telomeric g-quadruplexes: from human to tetrahymena...

Research ArticleTelomeric G-Quadruplexes From Human toTetrahymena Repeats

Erika DemkoviIovaacute1 7uboš Bauer1 Petra KrafIiacutekovaacute1 Katariacutena TluIkovaacute12

Petra Toacutethova1 Andrea Halaganovaacute1 Eva Valušovaacute3 and Viktor Viacuteglaskyacute1

1Department of Biochemistry Institute of Chemistry Faculty of Sciences P J Safarik University 04001 Kosice Slovakia2Department of Biological SciencesRNA Institute University at Albany SUNY Albany NY 12222 USA3Institute of Experimental Physics Slovak Academy of Sciences Watsonova 47 040 01 Kosice Slovakia

Correspondence should be addressed to Viktor Vıglasky viktorviglaskyupjssk

Received 30 July 2017 Revised 11 November 2017 Accepted 5 December 2017 Published 28 December 2017

Academic Editor Shozeb Haider

Copyright copy 2017 Erika Demkovicova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The human telomeric and protozoal telomeric sequences differ only in one purine base in their repeats TTAGGG in telomericsequences and TTGGGG in protozoal sequences In this study the relationship between G-quadruplexes formed from theserepeats and their derivatives is analyzed and compared The human telomeric DNA sequence G3(T2AG3)3 and related sequencesin which each adenine base has been systematically replaced by a guanine were investigated the result is Tetrahymena repeatsThe substitution does not affect the formation of G-quadruplexes but may cause differences in topology The results also show thatthe stability of the substituted derivatives increased in sequences with greater number of substitutions In addition most of thesequences containing imperfections in repeats which were analyzed in this study also occur in human and Tetrahymena genomesGenerally the presence of G-quadruplex structures in any organism is a source of limitations during the life cycle Therefore afuller understanding of the influence of base substitution on the structural variability of G-quadruplexes would be of considerablescientific value

1 Introduction

G-rich DNA sequences can form intra- and intermolecularG-quadruplexes based on the association of one or moreDNA strands The nucleotides which intervene between G-runs form loops of foldedG-quadruplex structures which canadopt a variety of different topological forms [1 2] When theguanine tracts are oriented in the same direction the double-chain reversal (propeller) loops link two adjacent parallelstrands to form a parallel structure [3] When the guaninetracts are oriented in opposite directions the edgewise ordiagonal loops link two antiparallel strands to form anantiparallel G-quadruplex [4] In antiparallel hybrid or so-called (3 + 1) structures a single strand is oriented in adifferent direction from the others [5ndash7] A novel (3+ 1) typefold which has recently been described by Marusic et alexhibits a conformation in which all three loop types occurin one conformation edgewise diagonal and double-chain

reversal loops [8] In addition intermolecular multimeric G-quadruplexes can be formed by the association of two ormorestrands [9]

These structures underline the high degree of G-quad-ruplex structural polymorphism a phenomenon which isdependent onmany different factors the length and sequenceof nucleic acid and environmental conditions present duringthe folding reaction such as the buffer pH stabilizing cationtemperature and the presence of agents causing dehydration[10ndash14] G-rich sequences with the propensity to form G-quadruplex structures can be located in many regions ofhuman genomic DNA especially in several biologicallyimportant regions including the end of linear eukaryotictelomeres [15 16] However putative G-rich sequences are notrandomly distributed within a genome such sequences pre-dominantly occur in protooncogene regions (which promotecell proliferation) and are depleted in tumour suppressorgenes (which maintain genomic stability) [17] It is very

HindawiJournal of Nucleic AcidsVolume 2017 Article ID 9170371 14 pageshttpsdoiorg10115520179170371

2 Journal of Nucleic Acids

Table 1 DNA oligodeoxynucleotides used in this study and their occurrence in human and Tetrahymena genomes

Name 120576a

(mMminus1 cmminus1) Sequenceb (51015840 rarr 31015840) Occurrence in genome (ID)Human Tetrahymena

HTR 2254 GGGTTAGGGTTAGGGTTAGGG Very high -HTR1 2221 GGGTTGGGGTTAGGGTTAGGG NG 0549151 M1162711HTR2 2221 GGGTTAGGGTTGGGGTTAGGG NG 0295331 -HTR3 2221 GGGTTAGGGTTAGGGTTGGGG NT 1876531 -HTR12 2188 GGGTTGGGGTTGGGGTTAGGG NW 0035710491 M116271HTR13 2188 GGGTTGGGGTTAGGGTTGGGG NC 0189262 M116271HTR23 2188 GGGTTAGGGTTGGGGTTGGGG NC 0189142 M116271HTR013 2300 GGGGTTGGGGTTAGGGTTGGGG - M116271HTR023 2300 GGGGTTAGGGTTGGGGTTGGGG NC 0189302 M116271HTR123 2154 GGGTTGGGGTTGGGGTTGGGG NC 0189122 AH0011122THR 2134 GGGGTTGGGGTTGGGGTTGGGG NG 0340201 Very highG4C2 2044 GGGGCCGGGGCCGGGGCCGGGG NG 0528101 -(AC)9 1719 (AC)9 ND ND(AC)18 3429 (AC)18 ND NDaMilimolar extinction coefficient at 257 nm bBasemodifications are underlined ND not determined

unlikely that these putative sequences can form in vivo anddirect evidence of their existence in living cells is still a topicof discussion [18ndash20] Undoubtedly the most extensivelystudied G-quadruplex forming sequences are those locatedat the 31015840-ends of human telomeres Telomeric sequences andspecialized nucleoprotein complexes which cap the ends oflinear chromosomes are essential for chromosomal stabilityand genomic integrity [21ndash23] Mammalian telomeres consistof tandem repeats of G-rich sequences d(TTAGGG)119899 Sev-eral kilobases of this sequence are double-stranded but morethan a hundred nucleotides remain unpaired and form single-stranded 31015840-overhangs [24] a state which would providefavourable conditions for the formation of one or more G-quadruplexes in vivo [22] The structure and stability oftelomeres play a significant role in the development of cancerand cell aging [25 26] There is also evidence that telomeresserve as a type of biological clock as telomere structuresappear to become shorter with each successive cell cycle Inimmortalized cells and in cancer cells however a telomeraseis activated to maintain the length of the telomere byreelongating the telomeric sequence at the chromosome ends[27 28] G-quadruplexes formed by single-stranded humantelomeric DNA have also been shown to inhibit the activityof telomerase [29] and this discovery has led to increasedinterest in the structures as attractive potential drug targets[30]

A broad range of studies of human telomeric G-quadru-plexes have been carried out using a wide variety of differenttechniques [1] To date high-resolution structures of fourdistinct folding topologies with three G-tetrad layers havebeen identified for the four human telomeric repeats [1ndash7]In addition an additional structure consisting of only twoG-tetrad layers has also been revealed which highlights thestructural polymorphism of telomeric G-quadruplexes [31]The structure of human telomeric DNA in crowded solu-tions has also been investigated by many authors [11] but

2 3

Tetrahymena

Human

10AGGGTTAGGGTTAGGGTTAGGG

GGGGTTGGGGTTGGGGTTGGGG

Figure 1 The HTR to THR conversion by substitutions of adeninefor guanine

this structure is likely to be a result of dehydration ratherthan molecular crowding [12 32 33] The great variety ofstructures identified to date can also be attributed to thepresence of flanking nucleotides outside the core sequenceG3(T2AG3)3 and the concentration of ions and to the use ofdifferent experimental methods and conditions [1 34]

A series of systematic studies concerning the sequencederivatives of human telomeric repeats were carried out byVorlıckova et al [35ndash38] and these earlier studies focusedon the substitution of guanine for adenine the introductionof abasic sites 8-oxoadenine replacing adenine and thesubstitution of 5-hydroxymethyluracil for thymine in telom-eric repeats were analyzed [39ndash41] However in this studyan opposite strategy is applied the substitution of adeninefor guanine (see Figure 1) The main aim is to achieve thetotal conversion of four human repeats to Tetrahymenarepeats which retain the ability to form intramolecularG-quadruplex Interestingly G-rich repetitions containingimperfections were also found in the human and Tetrahy-mena genome see Table 1 and Supporting Materials

In this study we examine the structures formed by theTetrahymena telomeric sequence dG4(T2G4)3 which differs

Journal of Nucleic Acids 3

from the human sequence by a single G-for-A replacementin each repeat [42] Since Gs are essential for the formationof G-quadruplexes we have systematically substituted eachof the three adenines for guanines in the TTA loops ofthe G-quadruplex-forming sequence G3(T2AG3)3 therebyincreasing the number of guanines by up to three guaninesper oligonucleotide Circular dichroism spectroscopy (CD)and polyacrylamide gel electrophoresis (PAGE) were used toobserve the effect of base substitution (s) on the formationthermal stability and conformation of G-quadruplexes Themeasurements were performed in the presence of both Na+and K+ ions and with concentrations of either PEG-200 oracetonitrile at 0 15 30 and 50wt at different temperaturesIn addition the formation of G-quadruplex structures wasverified and confirmed usingThiazole Orange (TO) TO is anexcellent DNA fluorescent probe for DNA structural formsbecause of its high fluorescence quantum yield [43] Thisligand stabilizes the G-quadruplex structure and can alsoinduce topological changes [44 45] The G-quadruplex-TOcomplex offers a characteristic profile of induced-circulardichroism spectrum in buffers containing sodium cations[44]

2 Materials and Methods

All experiments were carried out in a modified Britton-Robinson buffer (mRB) 25mM phosphoric acid 25mMboric acid 25mMacetic acid and supplemented by 50mMofKCl or NaCl PEG-200 (polyethylene glycol with an averagemolecular weight of 200) and acetonitrile (Fisher Slovakia)pH was adjusted by Tris to a final value of 70 Oligonu-cleotides with sequences shown in Table 1 were purchasedfromMetabion international AGThe lyophilized DNA sam-ples were dissolved in double-distilled water prior to use togive 1mMstock solutions Single-strandDNAconcentrationswere determined by measuring the absorbance at 260 nm athigh temperature (95∘C)

21 CD Spectroscopy CD and UV-vis spectra were measuredusing a Jasco model J-810 spectropolarimeter (Easton MDUSA) The temperature of the cell holder was regulated bya PTC-423L temperature controller Scans were performedover a range of 220ndash600 nm in a reaction volume of 300 120583lin a cuvette with a path length of 01 cm and an instrumentscanning speed of 100 nmmin 1 nm pitch and 1 nm band-width with a response time of 2 s CD data represents threeaveraged scans taken at a temperature range of 0ndash100∘CAll DNA samples were dissolved and diluted in suitablebuffers containing appropriate concentrations of ions anddehydrating agent The amount of DNA oligomers used inthe experiments was kept close to 25120583M of DNA strandconcentrationThe samples were heated at 95∘C for 5minutesthen allowed to cool down to the initial temperature beforeeachmeasurement CD spectra are expressed as the differencein the molar absorption of the right-handed and left-handedcircularly polarized light (Δ120576) in units of Mminus1sdotcmminus1 Themolarity was related to DNA oligomers A buffer baselinespectrumwas obtained using the same cuvette and subtractedfrom the sample spectra The thermal stability of different

quadruplexes was measured by recording the CD ellipticityat 295 and 265 nm as a function of temperature [14 46]The temperature ranged from 0 to 100∘C and the heatingrate was 025∘Cmin The melting temperature (119879119898) wasdefined as the temperature of themidtransition point119879119898 wasestimated from the peak value of the first derivative of thefitted curve DNA titration was performed with increasingconcentrations of TO TO was solubilized in DMSO toreach a final concentration of stock solution of 10mM Theconcentration of DNA and TO in 1mm quartz cell was30 120583M and 0ndash200120583M respectively and the increment of TOwas sim67 120583M Each sample was mixed vigorously for 3minfollowing the addition of TO CDUV spectra were measuredimmediately

22 Electrophoresis Samples consisting of 03120583l of 1mMstock solutions were separated using nondenaturing PAGEin a temperature-controlled electrophoretic apparatus(Z375039-1EA Sigma-Aldrich San Francisco CA) on 15acrylamide (19 1 acrylamidebisacrylamide) gels DNA wasloaded onto 13 times 16 times 01 cm gels Electrophoresis was run at10∘C for 4 hours at 125V (sim8Vsdotcmminus1) Each gel was stainedwith StainsAll (Sigma-Aldrich) The gel was also stainedusing the silver staining procedure in order to improve thesensitivity of the DNA visualization [44]

23 Fluorescence Spectroscopy The fluorescence spectra wereacquired with a Varian Cary Eclipse Fluorescence Spec-trophotometer at 22 plusmn 1∘C which was equipped with atemperature-controlled circulator A quartz cuvette with a3mm path length was used in all of the experiments In thefluorescence measurements the excitation and emission slitswere 5 nm and the scan speed was 240 nmmin 66120583M ofTO was titrated with DNA (33 66 and 132 120583M) in a mRBbuffer in both the presence and absence of monovalent metalcationsThemolar ratios betweenDNAand ligandwere 1 201 10 and 1 5 The excitation wavelength was adjusted to452 nm

3 Results and Discussion

31 Sequence Design and CD Spectra The sequence derivedfrom human telomeric sequence d(G3(T2AG3)3) and substi-tuted derivatives under different conditions are studied TheDNA sequences and the abbreviations used in this study aresummarized inTable 1 Points 1 2 and 3 indicate the positionsof the base substitution in the first second and third loops ofthe HTR sequence respectively Point 0 indicates a flankingguanine at the 51015840 end of the oligonucleotide Figure 1 In theDNA oligonucleotides derived from HTR the guanine (G)-for-adenine (A) in the TTA loop was substituted with theexpectation that the modified sequences would retain theability to form G-quadruplexes spontaneously albeit withdifferent topologies than those found in HTR sequencesThe HTR derivatives were analyzed in the presence of both50mM NaCl and KCl Figure 2 The first group representsoligonucleotides containing only single point mutations atdifferent positions HTR1 HTR2 and HTR3 (black linesin Figure 2) The second group represents oligonucleotides


minus3

0

4

HTR（４２1

（４２2

（４２3

240 260 280 300 320220Wavelength (nm)

Δ

(－minus1middotcＧ

minus1)

8

5

0

minus3

（４２12

（４２13

（４２23

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

10

5

0

THR（４２123（４２013

（４２023

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

HTR（４２1

（４２2

（４２3

minus2

0

2

4

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

minus2

0

2

4

（４２12

（４２13

（４２23

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

2

0

2

4

THR（４２123（４２013

（４２023

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

(a) (d)

(b) (e)

(c) (f)

Figure 2 CD spectra of oligonucleotides used in this study in a 25mMmodified Britton-Robinson buffer (pH 70) in the presence of 50mMKCl (andashc) and 50mMNaCl (dndashf)The HTR and THR spectra are shown in red and magenta respectively Each DNA sample was annealed at95∘C for 5min and then allowed to cool for sim1 h to the initial temperature at which the sample was kept at the beginning of the measurement[14]


containing two point mutations (spectra indicated with bluein Figure 2)The first two loops were modified in HTR12 thefirst and last loops were changed in HTR13 and the secondand third loops were modified in HTR23 OligonucleotidesHTR013 HTR023 and HTR123 contained three G-for-Asubstitutions (spectra in green) The spectrum and meltingtemperatures of the HTR012 sequence are very similar tothose of the HTR023 sequence (not shown in this study)while the HTR0123 sequence is equivalent to the THRsequence

The substituted sequences were also compared with theunmodified HTR and THR sequences In general terms eachof the guanine residues in any G-run could be involved in theformation of G-tetrads In the case of the formation of three-layered G-tetrad quadruplexes loop lengths were found tovary when the base substitution was introduced into theHTRsequence loops could consist of three or four nucleotidesdepending on the location and number of substitutionsHowever we cannot exclude the possibility of the formationof four-layered G-quadruplexes for sequences containingthree substitutions but it is important to note that such struc-tures would have to consist of at least one heteronucleotide-tetrad in which adenine is also present To date the 3D struc-ture of full-length THR sequences in presence of potassiumhas not been determined the only facet of the structurewhichis known is the tetrameric G-quadruplex structure formedfrom four shorter sequences d(TTGGGGT) (PDB 139D)[47] This structure consists of four G-tetrads and cannotbe stated as representing the real structure of a full-lengtholigonucleotide Nevertheless the 3D structure of THR hasbeen ascertained only in the presence of sodium (PDB 186D)[48] This structure consists of three stacked G-tetrads twoedgewise loops and one double-chain-reversal loop Despitethe fact that the sequences of THR andHTR differ at only oneof the six nucleotides their 3D topologies are quite differentbecause HTR in sodium adopts a three-G-tetrad structureconsisting of two edgewise loops and one central diagonalloop (PDB 143D) [4] However the HTR sequence can alsoadopt a stable basket-type conformation in the presence ofpotassiumconsisting of only twoG-tetrad layers (PDB 2KF8)[31]

Several naturally occurring HTR sequences have beenidentified to date Forms 1 (PDB 2HY9) and 2 (2JPZ) consistof three G-tetrads but the order of loops differs HTR formsone double-chain-reversal and two edgewise loops in bothforms [49 50] There is some similarity with THR G-quadruplexes which form in solution in the presence ofsodium [48] Form 3 is represented by a parallel G-quadru-plex with three double-chain-reversal loops (PBD 1KF1) [3]Recently Lim et al have also confirmed the structure of a 27-nt HTR derivative in the presence of sodium which differssignificantly from those mentioned above (PBD 2MBJ) [1]Although both known HTR structures solved in sodiumpossess the same relative strand orientations they differ in thehydrogen-bond directionalities and in the loop arrangementThe 2MBJ structure again consists of two edgewise and onedouble-chain-reversal loops

The sequence derived from the telomere of Oxytrichad[G4(T4G4)3] (PDB 201D and 230D) adopts a structure with

similar types of loops to those found in HTR in sodiumtwo edgewise and one central diagonal loops [4 51 52]However the Oxytricha sequence forms a four-layered G-tetrad quadruplex At the time of writing the solution struc-ture of Oxytricha sequence d[G4(T4G4)3] in K+ containingsolution had yet to be determined The main reason for thiscould be the fact that this sequence and THR in the presenceof potassium can adopt different topological forms whichcoexist in solution additional bands are observed duringelectrophoretic separation [14] Interestingly the four-layeredG-quadruplexes are very stable exhibiting particularly highmelting temperatures in the presence of potassium [14]Recently the structure of d(GGGGCC)4 in the presenceof potassium has also been determined the sequence con-tains cytosines instead of thymine residues and one 8-bromodeoxyguanosine (PDB 2N2D) [53]TheG-quadruplexstructure adopted by this sequence could be closely related tothat of THR in potassium This antiparallel structure is com-posed of four G-quartets which are connected by three edge-wise C-C loops CD spectra results show many signatures incommon with the THR sequence One of the cytosines inevery loop is stacked upon the G-quartet an arrangementwhich results is a very compact and stable structure Similarlythe melting temperature of the structure is higher than90∘C

It is generally accepted that CD spectroscopy is a veryuseful and cost-efficient method for offering a first glance atthe architecture of folded G-quadruplexes CD spectra of G-quadruplexes can be used to indicate whether the DNA hasfolded into a parallel or antiparallel conformation [36 54]

Although there are up to 25 generic folding topologiesof G-quadruplexes it is possible to classify the structuresinto three groups based on the sequence of glycosidic bondangles adopted by guanosines of the G-quadruplex [55]Group I consists of parallel G-quadruplexes with strandsoriented in the same direction and with guanosines of thesame glycosidic bond angles Parallel G-quadruplexes (GroupI) share the same characteristics irrespective of whether theycontain three or four loops an intense positive maximum atsim265 nm andminimum atsim240 nmGroups II and III consistof antiparallel G-quadruplexes Group II can be characterizedby guanosines of glycosidic binding angles in orientationssuch as anti-anti and syn-syn and also syn-anti and anti-synwhile Group III consists of stacked guanosines of distinctglycosidic bonding angles Antiparallel G-quadruplexes showa positive band at sim295 nm Positive and negative CD signalsat sim265 nm at sim240 nm respectively are characteristic forGroup II while Group III shows reverse peaks In contrastthe CD spectra of high ordered G-quadruplex architectureof Group III forms exhibit negative and positive signals at240 nm and sim265 nm respectively [55]

CD profiles corresponding to distinct G-quadruplex con-formations are determined empirically therefore the inter-pretation of CD spectra of unknown putative G-quadruplexsequences can be ambiguous A number of other factors canalso cause a degree of uncertainty over the evaluation ofCD spectra including for example the presence of mixedpopulations of various conformers andor the presence ofmultimeric conformations in solution [9 14 44 46]


CD measurements clearly show that the G-for-A substi-tutions had a considerable impact on the spectral profile ofeach sequence The presence of the G-quadruplex scaffoldformed from the unmodified HTR sequence is characterizedby a positive peak at sim295 nm with two shoulders at aroundsim270 and sim250 nm in the presence of potassium (Figure 2(a)red line) According to CD spectra these signatures arecharacteristic for Group II antiparallel G-quadruplexes Thisspectrum is indicative of the formation of a two-layeredbasket-type structure [31 55] The HTR sequence adoptsa clear antiparallel G-quadruplex conformation of GroupII type in the presence of sodium (Figure 2(d) red line)The structure is characterized by a large positive maximumat sim295 nm a smaller one near sim245 nm and a negativeCD peak at sim265 nm Previous studies have reported thatthese sequences form an intramolecular basket-type antipar-allel G-quadruplex [4] Every sequence shows a clear peakat sim295 nm which is characteristic of an antiparallel G-quadruplex topology The first set of oligomers with a singlesubstitution per oligonucleotide in the presence of potassiumshows two separated peaks at sim295 and sim265 nm the signalis dominant at 295 nm (spectra shown in black) HoweverTHR and HTR derivatives containing one or more G-for-Asubstitutions in the presence of potassium show an increaseof the peak at sim265 nm (Figures 2(b) and 2(c))This indicatesthe coexistence of more than one topological structure thatis both parallel and antiparallel configurations see also theelectrophoretic results in Figure 7 The structural polymor-phism was seen to increase with increasing numbers of Gs intheDNA sequenceTheCD signal atsim265 nm (spectra shownin green) was predominant for oligonucleotides containingthree substitutions (Figure 2(c))

In the presence of sodium only the HTR2 sequence witha substitution in the second loop exhibited a CD spectrumidentical to that of HTR although even this correspon-dence displayed lower amplitudes (spectra in dotted black inFigure 2(d)) HTR1 and HTR3 sequences with substitutionsin the first and third loop respectively also displayed apositive maximum at 295 nm but the negative peaks at265 nm were shallower and slightly shifted towards longerwavelengths in comparison to the results of the unmodifiedsequence Despite these differences they are nonethelesslikely to form G-quadruplexes of Group III Only the CDspectra of the THR sequence shows signatures of Group IItypes

Samples in the second group exhibited a positive maxi-mum at sim295 nm with a slight shift to lower wavelengths inthe case of HTR12 and HTR13 (Group III spectra shown inblue in Figure 2(b)) These two sequences displayed a lackof a negative peak at 265 nm and the smaller positive peakat around 245 nm was shifted slightly to longer wavelengths(Figure 2(e)) HTR23 shows a negative signal at sim245 nmand a positive signal at 265 and 295 nm results whichare indicative of the formation of Group II antiparallel G-quadruplexes

The CD spectra of HTR023 are close to those of GroupII G-quadruplexes while the CD of HTR013 and HTR123resemble those of Group III G-quadruplexes All sampleswith three mutations exhibited a positive maximum at

sim295 nm HTR013 exhibits negative signals at 235 and275 nm in the presence of sodium (Figure 2(f)) whileHTR023 shows positive signals at 265 and 295 nm

In general all the modified sequences in the presence ofboth Na+ and K+ were seen to differ to some degree from theHTR spectrum and were also found to differ from each otherThe varying CD spectral profiles from sample to sample are aresult of slight changes in G-quadruplex topology Howeverit was not possible to determine either the group or structureof the G-quadruplexes with any degree of certainty on thebasis of CD spectral profiles alone due to the coexistence ofvarious topological forms a finding which was confirmed bythe electrophoretic results discussed in Section 35

32 CD Spectra in the Presence of PEG-200 and AcetonitrileIn the presence of K+ the dehydrating agent PEG-200 isknown to induce a conformational change of telomeric G-quadruplexes primarily the transition from an antiparallelstructure to a parallel arrangement [2 9 11 13 14 56]Therefore the influence of PEG-200 and another dehydratingagent acetonitrile on CD spectral results and the stabilityof HTR derivatives were also investigated The represen-tative CD spectra of HTR and THR in the presence ofdifferent concentrations of both dehydrating agents (15 30and 50wt) and 50mM KCl are shown in Figure 3 Bothtypes of DNAs were found to form G-quadruplex structureswith a propeller-like parallel arrangement in the presenceof K+ However when the sequences were studied in thepresence of sodium with no potassium present no structuralconversions were observed this finding remained constantfor all of the studied HTR derivatives and THR Interestinglyat a PEG-200 concentration of 50wt the positive peaks at295 nm were found to disappear and a CD signal at 265 nmwas recorded which was sim2-fold higher than without thepresence of PEG-200 The same effect was observed foracetonitrile This is an intrinsic property of any converted G-quadruplex molecule In a recent study our group presenteda hypothesis which explains this fact the CD signal dependson the number and orientation of stacked glycosyl bonds[9 14 57] We have also previously shown that PEG-200causes the dimerization of HTR [9] therefore electrophoreticanalysis of the sequences in the presence of PEG-200 was alsoperformed Figure 8

The melting temperature of HTR and the vast majorityof G-quadruplex structures are known to increase in thepresence of PEG-200 In order to verify this fact the meltingtemperatureswere determined in the presence of PEG-200 onthe basis of CD melting curves The results are summarizedin Table 2 and clearly confirm that PEG-200 increases themelting temperatures of HTR derivatives In a methodologywhich has been used in our previous studies dual wavelengthmeasurements were performed for cases in which the spectradisplayed peaks at both 295 and 265 nm respectively [9 11]A 119879119898 of 632∘C was obtained in a mRB buffer containing50mM KCl as compared to a value of 504∘C in a bufferwith 50mM NaCl for HTR at 295 nm The overall picturewhich emerges from the thermodynamic data is that thestability of G-quadruplexes of HTR derivatives increases withincreased numbers of G-for-A substitutions in both Na+ and


10

minus2

5

0

PEG-200

CD

240 260 280 300 320220Wavelength (nm)

5030

150

(a)

Acetonitrile

240 260 280 300 320220Wavelength (nm)

0

5

10

CD

5030

150

(b)

CD

240 260 280 300 320220Wavelength (nm)

0

5

10

PEG-200

5030

150

(c)

240 260 280 300 320220Wavelength (nm)

CD

10

5

0

Acetonitrile

5030

150

(d)

Figure 3 CD spectra of HTR (a and b) and THR (c and d) oligomers in a 25mM mBR buffer (pH 70) in the presence of 50mM KCl (redlines) The samples contain either PEG 200 or acetonitrile 15 (vw) (green lines) 30 (vw) (light blue lines) and 50 (vw) (dark bluelines) The increase in the magnitude of CD signals at 265 with increasing concentrations of dehydrating agent is marked by an arrow inboth panels Each DNA sample was annealed at 95∘C for 5min and then allowed to cool overnight at 4∘CThe dashed red line represents thespectrum of THR without annealing

Table 2 Influence of PEG-200 on the melting temperatures of DNA oligomers in the presence of potassium and sodium ions

Oligomer119879119898 (∘C) in 50mM KCl + PEG-200 119879119898 (

∘C) in 50mM NaCl + PEG-2000 15 30 50 0 15 30 50

295265 295265 295265 295265 295265 295265 295265 295265HTR 632629 632ND 788ND ND898 504ND 512ND 528ND 640NDHTR1 718731 714763 643831 NDgt95 557ND 589ND 615ND 644NDHTR2 652659 642721 ND796 NDgt95 512ND 543ND 581ND 627NDHTR3 678686 693753 ND817 NDgt95 506ND 543ND 587ND 648716HTR12 695705 ND769 ND832 NDgt95 559ND 591ND 628ND 653721HTR13 722715 705791 ND864 NDgt95 537ND 562598 601632 ND735HTR23 813769 ND797 ND852 NDgt95 533ND 573ND 599630 644710HTR013 730800 ND790 ND879 NDgt95 525ND 563607 601633 ND727HTR023 707727 ND776 NDgt95 NDgt95 518545 560511 594602 ND738HTR123 739778 ND839 ND886 NDgt100 564ND 590ND 612640 ND757THR 831ND 877ND gt95 ND 574ND 597ND ND668 ND


K+ solutions The lowest 119879119898 value of HTR was recordedin both 50mM KCl and 50mM NaCl The 119879119898 of HTRderivatives was found to be higher in KCl than in NaCl All ofthe studied sequences show a higher 119879119898 value in the presenceof both dehydrating agents The results indicate that PEG-200 stabilizes G-quadruplexes with or without the A-for-Gmutation The proposed melting temperatures summarizedin Table 2 clearly demonstrate that both the number ofguanine residues in a G-tract and the nature of the stabilizingion are important determining factors in the thermal stabilityof G-quadruplexes

33 Titration Measurements Our group has recently devel-oped a new experimental methodology for the identificationof G-quadruplex forming sequences using the cyanine dyeThiazole Orange (TO) TO is an excellent DNA fluorescentprobe for various structural motifs due to its high fluo-rescence quantum yield [58] This experimental techniquecan also be used to investigate the hypothesis that HTRderivatives adopt G-quadruplex conformations TO interactswith various DNA secondary structures but it has a strongerbinding affinity to triplex and G-quadruplex structures thanto other structural motifs [43 45] Although TO is opticallyinactive TO-quadruplex complexes are chiral and display aunique profile of the induced CD (ICD) spectrum in thevisible region [44] Recently we have described the com-mon ICD features shared by many different G-quadruplexstructures The results of TO-quadruplex interaction are thepositive peaks at 265 and 295 nm (UV range) and the threepeaks in the visible region at sim512 sim492 and sim473 nm inthe solution either without the presence of metal cations orin presence of Na+ [44] TO facilitates the formation of G-quadruplex structures even without the presence of othercations but the adopted topology induced with TO can varyin comparison with the presence of sodium or potassium insolution the CD profile in the UV region can be different Acompletely different ICDprofile of the TO-DNAcomplexwasobserved for sequences unable to adopt G-quadruplex struc-ture [44] However other G-quadruplex ligands tested in ourlaboratory were not suitable for this purpose and providedambiguous results for example Thioflavin T porphyrinderivatives Hoechst 33342 andHoechst 33258Thismethod-ology is intended to be used as a supplementary techniquebecause it extends the possibilities of basic spectral methodsin terms of distinguishing G-quadruplex structures withoutthe use of more expensive and time-consuming methodsICD monitoring can be applied in different conditions butit is the most sensitive in solutions without the presence ofmetal cations it can also be applied with slightly reducedsensitivity in solutions containing Na+ or low concentrationsof K+ (lt5mM) It should be noted here that the interpretationof ICD profile at higher concentration of K+ is by no meansunambiguous Nevertheless we also performed the titrationexperiments in the presence of 50mM KCl because thiscondition is more biologically relevant

The results of titration analysis in the presence of 50mMNaCl are shown in Figure 4 The ICD results display theexpected positive signals at sim495 and sim510 nm and negativesignals at sim475 nm signatures which are characteristic for

G-quadruplexes In addition the G4C2 sequence was ana-lyzed because of its 3D structure in solutions containingpotassium As expected this oligonucleotide was also foundto form G-quadruplexes under the given conditions Signalscorresponding to those of antiparallel G-quadruplex struc-tures were also clearly detected in the UV region By increas-ing the concentration of TO the signals at 295 and 265 nmwere seen to decrease and increase respectively phenomenawhich are indicative of a conversion from antiparallel toparallel folding This effect was also observed under theinfluence of PEG-200 Figure 3

As was noted above titration measurements in 50mMKCl were also performed Figure 5 The signals observed inthe UV region clearly suggest that G-quadruplex structureswere formed in the presence of potassium but the effectof structural conversion was significantly suppressed Anintensive ICD signal with a maximum of around 500 nm isknown to correspond to the formation of complexes betweenDNA and ligands However there was a distinct lack of anyof the clear common features which are typically observedfor profiles obtained in the presence of sodium Interestinglythe ICD of the THR sequence was inverted and therefore wesuggest that the binding mode of TO with THR is differentfrom that with HTR derivatives Another explanation is thatTHR forms at least two distinct topological conformationsin solution and that one of these forms can bind with TOmore effectively As was reported in our previous study THRforms at least three different structures in the presence ofpotassium [14] we therefore decided to verify this hypothesisusing electrophoretic separation

It is important to exclude the potential side effect ofusing DMSO during TO titration experiments The stocksolution of TO contains DMSO a polar aprotic solvent whichmay produce an effect similar to that of PEG-200 [56] Theconcentration of DMSO used in our experiments did notexceed 45 wt In order to eliminate the dehydrating effectDMSO may cause in TO titration experiments titrationanalysis was also performed in the presence of DMSO aloneHowever no significant effect was observed at concentrationslower than 5wt in the absence of Na+ K+ and ionsNevertheless the presence of DMSO in solution containingK+ could explain the slight differences in ICD profiles atconcentrations of K+ greater than 5mM

34 Fluorescence Analysis DNA-TO complexes display aclear but wide absorption at around 500 nm A single positivepeak of TO was observed at 452 nm and this wavelength wasused for the excitation of the DNA-TO complex The fluo-rescence spectra of the HTR and THR sequences are shownin Figure 6 The measurements were performed in three dif-ferent environments (i) a mRB buffer without metal cations(ii) a mRB buffer supplemented with 50mM NaCl and (iii)mRB supplementedwith 50mMKCl For both oligomers thefluorescence enhancement achieved the highest yield in thesolutionwithoutmetal cationsThefluorescence yield ofHTRwas greater than the yield of THR in all three of the testedconditions The goal of this experiment was to demonstratethat the profile of fluorescence is not greatly dependent onthe sequence of DNA oligonucleotide for this ligand The


minus15

minus10

0

10

CD (m

deg)

minus10

0

20

10

minus10

0

10

0

CD (m

deg)

minus5

10

5

0

300 400 500 600220Wavelength (nm)

300 400 500 600220Wavelength (nm)

300 400 500 600220Wavelength (nm)

minus5

0

5

minus10

0

10

minus10

0

10

15

minus10

0

10

minus20

0

20

minus20

minus10

0

10

20

minus10

10

CD (m

deg)

minus10

0

10

CD (m

deg)

（４２1

（４２2

（４２3

（４２

（４２12

（４２13

（４２23

（４２013

（４２023

（４２123

G4C2 THR

Figure 4TheCD titration spectra of 27 120583MDNA sample with TO 0 25 5 and 75molar equivalents of TO represent by black green brownand red lines respectively Each sample was measured in a modified 25mMmBR buffer containing 50mMNaCl

fluorescence enhancement of TO can induce the formationof any type of G-quadruplex structure

35 Electrophoresis in the Presence of Na+ K+ and TONondenaturing polyacrylamide gel electrophoresis (PAGE)is an accessible technique which is used to supplementspectroscopic data when the presence of multiple species ofG-quadruplexes cannot readily be identified based on CDspectra alone The mobility of the DNA sample dependson many different factors including conformation chargeand molecular mass Electrophoretic separation can providevaluable information about the molecularity of G-quad-ruplexes Intramolecular G-quadruplexes have a compactstructure and thusmigrate faster through a cation-containinggel than their linear counterparts while intermolecular

G-quadruplexes migrate more slowly due to their highermolecular weight [9 14 44] Oligomers d(AC)9 d(AC)14and d(AC)18 were used as standards due to their lack ofsecondary structures These standards served as benchmarksin comparing the mobility of different electrophoretic pat-terns Since none of the sequences used were longer than22 nt the oligonucleotides which were observed to havemigrated faster than d(AC)9 could be identified as havingformed intramolecular G-quadruplexes It is also reasonableto assume that oligonucleotides which moved more slowlyor at a similar speed to d(AC)18 had adopted high-orderG-quadruplex structures Figure 7 shows the electrophoreticrecords of native 15 polyacrylamide gels illustrating therelative mobilities of the oligomers in the presence of 50mMNaCl and KCl at 10∘C (Figures 7(a) and 7(c)) In addition


minus5

0

10

20CD

(mde

g)

0

10

20

0

10

20

minus25

0

5

75

CD (m

deg)

0

5

15

CD (m

deg)

0

5

15

CD (m

deg)

0

2

4

minus5

0

10

15

minus5

0

10

20

minus5

0

10

15

minus5

0

10

20

0

15

300 400 500 600220Wavelength (nm)

250 400 500 600300Wavelength (nm)

250 400 500 600300Wavelength (nm)

（４２1

（４２2

（４２3

（４２

（４２13

（４２12

（４２23

（４２023

（４２013

（４２123

G4C2 THR

Figure 5 CD titration spectra of 27 120583MDNA sample with TO 0 25 5 and 75 molar equivalents of TO represented by black green brownand red lines respectively Each sample was measured in a modified 25mMmBR buffer containing 50mM KCl

the corresponding electrophoretic results where the gels andloading buffers contain 2 molar equivalents of TO are shownin Figures 7(b) and 7(d) In general some clear trends emergeGel electrophoresis performed in the presence of sodiumshows that all of the oligonucleotides had moved in onebulk with single bands migrating faster than d(AC)18 in eachcolumn This effect was also observed when TO was presentin the gelThese results indicate that all DNAoligonucleotidesform antiparallel intramolecularG-quadruplexes under theseconditions These results agree with the results obtained byCD spectroscopy It is important to note that intramolecularstructures had formed exclusively in the presence of sodiumdespite the introduction of mutations in HTR sequencesincreasing the possibility of the formation of different topolo-gies of G-quadruplexes The electrophoresis did not revealany significant anomalous mobility of oligomers sequences

with the same length were found to move more or lessequally

In contrast to sodium the presence of potassium led tothe formation of both intra and intermolecular arrangements(Figures 7(b) and 7(d)) In the first group the HTR1 quadru-plex with one substitution in the first loop exhibited thefastestmigrating band in comparison to that ofHTR A singlesmear bandwas also observed for theHTR2 sequence Smearstypically arise when two distinct conformers can be formeda slow isomerization between the two conformers duringthe electrophoretic separation is the main source of bandsmearing The mobility of the HTR and HTR3 sequenceswith substitutions in the third loop is similar The oligonu-cleotides containing two substitutions per oligomer displayedhigh levels of polymorphism These oligonucleotides formseveral coexisting conformers because each line contains


0

50

100

150

200

Fl i

nten

sity

600 800700500Wavelength (nm)

Figure 6 Fluorescence emission spectra (au) of TO in the presence of HTR (solid lanes) and THR (dashed lines) in mRB bufferThe spectrawithout the presence of metal cations with 50mMNaCl and 50mMKCl are represented by black blue and red lines respectively Themolarratio of DNA ligand is 1 5 Fluorescence emission of TO is shown in green

NaCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(a)

NaCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(b)

KCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(c)

KCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(d)

Figure 7 Electrophoretic records of studied DNA oligonucleotides Electrophoretic gel and buffer contained 50mMNaCl (a b) and 50mMKCl (b d) gels in (b) and (d) also contained 30 120583M TO 04 120583L of DNA from 1mM stock solution was applied to each electrophoretic well(sim3 120583M) The S-line represents the mobility of the mixture of oligonucleotides d(AC)9 d(AC)14 and d(AC)18


KCl + PEG-200

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

Figure 8 Electrophoretic record of DNA oligonucleotides in thepresence of PEG-200 Electrophoretic gel and buffer contained50mM KCl DNA samples were heated to 95∘C for 5min slowlycooled and then loaded into the electrophoretic wells 04 120583L ofDNA from 1mM stock solution was applied to each electrophoreticwell (sim3120583M) The loading buffer contained 50wt PEG-200

several bands moving at different rates Interestingly theHTR12 and HTR13 sequences displayed two faster well-recognized bands results which correspond to the formationof intramolecular conformers and slower bands representingmultimeric structures The addition of TO also caused thefastest conformers to coalesce and the slowest structuresto diminish HTR23 produced a faster intra- and slowerintermolecular species (dimer and tetramer) Surprisinglythe oligonucleotides with three substitutions per oligomerwere found to be slightly less polymorphic in comparisonwith the sequences containing two substitutions displayingonly bands with lower magnitudes corresponding to theformation of multiple-molecular G-quadruplexes in the caseof the HTR013 and HTR123 sequences TO was found toexert only a limited effect on the multimeric forms of theseoligonucleotides

36 Electrophoresis in the Presence of PEG-200 The depen-dence of HTR dimerization on PEG 200 concentration hasbeen analyzed in previous studies [9] The formation of bothintermolecular dimers and intramolecular monomers wasobserved in the buffer containing a PEG-200 concentration of15wtTheHTR derivatives containing 2 and 3 substitutionswere seen to convert readily to slower migrating dimericstructures even at lower concentrations of PEG-200 At aPEG-200 concentration of 50wt and 50mM KCl thecomplete structural conversion to a parallel dimeric G-quadruplex was induced Figures 3 and 8 This effect was notobserved in buffers that did not contain potassium [9 14]CD measurements at 50wt PEG-200 showed no signalat sim295 nm Based on our previous studies intermolecularspecies which migrate more slowly are indicative of theformation of dimers [2 9 14] The 3D structure of HTRcontaining a flanking sequence in an analogical conditionhas been determined using NMR [11] The results show

an intramolecular parallel G-quadruplex structure (PDB2LD8) but the overhanging nucleotides can cause a sterichindrance for the dimerization of this structure

4 Conclusion

In this study we clearly demonstrate that increasing thenumber of guanines in the loop regions of HTR sequencessupports the formation of G-quadruplex structures Anysubstitution of A-for-G increases the melting temperaturewhile the introduction of several substitutions was found tofacilitate the coexistence of several conformers in the pres-ence of potassium The systematic introduction of these sub-stitutions finally leads to the formation of sequences whichoccur in the Tetrahymena telomere In addition similarsequences were also found in the human genomeThese find-ings raise an interesting point Why does the Tetrahymenatelomere require sequences which can adopt such highlystable G-quadruplex structures In general very stable G-quadruplexes are usually a source of problems in cells duringthe life cycle of an organismTheTHR sequence ismore poly-morphic than HTR it forms two different monomeric andone dimeric conformers as has been shown here and in ourprevious studies [14] Our analysis focused on sequences con-sisting of fourG-runs without any overhanging nucleotides atboth termini this type of arrangement is not an ideal modelfor extrapolation to natural telomeric repeats which typicallyconsist of tens to thousands repeats

Our results demonstrate that all HTR derivatives includ-ingTHRcanbe converted fromantiparallel to parallel folds inthe presence of potassiumandPEG-200 ICD spectra indicatethat the bindingmode of TOwith THR in the presence of KClmight be different from those observed for HTR derivativesand this is a finding which could also be important forother molecules recognizing the THR structure in nature Itsuggests that the structure of THR shows some structuralfeatures which are different from those of HTR and HTRderivatives in the presence of potassium Confirmation of thebiological significance of this fact remains an open topic

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by the Slovak Research and Devel-opment Agency under Contracts nos APVV-0280-11 andAPVV-0029-16 European Cooperation in Science and Tech-nology (COSTCM1406) Slovak Grant Agency (1013116 and002UPJS-42015) and internal university grants (VVGS-PF-2017-251 and VVGS-2016-259)The authors thank G Cowperfor critical reading and correction of the manuscript

References

[1] K W Lim V C M Ng N Martın-Pintado B Heddi and AT Phan ldquoStructure of the human telomere in Na+ solutionAn antiparallel (2+2) G-quadruplex scaffold reveals additional


diversityrdquo Nucleic Acids Research vol 41 no 22 pp 10556ndash10562 2013

[2] V Vıglasky K Tluckova and Lrsquo Bauer ldquoThe first derivative ofa function of circular dichroism spectra Biophysical study ofhuman telomeric G-quadruplexrdquo European Biophysics Journalvol 40 no 1 pp 29ndash37 2011

[3] G N Parkinson M P H Lee and S Neidle ldquoCrystal structureof parallel quadruplexes from human telomeric DNArdquo Naturevol 417 no 6891 pp 876ndash880 2002

[4] Y Wang and D J Patel ldquoSolution structure of the humantelomeric repeat d[AG3(T2AG3)3] G-tetraplexrdquo Structure vol1 no 4 pp 263ndash282 1993

[5] A Ambrus D Chen J Dai T Bialis R A Jones and D YangldquoHuman telomeric sequence forms a hybrid-type intramolec-ular G-quadruplex structure with mixed parallelantiparallelstrands in potassium solutionrdquo Nucleic Acids Research vol 34no 9 pp 2723ndash2735 2006

[6] K N Luu A T Phan V Kuryavyi L Lacroix and D J PatelldquoStructure of the human telomere in K+ solution An intramo-lecular (3 + 1) G-quadruplex scaffoldrdquo Journal of the AmericanChemical Society vol 128 no 30 pp 9963ndash9970 2006

[7] A T Phan V Kuryavyi K N Luu and D J Patel ldquoStructure oftwo intramolecular G-quadruplexes formed by natural humantelomere sequences in K+ solutionrdquoNucleic Acids Research vol35 no 19 pp 6517ndash6525 2007

[8] M Marusic P Sket L Bauer V Viglasky and J Plavec ldquoSo-lution-state structure of an intramolecular G-quadruplex withpropeller diagonal and edgewise loopsrdquoNucleic Acids Researchvol 40 no 14 pp 6946ndash6956 2012

[9] P Tothova P Krafcıkova and V Vıglasky ldquoFormation of highlyordered multimers in G-quadruplexesrdquo Biochemistry vol 53no 45 pp 7013ndash7027 2014

[10] 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

[11] B Heddi and A T Phan ldquoStructure of human telomeric DNAin crowded solutionrdquo Journal of the American Chemical Societyvol 133 no 25 pp 9824ndash9833 2011

[12] M C Miller R Buscaglia J B Chaires A N Lane and J OTrent ldquoHydration is a major determinant of the G-quadruplexstability and conformation of the human telomere 31015840 sequenceof d(AG 3(TTAG3)3)rdquo Journal of the American Chemical Soci-ety vol 132 no 48 pp 17105ndash17107 2010

[13] D Miyoshi A Nakao and N Sugimoto ldquoMolecular crowdingregulates the structural switch of the DNA G-quadruplexrdquoBiochemistry vol 41 no 50 pp 15017ndash15024 2002

[14] V Vıglasky L Bauer and K Tluckova ldquoStructural features ofintra- and intermolecular G-quadruplexes derived from telom-eric repeatsrdquo Biochemistry vol 49 no 10 pp 2110ndash2120 2010

[15] J L Huppert and S Balasubramanian ldquoPrevalence of quadru-plexes in the human genomerdquo Nucleic Acids Research vol 33no 9 pp 2908ndash2916 2005

[16] A K Todd M Johnston and S Neidle ldquoHighly prevalentputative quadruplex sequence motifs in human DNArdquo NucleicAcids Research vol 33 no 9 pp 2901ndash2907 2005

[17] J Eddy andNMaizels ldquoGene function correlates with potentialfor G4 DNA formation in the human genomerdquo Nucleic AcidsResearch vol 34 no 14 pp 3887ndash3896 2006

[18] A Henderson Y Wu Y C Huang et al ldquoDetection of G-quadruplex DNA in mammalian cellsrdquo Nucleic Acids Researchvol 42 no 2 pp 860ndash869 2014

[19] R F Hoffmann Y M Moshkin S Mouton et al ldquoGuaninequadruplex structures localize to heterochromatinrdquo NucleicAcids Research vol 44 no 1 pp 152ndash163 2016

[20] V S Chambers G Marsico J M Boutell M Di Antonio G PSmith and S Balasubramanian ldquoHigh-throughput sequencingof DNA G-quadruplex structures in the human genomerdquoNature Biotechnology vol 33 no 8 pp 877ndash881 2015

[21] E H Blackburn ldquoTelomere states and cell fatesrdquo Nature vol408 no 6808 pp 53ndash56 2000

[22] M J McEachern A Krauskopf and E H Blackburn ldquoTelom-eres and their controlrdquo Annual Review of Genetics vol 34 pp331ndash358 2000

[23] N Grandin andM Charbonneau ldquoProtection against chromo-some degradation at the telomeresrdquo Biochimie vol 90 no 1 pp41ndash59 2008

[24] W E Wright V M Tesmer K E Huffman S D Levene andJ W Shay ldquoNormal human chromosomes have long G-richtelomeric overhangs at one endrdquo Genes amp Development vol 11no 21 pp 2801ndash2809 1997

[25] J A Londono-Vallejo ldquoTelomere instability and cancerrdquoBiochimie vol 90 no 1 pp 73ndash82 2008

[26] C B Harley ldquoTelomere loss mitotic clock or genetic timebombrdquoMutation Research DNAging vol 256 no 2ndash6 pp 271ndash282 1991

[27] A J Sfeir W Chai J W Shay andW EWright ldquoTelomere-endprocessing The terminal nucleotidesof human chromosomesrdquoMolecular Cell vol 18 no 1 pp 131ndash138 2005

[28] L M Colgin and R R Reddel ldquoTelomere maintenancemechanisms and cellular immortalizationrdquo Current Opinion inGenetics amp Development vol 9 no 1 pp 97ndash103 1999

[29] A M Zahler J R Williamson T R Cech and D M PrescottldquoInhibition of telomerase by G-quartet DNA structuresrdquoNature vol 350 no 6320 pp 718ndash720 1991

[30] S Neidle and G Parkinson ldquoTelomere maintenance as a targetfor anticancer drug discoveryrdquo Nature Reviews Drug Discoveryvol 1 no 5 pp 383ndash393 2002

[31] KW Lim S Amrane S Bouaziz et al ldquoStructure of the humantelomere in K + solution A stable basket-type G-quadruplexwith only twoG-tetrad layersrdquo Journal of theAmericanChemicalSociety vol 131 no 12 pp 4301ndash4309 2009

[32] R BuscagliaM CMillerW L Dean et al ldquoPolyethylene glycolbinding alters human telomere G-quadruplex structure by con-formational selectionrdquoNucleic Acids Research vol 41 no 16 pp7934ndash7946 2013

[33] L Petraccone A Malafronte J Amato and C GiancolaldquoG-quadruplexes from human telomeric DNA How manyconformations in PEG containing solutionsrdquo The Journal ofPhysical Chemistry B vol 116 no 7 pp 2294ndash2305 2012

[34] V Viglasky L Bauer K Tluckova and P Javorsky ldquoEvaluationof human telomeric G-quadruplexes The influence of over-hanging sequences on quadruplex stability and foldingrdquo Journalof Nucleic Acids vol 2010 Article ID 820356 2010

[35] MVorlıckovaM Tomasko A J Sagi K Bednarova and J Sagildquo8-Oxoguanine in a quadruplex of the human telomere DNAsequencerdquo FEBS Journal vol 279 no 1 pp 29ndash39 2012

[36] D Renciuk I Kejnovska P Skolakova K Bednarova J Mot-lova and M Vorlıckova ldquoArrangements of human telomereDNA quadruplex in physiologically relevant K+ solutionsrdquoNucleic Acids Research vol 37 no 19 pp 6625ndash6634 2009

[37] M Tomasko M Vorlıckova and J Sagi ldquoSubstitution of ade-nine for guanine in the quadruplex-forming human telomere


DNA sequence G3(T2AG3)3rdquo Biochimie vol 91 no 2 pp 171ndash179 2009

[38] M Vorlıckova J Chladkova I Kejnovska M Fialova and JKypr ldquoGuanine tetraplex topology of human telomere DNA isgoverned by the number of (TTAGGG) repeatsrdquo Nucleic AcidsResearch vol 33 no 18 pp 5851ndash5860 2005

[39] I Kejnovska K Bednarova D Renciuk et al ldquoClustered abasiclesions profoundly change the structure and stability of humantelomeric G-quadruplexesrdquo Nucleic Acids Research vol 45 no8 pp 4294ndash4305 2017

[40] H Konvalinova Z Dvorakova D Renciuk et al ldquoDiverseeffects of naturally occurring base lesions on the structure andstability of the human telomere DNA quadruplexrdquo Biochimievol 118 article no 4773 pp 15ndash25 2015

[41] M Babinsky R Fiala I Kejnovska et al ldquoLoss of loop adeninesalters human telomere d(AG(3)(TTAG(3))(3)) quadruplexfoldingrdquoNucleic Acids Research vol 42 no 22 pp 14031ndash140412014

[42] CWGreider and EH Blackburn ldquoA telomeric sequence in theRNA of Tetrahymena telomerase required for telomere repeatsynthesisrdquo Nature vol 337 no 6205 pp 331ndash337 1989

[43] I Lubitz D Zikich and A Kotlyar ldquoSpecific high-affinity bind-ing of thiazole orange to triplex and g-quadruplex DNArdquo Bio-chemistry vol 49 no 17 pp 3567ndash3574 2010

[44] P Krafcıkova E Demkovicova and V Vıglasky ldquoEbola virusderivedG-quadruplexesThiazole orange interactionrdquoBiochim-ica et Biophysica Acta (BBA) - General Subjects vol 1861 no 5pp 1321ndash1328 2016

[45] J Mohanty N Barooah V Dhamodharan S Harikrishna PI Pradeepkumar and A C Bhasikuttan ldquoThioflavin T as anefficient inducer and selective fluorescent sensor for the humantelomeric G-quadruplex DNArdquo Journal of the American Chem-ical Society vol 135 no 1 pp 367ndash376 2013

[46] J B Chaires ldquoHuman telomeric G-quadruplex Thermody-namic and kinetic studies of telomeric quadruplex stabilityrdquoFEBS Journal vol 277 no 5 pp 1098ndash1106 2010

[47] Y Wang and D J Patel ldquoSolution Structure of a Parallel-stranded G-Quadruplex DNArdquo Journal of Molecular Biologyvol 234 no 4 pp 1171ndash1183 1993

[48] Y Wang and D J Patel ldquoSolution structure of the Tetrahymenatelomeric repeat d(T2G4)4 G-tetraplexrdquo Structure vol 2 no 12pp 1141ndash1156 1994

[49] J Dai C Punchihewa A Ambrus D Chen R A Jones andD Yang ldquoStructure of the intramolecular human telomeric G-quadruplex in potassium solution A novel adenine triple for-mationrdquo Nucleic Acids Research vol 35 no 7 pp 2440ndash24502007

[50] J Dai M Carver C Punchihewa R A Jones and D YangldquoStructure of the hybrid-2 type intramolecular human telomericG-quadruplex in K+ solution Insights into structure poly-morphism of the human telomeric sequencerdquo Nucleic AcidsResearch vol 35 no 15 pp 4927ndash4940 2007

[51] Y Wang and D J Patel ldquoSolution Structure of the OxytrichaTelomeric Repeat d[G4(T4G4)3] G-tetraplexrdquo Journal ofMolec-ular Biology vol 251 no 1 pp 76ndash94 1995

[52] F W Smith P Schultze and J Feigon ldquoSolution structures ofunimolecular quadruplexes formed by oligonucleotides con-taining Oxytricha telomere repeatsrdquo Structure vol 3 no 10 pp997ndash1008 1995

[53] J Brcic and J Plavec ldquoSolution structure of a DNA quadruplexcontaining ALS and FTD related GGGGCC repeat stabilized by

8-bromodeoxyguanosine substitutionrdquo Nucleic Acids Researchvol 43 no 17 pp 8590ndash8600 2015

[54] J Kypr I Kejnovska D Renciuk and M Vorlıckova ldquoCirculardichroism and conformational polymorphism of DNArdquoNucleicAcids Research vol 37 no 6 pp 1713ndash1725 2009

[55] A I KarsisiotisNMHessari ENovellinoG P SpadaA Ran-dazzo and M Webba Da Silva ldquoTopological characterizationof nucleic acid G-quadruplexes by UV absorption and circulardichroismrdquo Angewandte Chemie International Edition vol 50no 45 pp 10645ndash10648 2011

[56] DMiyoshi T Fujimoto andN Sugimoto ldquoMolecular crowdingand hydration regulating of G-quadruplex formationrdquo Topics inCurrent Chemistry vol 330 pp 87ndash110 2013

[57] D M Gray J-D Wen C W Gray et al ldquoMeasured and cal-culated CD spectra of G-quartets stacked with the same oropposite polaritiesrdquoChirality vol 20 no 3-4 pp 431ndash440 2008

[58] CAllainDMonchaud andM-P Teulade-Fichou ldquoFRET tem-plated by G-quadruplex DNA A specific ternary interactionusing an original pair of donoracceptor partnersrdquo Journal ofthe American Chemical Society vol 128 no 36 pp 11890ndash118932006

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 DNA oligodeoxynucleotides used in this study and their occurrence in human and Tetrahymena genomes

Name 120576a

(mMminus1 cmminus1) Sequenceb (51015840 rarr 31015840) Occurrence in genome (ID)Human Tetrahymena

HTR 2254 GGGTTAGGGTTAGGGTTAGGG Very high -HTR1 2221 GGGTTGGGGTTAGGGTTAGGG NG 0549151 M1162711HTR2 2221 GGGTTAGGGTTGGGGTTAGGG NG 0295331 -HTR3 2221 GGGTTAGGGTTAGGGTTGGGG NT 1876531 -HTR12 2188 GGGTTGGGGTTGGGGTTAGGG NW 0035710491 M116271HTR13 2188 GGGTTGGGGTTAGGGTTGGGG NC 0189262 M116271HTR23 2188 GGGTTAGGGTTGGGGTTGGGG NC 0189142 M116271HTR013 2300 GGGGTTGGGGTTAGGGTTGGGG - M116271HTR023 2300 GGGGTTAGGGTTGGGGTTGGGG NC 0189302 M116271HTR123 2154 GGGTTGGGGTTGGGGTTGGGG NC 0189122 AH0011122THR 2134 GGGGTTGGGGTTGGGGTTGGGG NG 0340201 Very highG4C2 2044 GGGGCCGGGGCCGGGGCCGGGG NG 0528101 -(AC)9 1719 (AC)9 ND ND(AC)18 3429 (AC)18 ND NDaMilimolar extinction coefficient at 257 nm bBasemodifications are underlined ND not determined

unlikely that these putative sequences can form in vivo anddirect evidence of their existence in living cells is still a topicof discussion [18ndash20] Undoubtedly the most extensivelystudied G-quadruplex forming sequences are those locatedat the 31015840-ends of human telomeres Telomeric sequences andspecialized nucleoprotein complexes which cap the ends oflinear chromosomes are essential for chromosomal stabilityand genomic integrity [21ndash23] Mammalian telomeres consistof tandem repeats of G-rich sequences d(TTAGGG)119899 Sev-eral kilobases of this sequence are double-stranded but morethan a hundred nucleotides remain unpaired and form single-stranded 31015840-overhangs [24] a state which would providefavourable conditions for the formation of one or more G-quadruplexes in vivo [22] The structure and stability oftelomeres play a significant role in the development of cancerand cell aging [25 26] There is also evidence that telomeresserve as a type of biological clock as telomere structuresappear to become shorter with each successive cell cycle Inimmortalized cells and in cancer cells however a telomeraseis activated to maintain the length of the telomere byreelongating the telomeric sequence at the chromosome ends[27 28] G-quadruplexes formed by single-stranded humantelomeric DNA have also been shown to inhibit the activityof telomerase [29] and this discovery has led to increasedinterest in the structures as attractive potential drug targets[30]

A broad range of studies of human telomeric G-quadru-plexes have been carried out using a wide variety of differenttechniques [1] To date high-resolution structures of fourdistinct folding topologies with three G-tetrad layers havebeen identified for the four human telomeric repeats [1ndash7]In addition an additional structure consisting of only twoG-tetrad layers has also been revealed which highlights thestructural polymorphism of telomeric G-quadruplexes [31]The structure of human telomeric DNA in crowded solu-tions has also been investigated by many authors [11] but

2 3

Tetrahymena

Human

10AGGGTTAGGGTTAGGGTTAGGG

GGGGTTGGGGTTGGGGTTGGGG

Figure 1 The HTR to THR conversion by substitutions of adeninefor guanine

this structure is likely to be a result of dehydration ratherthan molecular crowding [12 32 33] The great variety ofstructures identified to date can also be attributed to thepresence of flanking nucleotides outside the core sequenceG3(T2AG3)3 and the concentration of ions and to the use ofdifferent experimental methods and conditions [1 34]

A series of systematic studies concerning the sequencederivatives of human telomeric repeats were carried out byVorlıckova et al [35ndash38] and these earlier studies focusedon the substitution of guanine for adenine the introductionof abasic sites 8-oxoadenine replacing adenine and thesubstitution of 5-hydroxymethyluracil for thymine in telom-eric repeats were analyzed [39ndash41] However in this studyan opposite strategy is applied the substitution of adeninefor guanine (see Figure 1) The main aim is to achieve thetotal conversion of four human repeats to Tetrahymenarepeats which retain the ability to form intramolecularG-quadruplex Interestingly G-rich repetitions containingimperfections were also found in the human and Tetrahy-mena genome see Table 1 and Supporting Materials

In this study we examine the structures formed by theTetrahymena telomeric sequence dG4(T2G4)3 which differs












minus3

0

4

HTR（４２1

（４２2

（４２3

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

8

5

0

minus3

（４２12

（４２13

（４２23

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

10

5

0

THR（４２123（４２013

（４２023

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

HTR（４２1

（４２2

（４２3

minus2

0

2

4

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

minus2

0

2

4

（４２12

（４２13

（４２23

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

2

0

2

4

THR（４２123（４２013

（４２023

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

(a) (d)

(b) (e)

(c) (f)





















10

minus2

5

0

PEG-200

CD

240 260 280 300 320220Wavelength (nm)

5030

150

(a)

Acetonitrile

240 260 280 300 320220Wavelength (nm)

0

5

10

CD

5030

150

(b)

CD

240 260 280 300 320220Wavelength (nm)

0

5

10

PEG-200

5030

150

(c)

240 260 280 300 320220Wavelength (nm)

CD

10

5

0

Acetonitrile

5030

150

(d)




∘C) in 50mM NaCl + PEG-2000 15 30 50 0 15 30 50











minus15

minus10

0

10

CD (m

deg)

minus10

0

20

10

minus10

0

10

0

CD (m

deg)

minus5

10

5

0

300 400 500 600220Wavelength (nm)

300 400 500 600220Wavelength (nm)

300 400 500 600220Wavelength (nm)

minus5

0

5

minus10

0

10

minus10

0

10

15

minus10

0

10

minus20

0

20

minus20

minus10

0

10

20

minus10

10

CD (m

deg)

minus10

0

10

CD (m

deg)

（４２1

（４２2

（４２3

（４２

（４２12

（４２13

（４２23

（４２013

（４２023

（４２123

G4C2 THR






minus5

0

10

20CD

(mde

g)

0

10

20

0

10

20

minus25

0

5

75

CD (m

deg)

0

5

15

CD (m

deg)

0

5

15

CD (m

deg)

0

2

4

minus5

0

10

15

minus5

0

10

20

minus5

0

10

15

minus5

0

10

20

0

15

300 400 500 600220Wavelength (nm)

250 400 500 600300Wavelength (nm)

250 400 500 600300Wavelength (nm)

（４２1

（４２2

（４２3

（４２

（４２13

（４２12

（４２23

（４２023

（４２013

（４２123

G4C2 THR






0

50

100

150

200

Fl i

nten

sity



NaCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(a)

NaCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(b)

KCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(c)

KCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(d)



KCl + PEG-200

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR





4 Conclusion





Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












minus3

0

4

HTR（４２1

（４２2

（４２3

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

8

5

0

minus3

（４２12

（４２13

（４２23

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

10

5

0

THR（４２123（４２013

（４２023

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

HTR（４２1

（４２2

（４２3

minus2

0

2

4

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

minus2

0

2

4

（４２12

（４２13

（４２23

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

2

0

2

4

THR（４２123（４２013

（４２023

240 260 280 300 320220Wavelength (nm)

Δ


minus1)

(a) (d)

(b) (e)

(c) (f)





















10

minus2

5

0

PEG-200

CD

240 260 280 300 320220Wavelength (nm)

5030

150

(a)

Acetonitrile

240 260 280 300 320220Wavelength (nm)

0

5

10

CD

5030

150

(b)

CD

240 260 280 300 320220Wavelength (nm)

0

5

10

PEG-200

5030

150

(c)

240 260 280 300 320220Wavelength (nm)

CD

10

5

0

Acetonitrile

5030

150

(d)




∘C) in 50mM NaCl + PEG-2000 15 30 50 0 15 30 50











minus15

minus10

0

10

CD (m

deg)

minus10

0

20

10

minus10

0

10

0

CD (m

deg)

minus5

10

5

0

300 400 500 600220Wavelength (nm)

300 400 500 600220Wavelength (nm)

300 400 500 600220Wavelength (nm)

minus5

0

5

minus10

0

10

minus10

0

10

15

minus10

0

10

minus20

0

20

minus20

minus10

0

10

20

minus10

10

CD (m

deg)

minus10

0

10

CD (m

deg)

（４２1

（４２2

（４２3

（４２

（４２12

（４２13

（４２23

（４２013

（４２023

（４２123

G4C2 THR






minus5

0

10

20CD

(mde

g)

0

10

20

0

10

20

minus25

0

5

75

CD (m

deg)

0

5

15

CD (m

deg)

0

5

15

CD (m

deg)

0

2

4

minus5

0

10

15

minus5

0

10

20

minus5

0

10

15

minus5

0

10

20

0

15

300 400 500 600220Wavelength (nm)

250 400 500 600300Wavelength (nm)

250 400 500 600300Wavelength (nm)

（４２1

（４２2

（４２3

（４２

（４２13

（４２12

（４２23

（４２023

（４２013

（４２123

G4C2 THR






0

50

100

150

200

Fl i

nten

sity



NaCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(a)

NaCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(b)

KCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(c)

KCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(d)



KCl + PEG-200

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR





4 Conclusion





Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















10

minus2

5

0

PEG-200

CD

240 260 280 300 320220Wavelength (nm)

5030

150

(a)

Acetonitrile

240 260 280 300 320220Wavelength (nm)

0

5

10

CD

5030

150

(b)

CD

240 260 280 300 320220Wavelength (nm)

0

5

10

PEG-200

5030

150

(c)

240 260 280 300 320220Wavelength (nm)

CD

10

5

0

Acetonitrile

5030

150

(d)




∘C) in 50mM NaCl + PEG-2000 15 30 50 0 15 30 50











minus15

minus10

0

10

CD (m

deg)

minus10

0

20

10

minus10

0

10

0

CD (m

deg)

minus5

10

5

0

300 400 500 600220Wavelength (nm)

300 400 500 600220Wavelength (nm)

300 400 500 600220Wavelength (nm)

minus5

0

5

minus10

0

10

minus10

0

10

15

minus10

0

10

minus20

0

20

minus20

minus10

0

10

20

minus10

10

CD (m

deg)

minus10

0

10

CD (m

deg)

（４２1

（４２2

（４２3

（４２

（４２12

（４２13

（４２23

（４２013

（４２023

（４２123

G4C2 THR






minus5

0

10

20CD

(mde

g)

0

10

20

0

10

20

minus25

0

5

75

CD (m

deg)

0

5

15

CD (m

deg)

0

5

15

CD (m

deg)

0

2

4

minus5

0

10

15

minus5

0

10

20

minus5

0

10

15

minus5

0

10

20

0

15

300 400 500 600220Wavelength (nm)

250 400 500 600300Wavelength (nm)

250 400 500 600300Wavelength (nm)

（４２1

（４２2

（４２3

（４２

（４２13

（４２12

（４２23

（４２023

（４２013

（４２123

G4C2 THR






0

50

100

150

200

Fl i

nten

sity



NaCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(a)

NaCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(b)

KCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(c)

KCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(d)



KCl + PEG-200

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR





4 Conclusion





Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










minus15

minus10

0

10

CD (m

deg)

minus10

0

20

10

minus10

0

10

0

CD (m

deg)

minus5

10

5

0

300 400 500 600220Wavelength (nm)

300 400 500 600220Wavelength (nm)

300 400 500 600220Wavelength (nm)

minus5

0

5

minus10

0

10

minus10

0

10

15

minus10

0

10

minus20

0

20

minus20

minus10

0

10

20

minus10

10

CD (m

deg)

minus10

0

10

CD (m

deg)

（４２1

（４２2

（４２3

（４２

（４２12

（４２13

（４２23

（４２013

（４２023

（４２123

G4C2 THR






minus5

0

10

20CD

(mde

g)

0

10

20

0

10

20

minus25

0

5

75

CD (m

deg)

0

5

15

CD (m

deg)

0

5

15

CD (m

deg)

0

2

4

minus5

0

10

15

minus5

0

10

20

minus5

0

10

15

minus5

0

10

20

0

15

300 400 500 600220Wavelength (nm)

250 400 500 600300Wavelength (nm)

250 400 500 600300Wavelength (nm)

（４２1

（４２2

（４２3

（４２

（４２13

（４２12

（４２23

（４２023

（４２013

（４２123

G4C2 THR






0

50

100

150

200

Fl i

nten

sity



NaCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(a)

NaCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(b)

KCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(c)

KCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(d)



KCl + PEG-200

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR





4 Conclusion





Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


minus5

0

10

20CD

(mde

g)

0

10

20

0

10

20

minus25

0

5

75

CD (m

deg)

0

5

15

CD (m

deg)

0

5

15

CD (m

deg)

0

2

4

minus5

0

10

15

minus5

0

10

20

minus5

0

10

15

minus5

0

10

20

0

15

300 400 500 600220Wavelength (nm)

250 400 500 600300Wavelength (nm)

250 400 500 600300Wavelength (nm)

（４２1

（４２2

（４２3

（４２

（４２13

（４２12

（４２23

（４２023

（４２013

（４２123

G4C2 THR






0

50

100

150

200

Fl i

nten

sity



NaCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(a)

NaCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(b)

KCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(c)

KCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(d)



KCl + PEG-200

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR





4 Conclusion





Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

50

100

150

200

Fl i

nten

sity



NaCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(a)

NaCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(b)

KCl

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(c)

KCl + TO

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR

(d)



KCl + PEG-200

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR





4 Conclusion





Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


KCl + PEG-200

（４２

1

（４２

2

（４２

3

（４２

12

（４２

13

（４２

23

（４２

013

（４２

023

（４２

123

S HTR

THR





4 Conclusion





Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

telomeric g-quadruplexes: from human to tetrahymena...

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