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Interaction of YOYO-1 with guanine-rich DNAShohini Ghosh Datta b , Christopher Reynolds b , Yugender K. Goud a & Bhaskar Datta aa Department of Chemistry , Indian Institute of Technology Gandhinagar , VGEC ComplexChandkheda, Ahmedabad , 382424 , Indiab Department of Chemistry , Missouri State University , 901 S. National Avenue, Springfield ,MO , 65897 , USAPublished online: 05 Jul 2013.

To cite this article: Journal of Biomolecular Structure and Dynamics (2013): Interaction of YOYO-1 with guanine-rich DNA,Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2013.807752

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Interaction of YOYO-1 with guanine-rich DNA

Shohini Ghosh Dattab, Christopher Reynoldsb, Yugender K. Gouda and Bhaskar Dattaa*aDepartment of Chemistry, Indian Institute of Technology Gandhinagar, VGEC Complex Chandkheda, Ahmedabad, 382424, India;bDepartment of Chemistry, Missouri State University, 901 S. National Avenue, Springfield, MO 65897, USA

Communicated by Ramaswamy H. Sarma

(Received 5 October 2012; final version received 20 May 2013)

The oxazole homodimer YOYO-1 has served as a valuable tool for the detection and quantification of nucleic acids.While the base specificity and selectivity of binding of YOYO-1 has been researched to some extent, the effect ofunorthodox nucleic acid conformations on dye binding has received relatively less attention. In this work, we attempt tocorrelate the quadruplex-forming ability of G-rich sequences with binding of YOYO-1. Oligonucleotides differing in thenumber of tandem G repeats, total length, and length of loop sequence were evaluated for their ability to form quadru-plexes in presence of sodium (Na+) or potassium (K+) ions. The fluorescence behavior of YOYO-1 upon binding suchG-rich sequences was also ascertained. A distinct correlation was observed between the strength and propensity of quad-ruplex formation, and the affinity of YOYO-1 to bind such sequences. Specifically, as exemplified by the oligonucleo-tides 5′-G4T2G4-3′ and 5′-G3TG3TG3-3′, sequences possessing longer G-rich regions and shorter loop sequencesformed stronger quadruplexes in presence of K+ which translated to weaker binding of YOYO-1. The dependence ofbinding of YOYO-1 on sequence and structural features of G-rich DNA has not been explored previously and such stud-ies are expected to aid in more effective interpretation of applications involving the fluorophore.

Keywords: YOYO-1; G-quadruplex; quadruplex topology; G-quartet dependence on monovalent cations; fluorescenceenhancement of intercalating dyes; nucleic acid staining reagents

1. Introduction

The enhanced ability to visualize and track biomoleculeshas formed the cornerstone of revolutionary growth inmolecular biology and biochemistry (Massoud &Gambhir, 2003; Miyakawa, Sawano, & Kogure, 2003;Prescher & Bertozzi, 2005). Over the past couple of dec-ades, advances in fluorescence technology have provideda more accessible route to tagging and imaging ofnucleic acids compared with radioactivity (Ballal,Cheema, Ahmad, Rosen, & Saha, 2009). In this regard,the discovery of small molecules that form stable andhighly fluorescent complexes with DNA has been abreakthrough in the development of DNA probes (Ahn,Costa, & Emanuel, 1996; Dragan et al., 2010; Glazer,Peck, & Mathies, 1990; Zipper, Brunner, Bernhagen, &Vitzthum, 2004). Such dye fluorophores have become anextremely versatile tool for studying the statistical–mechanical properties and biochemical interactions ofdouble-stranded DNA (Krishnamoorthy, Duportail, &Mely, 2002; Perkins, Smith, & Chu, 1994; Shimizu,Sasaki, & Tsuruoka, 2005).

YOYO-1 is a cyanine dye that is one of the mostcommonly used fluorophores to stain DNA (Glazer &Rye, 1992; Rye, Debora, Quesada, Mathies, & Glazer,1993; Rye et al., 1992). The dye is a homodimer ofoxazole yellow (YO) with the monomers bridged by abis-cationic linker (Figure 1). YO and YOYO-1 areassumed to bind DNA through intercalation and bis-inter-calation, respectively (Larsson, Carlsson, Jonsson, &Albinsson, 1994). They are almost nonfluorescent whenfree in solution, but exhibit a strong increase in fluores-cence quantum yield when bound to double-strandedDNA. The low background fluorescence from YOYO-1has made it an excellent probe for highly sensitive quanti-fication of DNA (Rye et al., 1993) visualizing DNAundergoing electrophoresis, (Rye, Drees, Nelson, &Glazer, 1993) analyzing DNA-ligand interactions(Kirschtein, Sip, & Kittler, 2000), and for imaging DNAmolecules (Hirons, Fawcett, & Crissman, 1994;Namasivayam, Larson, Burke, & Burns, 2002).

The use of YOYO-1 in staining and imaging DNAhas almost adopted a one-size-fits-all approach. While

*Corresponding author. Email: bdatta@iitgn.ac.in

Journal of Biomolecular Structure and Dynamics, 2013http://dx.doi.org/10.1080/07391102.2013.807752

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YOYO-1 has been used for detecting and staining sin-gle-stranded DNA, (Rye & Glazer, 1995) the role ofsequence and conformation in the binding interactions ofDNA oligomers has been explored to a limited extent(Abramo, Pitner, & McGown, 1998; Joseph, Taylor,McGown, Pitner, & Linn, 1996; Netzel, Nafisi, Zhao,Lenhard, & Johnson, 1995). In this context, the abilityof G-rich DNA and RNA sequences to form higher-orderstructures or quadruplexes is notable for its implicationon YOYO-1 binding. G-quadruplexes or tetraplexes arefour-stranded helical DNA or RNA structures arisingfrom the planar association of four guanines in a cyclichydrogen-bonding arrangement (Burge, Parkinson, Hazel,Todd, & Neidle, 2006; Gellert, Lipsett, & Davies, 1962;Howard, Frazier, & Miles, 1977; Huppert & Balasubra-manian, 2005; Williamson, 1994). G-rich regions areespecially prevalent at the ends of telomeric DNA in

eukaryotic chromosomes and in oncogene promoterregions (Paeschke, Simonsson, Postberg, Rhodes, &Lipps, 2005; Sundquist & Klug, 1989; Verma, Yadav,Basundra, Kumar, & Chowdhury, 2009). The G-richsequences found in telomeric and promoter regionscomprise tandem repeats and have short non-G tractsregularly interspersing the G ones (Hazel, Huppert, Bala-subramanian, & Neidle, 2004; Parkinson, Lee, & Neidle,2002; Phan, Luu, & Patel, 2006). The length of Grepeats and the length and identity of interspersing bases,as well as the nature of monovalent cations present insolution, are important determinants in quadruplex fold-ing and secondary structure (Cevec & Plavec, 2005;Cmugelj, Sket, & Plavec, 2003; Hazel et al., 2004;Risitano & Fox, 2004). In this regard, the formation ofquadruplexes comprising diverse folding topologies pre-sents an opportunity to test and possibly expand thescope for use of fluorophores such as YOYO-1. In par-ticular, a given G-rich DNA may fold into a number ofdifferent conformations, not all of which may be equallyaccessible for binding by the fluorophore.

We report here on the fluorescence behavior ofYOYO-1 in the presence of G-rich DNA sequences ofvarying lengths and compositions as we attempt to cor-relate the quadruplex-forming ability of the G-richsequences with the binding of YOYO-1. Quadruplexformation by the G-rich sequences was examined bycircular dichroism (CD) and UV-visible absorptionspectroscopy, while the interaction of YOYO-1 with theDNA oligomers was investigated through the fluores-cence enhancement of the dye. The G-rich sequencesstudied in this work (see Table 1) differ in their totallength, number of Gs in each tandem repeat unit, andnumber of Ts in interspersing regions. Our results showthat these variations affect the propensity and types ofquadruplex secondary structures that can be formed insolution which in turn affect the binding of YOYO-1.The nature of monovalent cation (Na+ or K+) used insolution clearly influences the ability of the G-richsequences to form quadruplexes of varying strengths.We show that small differences in the sequence andcomposition of G-rich DNA oligonucleotides in con-junction with specific monovalent cations can lead tosignificant differences in the fluorescence enhancement

Figure 1. Comparative fluorescence spectra of YOYO-1 inpresence of various G-rich oligonucleotides. Samples contain2 μM DNA and 1 μM YOYO-1 in presence of (A) 10mMNaCl and (B) 10mM KCl. Spectra were obtained by excitationat 488 nm.

Table 1. Sequences of G-rich oligonucleotides used in currentwork.

Name Sequence

G4T4-2 5′-GGG GTT TTG GGG TTT T-3′G4T4G4 5′-GGG GTT TTG GGG-3′G4T2G4 5′-GGG GTT GGG G-3′G3T3-2 5′-GGG TTT GGG TTT-3′G3T3G3 5′-GGG TTT GGG-3′G3TG3TG3 5′-GGG TGG GTG GG-3′TBA 5′-GGTTGGTGTGGTTGG-3′

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of YOYO-1. Our results expand the scope of usage ofYOYO-1 in the context of unusual DNA secondarystructures and highlight its potential use in distinguish-ing quadruplex morphologies.

2. Experimental

2.1. Materials

YOYO-1 was purchased from Invitrogen (Eugene, OR)and diluted to a 40 μM solution in DMSO. Dye aliquotsto be used for fluorescence spectroscopy were preparedimmediately prior to use to minimize photobleaching.Oligonucleotides were either purchased from IntegratedDNA Technologies (Coralville, IA, USA) and SigmaAldrich Chemical Co. (Bangalore, India) or synthesizedon an Applied Biosystems 392 DNA/RNA Synthesizerusing standard phosphoramidite chemistry, and reagentsfrom Glen Research (Sterling, VA). Oligonucleotideswere ammonia deprotected at 55 °C overnight and puri-fied using low-melting gel electrophoresis, or by usingreverse-phase cartridges on oligonucleotides synthesizedwith the hydrophobic dimethoxytrityl group. Extinctioncoefficients of the synthesized oligonucleotides were cal-culated by the nearest neighbor method (Tataurov, You,& Owczarzy, 2008). Absorbance values of purifiedsequences were obtained and used to calculate the work-ing concentrations of our DNA/RNA sequences.

2.2. Sample preparation

Samples containing 2 μM DNA were prepared in 20mMphosphate buffer pH 7.0. The samples contained either10mM NaCl or 10mM KCl and DNase-free water wasused to make up the total volume to 1mL. The G-richoligo samples were annealed by heating to 85–90 °C fol-lowed by gradual cooling to room temperature. A mixedsequence oligonucleotide 5′-GACTGCTAGTCTTACG-3′and its complementary sequence were used as a controlduplex for comparison of YOYO-1 fluorescence. In theexperiments concerning thrombin-binding aptamer (TBA)and D1, the oligos were annealed together in similar con-ditions as above, but with 100mM NaCl or 100mM KCl.

2.3. Fluorescence studies

YOYO-1 was added in 2.5 μL aliquots to oligonucleotidesamples for fluorescence studies. Emission spectra wererecorded on a Perkin Elmer LS 45 fluorescence spec-trometer by exciting YOYO-1 at 488 nm. Instrument slitwidths were controlled by the operating software andwere kept consistent at 5 nm. The intensity of fluores-cence at 510 nm was recorded as the maximum fluores-cence for most samples; in some cases, the intensity offluorescence at 515 nm was used in similar fashion. Allexperiments were performed in triplicate. Associationconstants were estimated from fluorescence titration datausing a previously reported method (Joseph et al., 1996).

2.4. UV-visible and CD spectroscopy

UV-thermal denaturation of G-rich oligonucleotidesamples was performed on a Perkin-Elmer Lambda 650spectrophotometer with a PTP 1 + 1 Peltier system tem-perature controller. LabTemp software on the instrumentwas used to set up UV-thermal denaturation experiments,where absorbance at 260 and 295 nm was recorded whileincreasing the temperature in 3 or 5 °C increments. Sam-ples were held at each temperature for five minutes toallow for better equilibration. CD spectroscopy was per-formed on a JASCO J-815 spectropolarimeter and allspectra were recorded at 25 °C. Samples containingDNA oligos, used for titration of YOYO-1 in fluores-cence studies, were prepared in identical fashion for UV-thermal denaturation and CD spectroscopic studies.

3. Results and discussion

We investigated the interaction of G-rich DNA oligonu-cleotides with YOYO-1 in the context of (a) compositionof tandem repeats in the sequences, (b) the role of mono-valent cations, Na+ and K+ and, (c) stability of quadru-plexes being formed. Previous research in this area albeitlimited has pointed to YOYO-1 being sensitive to con-formational differences among DNA sequences (Abramoet al., 1998; Simon, Abramo, Sell, & McGown, 1998).The G-rich DNA sequences used in the present work areshown in Table 1. The oligonucleotides varied in thenumber of repeating Gs, the number of interspersed Ts,as well as the total length of the sequence. First, the abil-ity of YOYO-1 to bind with G-rich DNA sequences wasinvestigated by measuring fluorescence enhancement ofthe dye. As shown in Figure 1(A), the intensity of fluo-rescence emission of YOYO-1 in presence of the G-richsequences is lower than a mixed base duplex of compa-rable length. Notably, the intensity of fluorescence emis-sion of YOYO-1 upon binding G-rich DNAs changesdepending on the identity of the sequence and the pres-ence of Na+ or K+. We investigated the effect of Na+

and K+ on the G-rich sequences employed in the currentwork by UV-thermal denaturation and CD spectroscopy.The number and size of tandem repeating units in G-richsequences is known to affect the topology of G-quadru-plex formation. As shown in Figure 2(A), G4T4G4 andG3T4G3 display distinctly different CD spectra. The CDspectral signatures of DNA secondary structures, includ-ing quadruplexes, have been comprehensively analyzedpreviously in terms of their folding topologies (Reid &Chaires, 2003; Vorlickova, Kejnovska, Bednarova,Renciuk, & Kypr, 2012). In particular, parallel and anti-parallel quadruplexes have been distinguished based ontheir maximum and minimum CD bands (Paramasivan,Rujan, & Bolton, 2007; Williamson, 1993). Antiparallelquadruplexes show maxima and minima at 295 and

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265 nm, respectively, while parallel quadruplexes displaymaxima and minima at 260 and 240 nm (Balagurumoor-thy, Brahmachari, Mohanty, Bansal, & Sasisekharan,1992; Giraldo, Suzuki, Chapman, & Rhodes, 1994). Theprominent role of monovalent cations in quadruplex for-mation is evident from the differences in intensity of CDbands observed for G4T4G4 and G4T2G4 in Na+ vs. K+

(Figure 2(B)). The oligonucleotide G3TG3TG3 exhibitsCD maxima and minima at 260 and 240 nm, respec-tively, indicating the formation of a parallel quadruplex.In contrast, the CD spectrum of G3T3G3 annealed inpresence of Na+ or K+ does not show any prominentbands. The role of monovalent cations is also discernibleby comparison of CD spectra of G4T4-2 in presence ofNa+ vs. K+. The maxima at 295 nm observed in presenceof Na+ persists in presence of K+, while the minima at260 observed in presence of Na+ is weaker in presenceof K+ (see Figure 2(B)). Though such a change does not

indicate a clear transformation of an antiparallel quadru-plex into a parallel quadruplex, it does imply a subtlechange in the topology of the quadruplex even withinthe antiparallel motif (Paramasivan et al., 2007).

The differences in the magnitude and nature of CDbands for the various G-rich sequences used can be cor-related to the binding behavior of YOYO-1 towardsthose oligonucleotides. Addition of YOYO-1 to separatesamples of G4T4-2 annealed in presence of Na+ and K+,followed by measurement of fluorescence emission ofthe dye, produces very different results (Figure 3(A)).The fluorescence of YOYO-1 after each addition in sucha titration is indicative of the amount of dye bound tothe specific G-rich DNA, since YOYO-1 exhibits negli-gible fluorescence when free in solution. In case ofG4T4-2, the magnitude of fluorescence is almost two- tothree-fold less in presence of K+ compared to Na+.Interestingly, the oligonucleotide G4T4G4 exhibits verysimilar binding behavior with YOYO-1 in presence ofNa+ and K+ till at least 0.5 μM of the dye (Figure 3(B)).This oligonucleotide had a comparatively smaller differ-ence in its CD spectrum between samples containingNa+ and K+, in contrast to the differences observed forG4T4-2. In this regard, the YOYO-1 binding behavior ofG4T2G4 is very different from that of G4T4G4. Thequadruplexes formed by the two oligos differ in theirtopologies as well as their thermal stabilities. WhileG4T4G4 folds into antiparallel quadruplexes in presenceof Na+ and K+, G4T2G4 forms parallel quadruplexespreferring K+ over Na+ (see Figure 2). Notably, the dif-ference in thermal stability of the quadruplex formed byG4T2G4 in presence of K+ vs. Na+ is much more com-pared to the same difference for G4T4G4 (see Table 2and Figure S1). The quadruplex formed by G4T2G4 inpresence of K+ displays much weaker YOYO-1 binding,as deduced from the lower intensities of fluorescence ofthe dye compared to that in presence of Na+. Thebinding of YOYO to G4T2G4 is thus dependent on theidentity of the monovalent cation in solution. In particu-lar, quadruplex formation by G4T2G4 in presence of K+

leads to stronger secondary structures than in presence ofNa+. The weaker binding of YOYO-1 to G4T2G4 inpresence of K+ points to the strength and natureof quadruplex as being important determinants of dye-binding behavior.

The correlation between quadruplex formation by G-rich sequences with the ability of YOYO-1 to bind is alsoevident from the fluorescence titration behavior of G3T3-2 (Figure 4(A)). This oligonucleotide does not displayany CD bands in presence of Na+ and exhibits one of thehighest magnitudes of fluorescence among the G-richsequences investigated. G3T3-2 only shows very weakCD in presence of K+ with a Tm< 10 °C compared toones formed by G4T4G4 (see Table 2). While the bindingof YOYO-1 with G3T3-2 is marginally weaker in

Figure 2. CD spectral characterization of G-rich DNA studiedin the present work. Samples contain 2 μM DNA in presenceof (A) 10mM NaCl and (B) 10mM KCl.

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presence of K+ compared to Na+, the difference is lessstark compared to that observed for G4T4-2 (vide supra).The strength of the quadruplex secondary structure is thusan important factor in determining the binding capabilityof YOYO-1. Further, the dye-binding behavior of the oli-gonucleotide G3T3G3 also points to the propensity andstrength of quadruplex formation as important determi-nants in YOYO-1 binding. Quadruplex formation byG3T3G3 is not favored either by Na+ or by K+, and asexpected, the YOYO-1 binding profile is nearly identicalin the two cases (Figure 4(B)). The correlation betweenquadruplex formation and YOYO-1 binding is reinforcedby the binding behavior of YOYO-1 with G3TG3TG3(see Figure 4(C)). This G-rich oligonucleotide folds intosimilar quadruplexes in presence of Na+ and K+ as ascer-tained from their CD spectra (see Figure 2). However, thedifference in their thermal stabilities manifests in an enor-mous difference in their dye-binding capability (seeTable 2 and Figure S1). While the quadruplex formed byG3TG3TG3 in presence of K+ is saturated by .5 μMYOYO-1, in contrast, the quadruplex formed in presenceof Na+ continues to bind YOYO-1 at 1 μM of the dye. Incase of G3TG3TG3, the parallel quadruplex motif isfavored by both Na+ and K+ and hence cannot be theprinciple factor governing the binding of the dye. Instead,the stability of the quadruplex as measured by its thermaldenaturation appears to have a stronger correlation withthe ability of YOYO-1 to bind (see Table 2). We haveused the fluorescence titrations to determine associationconstants of YOYO-1 with the G-rich oligos used in thiswork (Table 3). In particular, the distinct differences inbehavior of G4T2G4 and G3TG3TG3 towards YOYO-1binding is substantiated by the corresponding associationconstants in presence of Na+ and K+. The binding affinityof YOYO-1 for all the G-rich sequences is weaker com-pared to the known binding affinity (1010–1012M�1)towards duplex DNA (Glazer & Rye, 1992; Larssonet al., 1994). Notably, sequences that fold into strong andstable quadruplexes display weaker association constantswith YOYO-1.

Figure 3. Fluorescence enhancement of YOYO-1 uponaddition to oligonucleotides possessing three G repeats, (A)G4T4-2, (B) G4T4G4 and (C) G4T2G4 in presence of,respectively Na+ (�) and K+ (○).

Table 2. Melting temperatures of G-rich oligonucleotides usedin Na+ and K+.

Name

Tm (°C)

Na+ K+

G4T4-2 57.6 ± .4 >90G4T4G4 42.6 ± .4 46.8 ± .5G4T2G4 31.1 ± .6 58.5 ± .2G3T3-2 <10 <10G3T3G3 <10 <10G3TG3TG3 43.5 ± .2 >90TBA 47.5 ± .6 >90

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The distinctively weaker binding of YOYO-1 to theG-rich sequences studied in this work raises the questionof its fluorescence behavior being able to distinguishquadruplex vs. duplex species. We addressed this issueby exploring the fluorescence binding behavior ofYOYO-1 with the TBA (Bock, Griffin, Latham,Vermaas, & Toole, 1992). The ionic strength dependenceof the quadruplex formed by TBA has been reported byus previously (Datta & Armitage, 2001). Notably, theidentity of monovalent cations in solution (Na+ or K+) aswell as their concentration have a marked effect on thestability of the quadruplex. As shown in Figure 5,YOYO-1 exhibits greater fluorescence emission for TBAin presence of Na+ compared to K+. This is consistentwith the overall pattern observed so far in terms of moreprominent quadruplex character being unsupportive ofYOYO-1 binding. Next, in samples containing TBAannealed with D1 (5′-CCACACC-3′), an oligonucleotidecomplementary to the central region of TBA; YOYO-1binding was much more when the sample contained Na+.In contrast, TBA annealed with D1 in presence of K+

produced a fluorescence spectrum that was nearly identi-cal to that obtained for TBA alone in K+. Under the con-ditions of this experiment, D1 is unable to form a duplexwith TBA in the presence of K+ and this is revealed bythe weaker binding of YOYO-1. These results are furtherevidence that YOYO-1 binds duplexes with strongeraffinity and are able to distinguish between duplex andquadruplex structures based on the inferior binding tothe latter. The dye-binding behavior with TBA was fur-ther explored by fluorescence titrations (see Figure S2),which confirmed the lower affinity of YOYO-1 for TBAin presence of K+ compared to Na+.

The contribution of sequence traits and identity ofmonovalent cation towards quadruplex formation andstability and in turn towards YOYO-1 binding is alsoclearly evident from the CD spectra of the various oligo-nucleotides measured after addition of YOYO-1. Asshown in Figure 6, the CD intensities of G4T4-2 andG4T4G4 are adversely affected by YOYO-1 binding.Interestingly upon YOYO-1 binding, G4T4G4 in K+

appears to lose its secondary structure completely in con-trast to its fate in presence of Na+. The changes in CDbands of the G-rich DNA upon addition of YOYO-1 areindicative of changes in helicity in the original DNAstructures upon binding of YOYO-1. In particular, inter-calation within or stacking of YOYO-1 on G-quartetslikely weakens the hydrogen-bonding network, therebydisrupting the quadruplex secondary structures thatexisted. The parallel quadruplexes formed by G4T2G4and G3TG3TG3 are affected to a lesser degree asinterpreted from the CD intensities (comparing Figures 2

Figure 4. Fluorescence enhancement of YOYO-1 uponaddition to oligonucleotides possessing three G repeats, (A)G3T3-2, (B) G3T3G3 and (C) G3TG3TG3 in presence of,respectively Na+ (�) and K+ (○).

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and 6). In this regard, the antiparallel quadruplex(es)formed by G4T4G4 and G4T4-2 may be considered tobe more conducive to binding by YOYO-1 compared tothe secondary structures that deviate from the antiparalleltopology. The subtle difference in behavior between theparallel and antiparallel quadruplexes studied as part ofthis work, illustrates the importance of quadruplex topol-ogy within the broader context of strength and propen-sity for formation of quadruplexes. This aspect iscurrently being investigated further by testing appropriatesequences and other commonly used intercalating dyes.

4. Conclusions

Nucleic acid staining dyes have revolutionized the waysin which nucleic acids can be used and applied,especially in a biomedical context. The fluorescenceenhancement of intercalating chromophores is one of themost common premises underlying dye/conjugate-basedmonitoring of interactions involving nucleic acids. In this

context, a one-size-fits-all approach appears to predomi-nate the use of such chromophores. While nucleic acidsdo not present the structural diversity observed inproteins, the range of biologically relevant structures ismuch more than just the double-helical B-form of DNA.The current investigation sheds light on the effect of theG-quadruplex secondary structure on intercalative dyebinding. The results are of considerable interest andvalue both due to the specific chromophore investigatedas well as the observed prevalence of guanine-richsequences in genomic DNA. We observe a strong corre-lation between the strength and propensity of quadruplexformation and the ability of YOYO-1 to bind suchsequences. Specifically, the oligos that form strongquadruplexes allow a smaller enhancement in YOYO-1fluorescence and a weaker binding affinity with YOYO-1 compared to ones that are unable to or fold intoweaker quadruplex structures. This correlation appears to

Table 3. Association constants of YOYO-1 for G-richoligonucleotides.

Name

Ka (M�1)

Na+ K+

G4T4-2 2.7 (±.4)� 107 8.2 (±.05)� 106

G4T4G4 1.8 (±.07)� 107 1.5 (±.3)� 107

G4T2G4 2.3 (±.2)� 107 7.5 (±.08)� 105

G3T3-2 6.3 (±.04)� 107 6.1 (±.2)� 107

G3T3G3 2.4 (±.08)� 107 2.5 (±.1)� 107

G3TG3TG3 5.9 (±.1)� 107 8.3 (±.1)� 105

TBA 1.1 (±.06)� 107 7.6 (±.03)� 105

Figure 5. Comparative fluorescence spectra of YOYO-1 inpresence of TBA under various conditions. Samples contained2 μM TBA and 1 μM YOYO-1 along with 100mM NaCl,100mM KCl, and/or 2 μM D1 as indicated in the legend.Spectra were obtained by excitation at 488 nm.

Figure 6. CD spectral characterization of G-rich DNA used inpresent work upon binding YOYO-1. Samples contain 2 μMoligonucleotides and 1 μM YOYO-1 in presence of (A) 10mMNa+ and (B) 10mM K+.

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be independent of the topology of quadruplex formationwithin the sequences studied. CD and UV-thermal dena-turation experiments reveal that the factors that promotestrong quadruplexes such as presence of potassium ionsand specific sequence traits also effectively blockYOYO-1 from binding with oligos. These results arenotable due to the implications inherent in using YOYO-1 for diverse staining applications involving nucleicacids (Anazawa, Matsunaga, & Yeung, 2002; Gichner,Mukherjee, & Veleminsky, 2006; Marie, Vaulot, &Partensky, 1996). In particular, sequence information onthe nucleic acids being used for dye staining along withknowledge of experimental conditions in such studiescould allow for more effective interpretation of dye-stain-ing results. Conversely, the observation of significantlyvariable fluorescence upon use of YOYO-1 could beattributed to factors such as quadruplex secondarystructures. Similar knowledge of the behavior of otherchromophores is expected to help interpret and optimizetheir use, and are currently being investigated by us.

Supplementary material

The supplementary material for this paper is availableonline at http://dx.doi.10.1080/07391102.2013.807752.

Acknowledgment

B.D. thanks Indian Institute of Technology Gandhinagar for asummer research internship that supported Y.K.G.

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