutationof bases at the loops produces dramatic changes in the confor-mational equilibria The assumption that four guanine tractsof equal length produce conformational homogeneity maynot be true andmust be checked bymeans of complementarytechniques such as SEC

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors acknowledge funding from the Spanish Gov-ernment (CTQ2012-38616-C02-02 andCTQ2010-20541-C03-01)

References

[1] J L Mergny J Gros A D Cian et al ldquoEnergetics kinetics anddynamics of quadruplex foldingrdquo in Quadruplex Nucleic AcidsS Neidle and S Balasubramanian Eds pp 31ndash72 The RoyalSociety of Chemistry Cambridge UK 2006

[2] J Jaumot and R Gargallo ldquoExperimental methods for studyingthe interactions between G-quadruplex structures and ligandsrdquoCurrent Pharmaceutical Design vol 18 no 14 pp 1900ndash19162012

[3] D Miyoshi A Nakao and N Sugimoto ldquoStructural transi-tion from antiparallel to parallel G-quadruplex of d(G

4T4G4)

induced by Ca2+rdquoNucleic Acids Research vol 31 no 4 pp 1156ndash1163 2003

[4] G Biffi D Tannahill J McCafferty and S BalasubramanianldquoQuantitative visualization of DNAG-quadruplex structures inhuman cellsrdquo Nature Chemistry vol 5 pp 182ndash186 2013

[5] H Fernando A P Reszka J Huppert et al ldquoA conservedquadruplexmotif located in a transcription activation site of thehuman c-kit oncogenerdquo Biochemistry vol 45 no 25 pp 7854ndash7860 2006

[6] S-T D Hsu P Varnai A Bugaut A P Reszka S Neidleand S Balasubramanian ldquoA G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex havingasymmetric G-tetrad dynamicsrdquo Journal of the American Chem-ical Society vol 131 no 37 pp 13399ndash13409 2009

[7] V Kuryavyi A T Phan and D J Patel ldquoSolution structuresof all parallel-stranded monomeric and dimeric G-quadruplexscaffolds of the human c-kit2 promoterrdquoNucleic Acids Researchvol 38 no 19 pp 6757ndash6773 2010

[8] M C Miller H T Le W L Dean P A Holt J B Chairesand J O Trent ldquoPolymorphism and resolution of oncogene pro-moter quadruplex-forming sequencesrdquo Organic and Biomolec-ular Chemistry vol 9 no 22 pp 7633ndash7637 2011

[9] H T Le M C Miller R Buscaglia et al ldquoNot all G-quadruplexes are created equally an investigation of thestructural polymorphism of the c-Myc G-quadruplex-formingsequence and its interaction with the porphyrin TMPyP4rdquoOrganic ampBiomolecular Chemistry vol 10 pp 9393ndash9404 2012

[10] L Spindler M Rigler I Drevensek-Olenik N Marsquoani Hessariand M Webba da Silva ldquoEffect of base sequence on G-wireformation in solutionrdquo Journal of Nucleic Acids vol 2010Article ID 431651 8 pages 2010

[11] W H Braunlin I Giri L Beadling and K J BreslauerldquoConformational screening of oligonucleotides by variable-temperature high performance liquid chromatographydissecting the duplex-hairpin-coil equilibria ofd(CGCGAATTCGCG)rdquo Biopolymers vol 74 no 3 pp221ndash231 2004

[12] P Bucek J Jaumot A Avino R Eritja and R GargalloldquoPH-modulatedWatson-Crick duplex-quadruplex equilibria ofguanine-rich and cytosine-rich DNA sequences 140 base pairsupstream of the c-kit transcription initiation siterdquo Chemistryvol 15 no 46 pp 12663ndash12671 2009

[13] N Khan A Avino R Tauler C Gonzalez R Eritja and RGargallo ldquoSolution equilibria of the i-motif-forming regionupstream of the B-cell lymphoma-2 P1 promoterrdquo Biochimievol 89 no 12 pp 1562ndash1572 2007

[14] W A Kibbe ldquoOligoCalc an online oligonucleotide propertiescalculatorrdquoNucleic Acids Research vol 35 ppW43ndashW46 2007

[15] S Fernandez R Eritja A Avino J Jaumot and R GargalloldquoInfluence of pH temperature and the cationic porphyrin

ISRN Biochemistry 7

TMPyP4 on the stability of the i-motif formed by the 51015840-(C3TA2)4-31015840 sequence of the human telomererdquo InternationalJournal of Biological Macromolecules vol 49 no 4 pp 729ndash7362011

[16] N Smargiasso F Rosu W Hsia et al ldquoG-quadruplex DNAassemblies loop length cation identity and multimer forma-tionrdquo Journal of the American Chemical Society vol 130 no 31pp 10208ndash10216 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Biochemistry 7

TMPyP4 on the stability of the i-motif formed by the 51015840-(C3TA2)4-31015840 sequence of the human telomererdquo InternationalJournal of Biological Macromolecules vol 49 no 4 pp 729ndash7362011

[16] N Smargiasso F Rosu W Hsia et al ldquoG-quadruplex DNAassemblies loop length cation identity and multimer forma-tionrdquo Journal of the American Chemical Society vol 130 no 31pp 10208ndash10216 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology